[go: up one dir, main page]

WO2022249186A1 - Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof - Google Patents

Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof Download PDF

Info

Publication number
WO2022249186A1
WO2022249186A1 PCT/IL2022/050559 IL2022050559W WO2022249186A1 WO 2022249186 A1 WO2022249186 A1 WO 2022249186A1 IL 2022050559 W IL2022050559 W IL 2022050559W WO 2022249186 A1 WO2022249186 A1 WO 2022249186A1
Authority
WO
WIPO (PCT)
Prior art keywords
gdh
fgm
fad
seq
recombinant protein
Prior art date
Application number
PCT/IL2022/050559
Other languages
French (fr)
Inventor
Lital Alfonta
Raz Zarivach
Jennifer GRUSHKA
Itay ALGOV
Original Assignee
B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US17/332,718 external-priority patent/US20210292804A1/en
Application filed by B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University filed Critical B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University
Priority to IL308859A priority Critical patent/IL308859A/en
Priority to EP22810794.2A priority patent/EP4352211A1/en
Publication of WO2022249186A1 publication Critical patent/WO2022249186A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • C07K14/80—Cytochromes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/145—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue
    • A61B5/1468—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means
    • A61B5/1486—Measuring characteristics of blood in vivo, e.g. gas concentration or pH-value ; Measuring characteristics of body fluids or tissues, e.g. interstitial fluid or cerebral tissue using chemical or electrochemical methods, e.g. by polarographic means using enzyme electrodes, e.g. with immobilised oxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
    • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
    • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
    • Y02E60/30—Hydrogen technology
    • Y02E60/50—Fuel cells

Definitions

  • the present invention relates to recombinant proteins, methods of using the same such as for direct electron transfer in bio-electrochemical applications.
  • Redox enzymes are proteins that participate in biocatalytic processes which involve electron transfer (ET). Depending on their redox potential, enzyme mediated redox reactions may be used in anodes and cathodes of biofuel cells as well as in biosensing applications. For the utilization of most redox enzymes in such devices, a mediator molecule should be used to mediate the ET between the enzyme and the electrode. The use of an external redox mediator results in a potential loss as well as in low power outputs.
  • E electron transfer
  • Redox mediator molecules introduce two major challenges to the system: the first is having a middle point potential value that affords an efficient electron transfer from an enzyme to the electrode, which results in insufficient energy production. The other, is the need for diffusion of the mediator molecule through solution towards the electrode which might cause an additional loss of energy.
  • the present invention relates to a recombinant protein having superior FAD-GDH (flavin- adenine dinucleotide glucose dehydrogenase) activity and methods of using the same.
  • a recombinant protein comprising (a) alpha subunit of an FAD-GDH; and (b) a minimal c-type cytochrome peptide, comprising an amino acid sequence comprising a non-canonical amino acid (ncAA), wherein the amino acid sequence is set forth in SEQ ID NO: 20 or SEQ ID NO: 22, and wherein the ncAA is covalently bound to a mediator molecule being represented by Formula I: wherein n is an integer in a range from 1 to 5.
  • polynucleotide comprising a nucleic acid sequence encoding the polypeptide disclosed herein.
  • an expression vector or a plasmid comprising the polynucleotide disclosed herein.
  • transgenic or a transfected cell comprising any one of: (a) the recombinant protein disclosed herein; (b) the polynucleotide disclosed herein; (c) the expression vector or plasmid disclosed herein; or (d) both (a) and (b).
  • an extract obtained or derived from the transgenic or transfected cell disclosed herein is provided.
  • composition comprising any one of: (a) the recombinant protein disclosed herein; (b) the polynucleotide disclosed herein; (c) the expression vector or plasmid disclosed herein; (d) the transgenic or transfected cell disclosed herein; (e) the extract disclosed herein; or (f) any one of (a) to (d), and an acceptable carrier.
  • an electrode coupled to the polypeptide disclosed herein, wherein coupled is by non-covalent interactions.
  • a device comprising the electrode disclosed herein.
  • a method for determining presence, concentration, or both, of an analyte in a liquid medium comprising: (i) providing the device disclosed herein; (ii) contacting the device with the liquid medium; (iii) measuring the electric signal generated between a cathode and an anode, the electric signal being indicative of any one of the presence of the analyte, the concentration of the analyte, and both; and (iv) determining the presence, concentration, or both, of the analyte in the liquid medium based on the electric signal measured in step (c).
  • the ncAA comprises Propargyl-lysine (PrK).
  • n 2
  • the mediator molecule is characterized by having a diethylene oxide group having a length of 6.6A at most.
  • the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
  • the polynucleotide is operably linked to a regulatory element.
  • the regulatory element is a T7 promoter.
  • the expression vector or plasmid further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH.
  • the nucleic acid sequence encoding the gamma subunit of an FAD-GDH is operably linked to a regulatory element.
  • the transgenic or transfected cell is a prokaryotic cell.
  • the analyte comprises glucose
  • Figure 1 includes a 3D model of Flavin-adenine dinucleotide dependent glucose dehydrogenase FAD-GDH-MCD (FGM) based on the structure of GDH, predicted by homology to formate oxidase using Swiss-model (3q9t) and on the structure of MCD from MamP crystal structure (4jj0).
  • FAD-GDH from Burkholderia cepacia is presented with its FAD binding motif (orange, " 1 ") and the FAD co-factor MCD model (light orange, "2”) and the linker (grey, "3") were cut from mamP known 3D structure and include the Heme binding motif (red, "4") and heme molecule (pink, "5"). Heme and FAD molecules were attached to the protein model manually using PyMOL;
  • Figures 2A-2B include micrographs showing Coomassie stained sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis of FGM and GDH elution fractions resulting from IMAC purification.
  • FGM and GDH catalytic sub-units are shown between 60 to 75 kDa. Band at ca. 13 kDa in both lanes correlates to FAD-GDH g subunit (2A); and in-gel heme staining showing the presence of a heme molecule in FGM compared to its absence in GDH (2B, left panel) and anti 6xhis- tag western blot verifying the full-length protein expression;
  • Figures 3A-3D include graphs showing 2,6-dichloroindophenol (DCIP) reduction assay comparing the oxidation of D-glucose by both FGM ( ⁇ ) and GDH ( ⁇ ) (3A) and Heme activity measurements verifying the presence of a porphyrin in FGM compared to GDH.
  • DCIP 2,6-dichloroindophenol
  • SWV square-wave voltammetry
  • Figure 6 includes a map of expressed FGM.
  • Figures 8A-8C include graphs showing GCE/FGM selectivity test that was performed by adding 3.6 mM glucose followed by two sequential additions of sugars in their relevant physiological concentration - 1.67 and 3.3 mM galactose 0.3 and 0.6 mM lactose ( ⁇ ), 2.9 and 5.8 mM maltose (red — ) and 1.67 and 3.3 mM xylose (— ) (8A); other molecules interference was tested by adding 3.6 mM glucose followed by two additions of 0.17mM ascorbic acid and 0.2 mM acetaminophen at an applied potential of 0.0 mV (8B); and 300 mV vs. Ag/AgC (8C);
  • Figure 9 includes a 3D model of FAD-GDH from B. cepacia (green) is presented with its FAD binding motif (orange, "1") and the FAD co-factor (blue, "2").
  • MCD model cyan, "3" and the linker (grey, "4") were cut from MamP known 3D structure and include the heme molecule (pink, "5").
  • the fusion shown in this figure was manually generated using PyMOL software. ncAA possible incorporation sites were colored - proximity to FAD (red, "6"), to MCD (yellow, "7”), distant from both FAD and MCD (black, "8”);
  • Figures 10A-10B include non-limiting pyrene-azide linker structures with different lengths (10A) and 5-etramethylrhodamine (TAMRA)-azide chemical structure (10B);
  • Figures 11A-11E include FGM structural model: a 3D model of FGM was modeled by fusing the crystal structure of FAD-GDH from Burkholderia cepacia (g-subunit in silver, a-subunit in gold, PDB ID: 6A2U) to the crystal structure of MCR2 domain from MamP crystal structure in dark green (PDB ID: 4JJ0) (11A). Dimensions of protein’s height and width were estimated using UCSF chimera software.
  • PrK Propargyl-L-lysine
  • FAD co-factor is blue
  • heme is dark red
  • Chemical structures of PrK (1), PDAz (2) and PCA (3) 11B
  • distance measurements between S247PrK residue and the FAD co-factor left panel
  • between the T558PrK residue and the heme domain (right panel)
  • 11C schematic illustration of the glassy-carbon electrode modification procedure
  • 11D schematic illustration of the expected orientation of FGM- S247PDAz, FGM-T558PDAz and FGM-S247PCA on the electrode surface (HE);
  • Figures 12A-12C include protein expression verification gel micrographs and a graph: anti his- tag western blot of crude lysates from bacterial expression system with the supplementation (+) and without (-) of 2 mM PrK (12A); in-gel heme staining of FGM-S247PrK and FGM-T558PrK (12B); and a graph of DCIP FAD-GDH activity assay for glucose oxidation (12C). Curves showing the reduction of DCIP by FGM-S247PrK (square), GDH-S247PrK (triangle) and FGM-T558PrK (circle);
  • Figure 13 includes a gel micrograph verification of PrK incorporation using TAMRA-Az. 6 m M FGM-S247PrK, GDH-S247PrK and FGM-T558PrK were conjugate to TAMRA-Az using click reaction. The conjugated protein was loaded into SDS-PAGE and a fluorescent image of the gel was taken. PM2700 was used as the protein size marker;
  • Figure 14 includes graphs showing cyclic voltammograms in different scan-rates in the absence of glucose and peak current vs. scan rate plots. An arrow marks the relevant peak of each variant. The relation between the peak current and the scan rate was analyzed by simple linear regression and the R 2 is presented;
  • Figures 15A-15B include plasmid maps of pec86-pylOTS plasmid (15A) and pETDuet- FGMS247TAG expression plasmid (15B);
  • Figure 16 includes images of atomic force microscopy (AFM) measurements of highly oriented pyrolytic graphite (HOPG) after incubation using different linkers. Measurement of the height of the HOPG surface after incubation with acetate buffer (left panel), FGM-S247PDAz (middle panel) and FGM-S247PCA (right panel). The square and line on the right panel indicate the area which its height was analyzed and is presented in the graphs below the respective image, arrows indicate a single protein on the HOPG surface. 200 nm scale bars are represented by white lines. Representative 3D images of the same modified surfaces are present. All images are of the same scale and measured areas of lpmxlpm;
  • Figure 17 includes a graph of differential pulse voltammetry (DPV) curves of EDC-NHS+PCA coupling reaction mixture (1) and click reaction mixture (2) that lacks the protein sample.
  • DUV differential pulse voltammetry
  • Figures 18A-18H includes graphs of electrochemical characterization of wired FGM and GDH: DPV measurements of FGM-S247PDAz (2) and FGM-S247PCA (1) under argon in the absence of glucose (18A).
  • Peaks are indicated by arrows; linearized multistep amperometry current decay plot of FGM-S247PDAz (circle), FGM-T558PDAz (diamond), GDH-S247PDAz (triangle) and FGM- S247PCA (square) (18B); CVs of FGM-S247PDAz, FGM-T558PDAz, GDH-S247PDAz and FGM- S247PCA in high scan rates before (1) and after (4, 5, 3 and 2, respectively) the addition of 5 mM glucose (18C); CV of heme -binding domain attached to the electrode surface before (1) and after (2) the addition of 5 mM of glucose (18D); CVs in scan rate of 10 mV/sec for FGM-S247PDAz, FGM-T558PDAz, GDH-S247PDAz and FGM-S247PCA before the addition of 5 mM glucose (1), and after the addition of 5 mM glucose (3, 5, 4 and
  • Figures 19A-19B include graphs showing linear range of FGM-T558PDAz (19A) and FGM- S247PCA (19B) current response towards glucose;
  • Figure 20 includes anti-His tag western blot analysis of FGM expressed from pTrc plasmid (1) vs. the use of a pETDuet plasmid (2);
  • Figure 21 includes FGM 3D model structure with arrows indicating the ncAA incorporation sites
  • Figures 22A-22C include gel micrographs and a graph showing ncAA incorporated FGM variants characterization: FGM variants after ‘click’ with a fluorescent marker (22A), in-gel heme staining of protein concentrated elution samples (22B) and a graph of FAD-GDH glucose oxidation activity assay (22C);
  • Figures 23A-23C include graphs and a chemical structure elucidation showing site-specific ‘wiring’ verification: Cyclic voltammetry (CV) of electrodes after incubation with a ‘clicked’ FGM and S247PrKFGM using a pyrene-azide linker (23A); FGM - blue and red curves, S247PrKFGM - green and purple curves; CV of ‘clicked’ S247PrKFGM vs. S247PrKFGM entrapped under a dialysis membrane. Entrapped enzymes: blue and red curves, ‘clicked’ enzymes: green and purple curves (23B); and pyrene-di-ethylene oxide-azide linker chemical structure (23C);
  • Figures 24A-24B include graphs showing click reaction effect on S247PrKFGM: CV of S247PrKFGM + pDAz no glucose (green), S247PrKFGM + pDAz 5 mM glucose (purple), S247PrKFGM without pDAz no glucose (blue), S247PrKFGM without pDAz 5 mM glucose (red); and chronoamperometry of S247PrKFGM without pDAz (blue), S247PrKFGM + pDAz (red) (24A) and S247PrKFGMpDAz specificity test (24B); and
  • Figure 25 includes a graph showing CV of S247PrKFGM response to 5 mM glucose in different scan rates.
  • FAD- GDH flavin-adenine dinucleotide glucose dehydrogenase
  • the present invention is directed to a polypeptide comprising a non-canonical amino acid (ncAA). According to some embodiments, the present invention provides an electrode coupled to the polypeptide of the present invention.
  • ncAA non-canonical amino acid
  • the present invention is based, in part, on the surprising finding that using a site-specific incorporation of the ncAA, allows for a of a specific orientation towards the electrode. By controlling the incorporation of the ncAA and orientation, the catalytic current can be improved in response to glucose.
  • the wiring of the polypeptides through different sites result in a significant effect on their electron transfer (ET) characteristics and their ability to communicate with an electrode.
  • the site-specific wiring of the polypeptides allows higher catalytic currents and lower onset potential, indicating highly efficient ET that is gained due to the site-specific wiring.
  • fusing the enzyme with a minimal cytochrome domain (MCD), instead of large cytochrome, allows to shorten the enzyme-electrode distance and improve the direct electron transfer (DET) capabilities of the enzyme.
  • MCD minimal cytochrome domain
  • DET direct electron transfer
  • the fusion enzyme communicated with an electrode directly, without the use of a mediator molecule. Direct electron transfer between the redox enzyme and an electrode resulted in enhancement of chemical detection.
  • the disclosed recombinant enzyme showed a substantially reduced redox potential, e.g., from +400 mV to 0 mV, thereby improving enzyme selectivity in various electrochemical applications including glucose sensing and monitoring as well as an electromotive force in a bio-electrochemical power device.
  • the ncAA comprises Propargyl-lysine (PrK).
  • the ncAA is covalently bound to a mediator molecule comprising polycyclic aromatic system.
  • the ncAA is covalently bound to a mediator molecule, wherein the mediator molecule is represented by Formula I:
  • n is an integer in a range from 1 to 5.
  • n is an integer in a range from 1 to 4, 1 to 3, 2 to 5, 2 to 4, or 2 to 3, including any range therebetween. Each possibility represents a separate embodiment of the invention. [065] In some embodiments, n equals 3. In some embodiments, n equals 2.
  • polypeptide As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to two or more amino acids linked together.
  • polypeptide As used herein, the terms “polypeptide”, “peptide”, “protein”, and “amino acid sequence” as used herein refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the term “peptide”, “oligopeptide”, “polypeptide”, or “protein”.
  • the mediator molecule comprises a diethylene oxide arm or group.
  • the mediator molecule is a linker.
  • the terms” mediator molecule” and “linker: are used herein interchangeably.
  • the diethylene oxide arm or group has a length of 2-8 A, 3-8 A, 4-8 A, 5- 8 A, 6-8 A, 6-7 A, 4-6.5 A, 5.5-7.5 A, or 5.8-6.6 A. each possibility represents a separate embodiment of the invention.
  • the diethylene oxide arm or group has a length of not more than 5 A, 5.2 A, 5.4 A, 5.6 A, 5.8 A, 6.0 A, 6.1 A, 6.2 A, 6.3 A, 6.4 A, 6.6 A, 6.8 A, 7.0 A, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
  • diethylene oxide arm or group length as used herein, comprises total length, average length, or maximal length, of the diethylene oxide arm or group.
  • the present invention provides a polynucleotide comprising a nucleic acid sequence encoding the polypeptide described herein.
  • the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
  • the polynucleotide comprises the nucleic acid sequence:
  • the polynucleotide comprises the nucleic acid sequence:
  • the polynucleotide comprises the nucleic acid sequence:
  • AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG SEQ ID NO: 7
  • polynucleotide and “nucleic acid” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA, or any combination thereof.
  • Polynucleotides encoding polypeptides may be obtained from any source including, but not limited to, a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Accordingly, polynucleotides encoding a polypeptide can be conveniently obtained from a cDNA library prepared from human tissue. The polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
  • the polynucleotide is codon optimized to facilitate or increase translation efficiency in a host cell. In some embodiments, the polynucleotide is codon optimized for expression in a microorganism cell. Methods for codon optimization are common and would be apparent to one of ordinary skill in the art, as well as codon preference of various types of cells, e.g., of E. coli.
  • a polynucleotide comprising: (a) a first regulatory element operably linked to the alpha catalytic subunit of the FGM expression gene; and (b) a second regulatory element operably linked to the gamma catalytic subunit of the FGM expression gene.
  • the regulatory element is a promoter
  • the first regulatory element and the second regulatory element are identical. In some embodiments, the first regulatory element and the second regulatory element is a bacteriophage. In some embodiments, the first regulatory element and the second regulatory element is a T7 phage.
  • the polynucleotide is a DNA molecule.
  • a vector or a plasmid comprising the polynucleotide of the invention.
  • the expression vector or plasmid further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH.
  • each of the polynucleotide encoding the polypeptide of the invention, and the nucleic acid sequence encoding the gamma subunit of an FAD-GDH are operably linked to a separate regulatory element.
  • the expression vector further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH, wherein each of the polynucleotide encoding the polypeptide of the present invention, and the nucleic acid sequence encoding the gamma subunit of an FAD-GDH are operably linked to a separate regulatory element.
  • the regulatory element is a promoter.
  • the promoter is a T7 promoter.
  • transgenic or a transfected cell comprising: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; or d) any combination of (a) to (c).
  • the cell is a prokaryotic cell. In one embodiment, the cell is a bacterial cell.
  • an extract obtained or derived from the cell disclosed herein comprises: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; d) any combination of (a) to (c).
  • composition comprising: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; d) the cell disclosed herein; e) the extract disclosed herein; f) any combination of (a) to (e); and g) and an acceptable carrier.
  • coupled is by non-covalent interactions.
  • non-covalent interactions refer to pi-pi stacking interactions between the pyrene groups of the mediator molecule represented by Formula I and the electrode surface.
  • pi-pi stacking refers to attractive, non-covalent interactions between aromatic rings.
  • a device comprising the electrode described hereinabove.
  • a method for determining an analyte in a liquid medium comprising: (i) providing the device described hereinabove; (ii) contacting the device with the liquid medium; (iii) measuring the electric signal generated between the cathode and the anode, the electric signal being indicative of the presence and/or the concentration of the analyte; and (iv) determining the analyte based on the electric signal.
  • the analyte comprises glucose.
  • a method for transferring an electron to an electrode comprising coupling the polypeptide described herein to an electrode, thereby transferring an electron to the electrode.
  • a method for quantifying the amount of a reporter in a sample having a first detectable range of light absorbance in an oxidized state a second range of light absorbance in a non-oxidized state comprising: (a) contacting polypeptide described herein with the reporter in a non-oxidized state; and (b) measuring the amount of the reporter in an oxidized state, thereby quantifying the amount of a reporter in a sample.
  • the first detectable range of light absorbance is detectable in visible light and the second range of light absorbance is non-detectable in visible light.
  • the reporter is 2,6-Dichloroindophenol. In some embodiments, the 2,6-Dichloroindophenol is coupled to glucose.
  • a method for expression of the polypeptide described herein comprising expressing a first subunit from a first regulatory element and expressing a second subunit from a second regulatory element, thereby expressing the polypeptide described herein.
  • a first subunit is an alpha catalytic subunit of the FGM.
  • a second subunit is a gamma catalytic subunit of the FGM.
  • the regulatory element is a promoter
  • the first regulatory element and the second regulatory element are identical. In some embodiments, the first regulatory element and the second regulatory element is a bacteriophage. In some embodiments, the first regulatory element and the second regulatory element is a T7 phage.
  • the present invention provides, in some embodiments, a recombinant protein comprising: (a) an alpha subunit of an FAD-GDH; and (b) a minimal cytochrome peptide.
  • the alpha subunit of the FAD-GDH is derived or recovered from a prokaryotic cell. In one embodiment, the alpha subunit of the FAD-GDH is derived or recovered from a bacterial cell. In one embodiment, the alpha subunit of the FAD-GDH is Burkholderia cepacian alpha subunit of FAD-GDH. In one embodiment, the alpha subunit of the FAD-GDH of the present invention is derived from a thermostable enzyme, an oxygen independent enzyme, or both.
  • thermostable enzyme refers to an enzyme that is relatively stable to heat.
  • the thermostable enzymes can withstand the high temperature incubation used to remove the modifier groups, typically, but not exclusively, greater than 50° C, without suffering an irreversible loss of activity.
  • the recombinant protein further comprises the gamma subunit of an FAD- GDH.
  • the invention provides a composition comprising or consisting the recombinant protein with or without the gamma subunit of an FAD-GDH.
  • the invention provides a composition comprising or consisting the recombinant protein and the gamma subunit of an FAD-GDH.
  • the invention provides a composition comprising or consisting at least two different proteins: (a) the recombinant protein; and (b) the gamma subunit of an FAD-GDH.
  • the at least two different proteins are unbound.
  • the gamma subunit is from the same FAD-GDH as the alpha subunit.
  • the gamma subunit is from a different FAD-GDH as the alpha subunit.
  • the gamma subunit is from the same or different FAD-GDH as the alpha subunit.
  • the recombinant protein further comprises a minimal cytochrome peptide.
  • the minimal cytochrome peptide is a natural peptide.
  • the minimal cytochrome peptide comprises a non-natural peptide.
  • the recombinant protein is devoid of the gamma subunit of the FAD-GDH.
  • the minimal cytochrome peptide comprises a c-type cytochrome domain. In one embodiment, the minimal cytochrome peptide does not comprise a b-type cytochrome domain. In one embodiment, the minimal cytochrome peptide comprises a c-type cytochrome domain MCR-2 from a MamP protein. In one embodiment, the minimal cytochrome peptide comprises a magnetotactic bacterius minimal cytochrome peptide. In one embodiment, the minimal cytochrome peptide is a magnetotactic bacterius minimal cytochrome peptide.
  • the minimal cytochrome peptide is a peptide comprising or consisting of 11 to 30 amino acids. In one embodiment, the minimal cytochrome peptide is a peptide comprising or consisting of 11 to 24 amino acids. In some embodiments, the minimal domain is a naturally occurring cytochrome. In some embodiments, the minimal domain is a synthetic cytochrome.
  • the minimal cytochrome peptide is a cytochrome peptide (e.g., c-type cytochrome) comprising or consisting of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, including any range therebetween.
  • the peptide comprises cytochrome functionality.
  • the peptide comprises ET functionality.
  • the minimal cytochrome peptide is linked to the amino terminus of the alpha subunit of an FAD-GDH.
  • the minimal cytochrome peptide is linked to the carboxy terminus of the alpha subunit of an FAD-GDH.
  • the minimal cytochrome peptide is linked to the amino terminus of the gamma subunit of an FAD-GDH. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus of the gamma subunit of an FAD-GDH.
  • the minimal cytochrome peptide is linked to the subunit of an FAD-GDH directly or indirectly. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus of the subunit of an FAD-GDH directly or indirectly.
  • the recombinant protein further comprises a linker. In one embodiment, the recombinant protein, further comprises a peptide linker. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus or the amino terminus of the alpha subunit of an FAD-GDH via a peptide linker. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus or the amino terminus of the gamma subunit of an FAD-GDH via a peptide linker.
  • the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 5 to 20 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 8 to 18 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 12 to 15 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 5 to 15 amino acids. In some embodiments, the linker is between 1 and 10, 1 and 9, 1 and 8, 1 and 7, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 3 and 10, 3 and 9, 2 and 8 or 2 and 7 amino acids in length.
  • the linker comprises 30% to 60% glycine. In some embodiments, the linker comprises 30% to 60% serine. In some embodiments, the linker is hydrophilic. In some embodiments, the linker does not cause steric hinderance. In some embodiments, the linker is a flexible linker. In some embodiments, the linker does not interfere with maturation of the porphyrin binding MCD. In some embodiments, the linker does not interfere with enzymatic activity of the other subunits. In some embodiments, the linker is not so short that the other subunit interferes with maturation of the porphyrin binding MCD. In some embodiments, the linker retains the subunits in close enough proximity to allow electron transfer.
  • the linker has a length of up to 20 A, up to 25 A, or up to 30 A. In some embodiments, the linker has a length of greater than or equal to 5 A, 10 A, 12 A, 15 A or 17 A.
  • Non-limiting exemplary linker is a peptide comprising GSGYGSG. In some embodiments, the linker comprises or consists of SEQ ID NO: 24. In some embodiments, the linker comprises or consists a non-peptide backbone.
  • the linker is encoded by a DNA sequence comprising or consisting the nucleotide sequence: GAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCG (SEQ ID NO: 4). It will be understood by a skilled artisan that synonymous substitutions may be made to this sequence.
  • the linker is encoded by a DNA sequence comprising or consisting of a nucleotide sequence synonymous with SEQ ID NO: 4).
  • a shorter linker e.g. shorter than to 5 A
  • a longer linker e.g., longer than 30 A
  • the recombinant protein further comprises a short tag peptide (3 to 20 amino acids long).
  • the short tag peptide is his tag.
  • the tag is a 6x his tag.
  • Protein tags are well known in the art and any tag that does not interfere with the function (redox and ET) of the recombinant protein may be used.
  • the short tag is between 1 and 30, 1 and 25, 1 and 20, 1 and 15, 1 and 10, 2 and 30, 2 and 25, 2 and 20, 2 and 25, 2 and 10, 3 and 30, 3 and 25, 3 and 20, 3 and 15 or 3 and 10 amino acids in length. Each possibility represents a separate embodiment of the invention.
  • the recombinant protein has a molecular weight in the range of 58 to 75 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 60 to 70 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 63 to 65 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 62 to 68 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 63 to 65 kDa.
  • amino acid sequence of the recombinant protein comprises or consists of the following sequence:
  • amino acid sequence of the recombinant protein comprises or consists of the following sequence:
  • amino acid sequence of the recombinant protein comprises or consists of the following sequence:
  • an electrode carrying or coupled to a recombinant protein comprising A, B, C, and D, wherein: A is a cofactor of a redox enzyme; B is a redox enzyme; C is a linker moiety; and D is an electron transfer (ET) domain that is configured to transfer electrons between the electrode and A.
  • the ET comprises a cytochrome.
  • the A, B, C, and D are linked to each other under the following order: A-B- C-D.
  • the cofactor when not bound to a linker moiety, comprises at least one pair of hydroxyl groups.
  • a device comprising the electrode.
  • the electrode comprises a material selected from, without being limited thereto, graphite and glassy carbon electrode (GCE).
  • GCE glassy carbon electrode
  • the electrode is made of or coated by an electrically conducting substance, such as, without being limited thereto gold, platinum, silver, conducting glass such as indium tin oxide (ITO).
  • an electrically conducting substance such as, without being limited thereto gold, platinum, silver, conducting glass such as indium tin oxide (ITO).
  • the term “chemically attach to” it is meant to refer to being attached via a covalent bond.
  • the term “coupled” refers to a physical attachment, such that the two are bonded together.
  • the bond is a covalent bond.
  • the bond is a synthetic bond.
  • the bond is a chemical bond.
  • by “chemically attach to” it is meant to refer to being attached to (“wiring") the electrode via a synthetic linker (also referred “electrode linker” or “mediator molecule” or “mediator”) being configured to link the recombinant protein to an electrode.
  • the wiring can be obtained by a non-specific process, by using a chemical modification and conductive matrices such as, without limitation, graphene oxide and multi -walled carbon nanotubes.
  • the wiring is a site-specific wiring, performed e.g., by inserting at least one non-canonical amino acid in a desired site of one of the groups (A to D) that, optionally, covalently links to a moiety that binds to an electrode as described herein.
  • the recombinant protein disclosed herein e.g., in the form of A-B-C-D
  • DET direct electron transfer
  • the distance between the enzyme’s active site and the electrode is as short as a few Angstroms (e.g., 1 to 20 A). In some embodiments, the distance is between 0-20, 0-19, 0-18, 0-17, 0-16, 0-15, 0-14, 0-13, 0-12, 0-11, 0-10, 1-20, 1-19, 1-18, 1- 17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 3-10, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, or 3-10 A.
  • Each possibility represents a separate embodiment of the invention.
  • the ET domain when linked to the enzyme, is minimal so as not to introduce additional insulation to the system by a complex proteinaceous matrix.
  • the ET domain is linked by a flexible linker. As described herein, the flexibility allows avoiding interruption of the catalytic redox activity.
  • the minimal domain is not more than 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 amino acids. Each possibility represents a separate embodiment of the invention.
  • the cofactor is selected from, without being limited thereto, FAD, NAD+, and NADP+.
  • the redox enzyme refers to an enzyme that can catalyze a redox reaction.
  • the redox enzyme may be selected from, without being limited thereto, oxidase, dehydrogenase, and malic enzyme (e.g., malate dehydrogenase).
  • the redox enzyme is selected from an oxidase, a dehydrogenase, a reductase, a peroxidase, a glyoxalase, a hydroxylase and a malic enzyme.
  • the redox enzyme is sugar dehydrogenase.
  • the sugar is glucose.
  • enzyme is alcohol dehydrogenase.
  • the redox enzyme in the immobilized group is characterized by a redox potential of less than 50 mV. In one embodiment, the redox enzyme in the immobilized group is characterized by a redox potential of 50 mV, 40 mV, 30 mV, 20 mV, 10 mV, 0 V, -10 mV, -20 mV, -30 mV, -40 mV, -50 mV, -60 mV, -70 mV, -80 mV, -90 mV, -100 mV, -110 mV, -120 mV, -130 mV, -140 mV, -150 mV, -160 mV, -170 mV, -180 mV, -190 mV, or -200 mV (induced potential vs. Ag/AgCl)
  • the dehydrogenase is selected from, without being limited thereto, alcohol dehydrogenase, glutamic acid dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and glycerol dehydrogenase.
  • the linker moiety comprises a peptide.
  • the peptide comprises serine.
  • the linker moiety comprises a short peptide e.g. having 5 to 20 amino acids, or 5 to 15 amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, including any range therebetween.
  • the linker is selected from any embodiments of a linker described hereinabove.
  • the linker moiety is a flexible linker.
  • the linker moiety is hydrophilic.
  • the linker moiety is synthetic.
  • the linker moiety is not from cellobiose dehydrogenase.
  • the linker moiety is not from pyranose dehydrogenase.
  • the linker moiety does not interfere with enzyme function and/or DET.
  • a non-limiting example of a linker is a peptide comprising GSGYGSG.
  • the linker is characterized by a length of: 5 to 40, or 20 to 30 A, e.g., 5, 10, 15, 20, 25, 30, 35, or 40A, including any value and range therebetween.
  • the electron transfer domain comprises a cytochrome, e.g., MCD.
  • the electron transfer domain comprises a cytochrome c. In some embodiments, the electron transfer domain does not comprise a cytochrome b. In some embodiments, the recombinant protein is not a naturally occurring protein.
  • the device is a biosensor.
  • biosensors are analytical devices that combine a biological material (e.g., tissues, microorganisms, enzymes, antibodies, nucleic acids etc.) or a biologically-derived material with a physicochemical transducer or transducing microsystem.
  • the device comprises or is configured to attach to an electronic circuitry for energizing the electrode and measuring the response.
  • the biosensor is for measuring the concentration of a sugar in a medium.
  • the biosensor is for measuring the concentration of glucose in a medium.
  • the biosensor is for measuring the concentration of alcohol in a medium.
  • the medium is a bodily fluid.
  • the bodily fluid is selected from at least one of: blood, serum, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, urine, breast milk, interstitial fluid, and stool.
  • the biosensor is capable of measuring whole blood, serum or plasma glucose without dilution or sample processing, which can evaluate the current status of glucose or an individual suffering from diabetes.
  • the bodily fluid is undiluted.
  • the bodily fluid is diluted with a buffer.
  • the biosensor comprises an anode compartment and a cathode compartment, the compartments being in fluid communication, wherein the anode compartment comprises an anode electrode and a substrate; and wherein the cathode compartment comprises a cathode electrode, the anode electrode and cathode electrode in electrical communication.
  • the electrode disclosed herein is the anode.
  • the present invention provides a method for determining an analyte in a liquid medium, the analyte being capable to undergo a biocatalytic oxidation or reduction reaction in the presence of an oxidizer or a reducer, respectively, the method comprising:
  • the method is for determining the presence of the analyte in the medium. In some embodiments, the method is for determining the concentration of the analyte in the medium.
  • the liquid medium is, for example, a body fluid e.g. blood, lymph fluid or cerebro-spinal fluid
  • the method comprises inserting the biosensor into the body and bringing it into contact with the body fluid and determining the analyte in the body fluid within the body.
  • body fluids or any other analyte may be tested non-invasively, and in such cases the method may comprise adding a buffer to the fluid.
  • the buffer has pH 4 to 8. In some embodiments, the buffer has pH 5 ( ⁇ 1).
  • the contacting is in vivo. In some embodiments, the contacting is ex vivo. In some embodiments, a detected electrical signal indicates the analyte is present. In some embodiments, the greater the electrical signal the greater the concentration of analyte. In some embodiments, the electrical signal is compared to a predetermined standard that indicates the concentration of the analyte based on the electrical signal.
  • Non-limiting examples of analytes are sugar molecules e.g., galactose, lactose, maltose and xylose glucose, fructose, maltose; lactate; bilirubin; alcohols or amino acids.
  • sugar molecules e.g., galactose, lactose, maltose and xylose glucose, fructose, maltose; lactate; bilirubin; alcohols or amino acids.
  • a method for transferring an electron to an electrode comprising coupling the disclosed recombinant protein in an embodiment thereof to an electrode, thereby transferring an electron to an electrode.
  • coupling is in the absence of a mediator molecule.
  • amino acid sequence of the recombinant protein comprises or consists of the following sequence:
  • amino acid sequence of the recombinant protein comprises or consists of the following sequence:
  • XI is R.
  • X2 is S.
  • X3 is D.
  • X4 is T.
  • X5 is P.
  • one or more amino acids selected from XI to X5 comprise at least one non-canonical amino acid (ncAA) residue.
  • non-canonical amino acid residue refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, b-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains.
  • Non-canonical amino acid residues may be incorporated into a peptide within the scope of the invention by employing known techniques of protein engineering that use recombinantly expressing cells.
  • one or more from XI to X5 are present in proximity to the protein domain selected from, without being limited thereto: FAD binding domain, or MCD. In some embodiments one or more from XI to X5 are present in a site that is distant from either FAD domain or MCD.
  • proximity it is meant to refer to a distance of 10 to 25 A, e.g., 10, 15, 20, or 25 A, including any value and range therebetween.
  • distal it is meant to refer to a distance of 30 to 100 A, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 A.
  • the ncAA is a clickable ncAA.
  • ncAA By “clickable ncAA” it is meant to refer to ncAA attachable to another group or moiety by biorthogonal chemical mechanism.
  • the ncAA comprises Propargyl -lysine (PrK).
  • the ncAA has attached thereto an electrode linker (mediator) configured to couple the recombinant protein to an electrode.
  • an electrode linker (mediator) configured to couple the recombinant protein to an electrode.
  • the linker comprises an aromatic group.
  • the aromatic group comprises polycyclic aromatic hydrocarbon system.
  • polycyclic aromatic hydrocarbon system it is meant to refer to a system comprising e.g., 3, 4, 5, or 6, fused benzene rings, which, in some embodiments, is in the form in a flat aromatic system.
  • the aromatic group is selected from, without being limited thereto, pyrene, perylene, benzopyrene, oxoperylene, rubrene, perylene bisimide, styrene, anthracene, tetracene, pentacene, or any derivative thereof.
  • the aromatic group comprises pyrene or a derivative thereof.
  • the linker further comprises an azide group.
  • the azide group allows to bind to the PrK, for example, via an alkyne group, e.g., by “click” chemistry.
  • the aromatic system e.g., pyrene group
  • the azide group is present at the other pole of the mediator.
  • the two groups are connected to each other by an alkyl oxide, for example, and without being limited thereto, tri-ethylene oxide, di ethylene oxide or mono -ethylene oxide.
  • the mediator is characterized by a length of 3 to 9 or 4 to 8 A, for example 3, 4, 5, 6, 7, 8, or 9 A, including any value and range therebetween.
  • the recombinant protein (e.g., the cytochrome domain) is bound to a porphyrin comprising a metal. In one embodiment, the recombinant protein is bound to a compound of formula I:
  • R is any electron donor, or a compound as further described herein, wherein the compound of formula I is bound to a metal.
  • the recombinant protein but not the gamma subunit is bound to a porphyrin as described herein.
  • R represents, independently and in each occurrence, a substituent selected from the group consisting of: alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, phosphonate, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, amine, alkanoamine, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamide, or is absent.
  • R represents, independently and in each occurrence, hydrogen.
  • R represents -(Ci-C 6 )alkyl. In some embodiments, R represents -(Ci- C 6 )alkoxy. In some embodiments, R represents -(Ci-C 6 )alkylthio. In some embodiments, R represents - (Ci-C 6 )alkylsulfinyl. In some embodiments, R represents -(Ci-C 6 )alkylsulfonyl. In some embodiments, R represents -[(Cl-C6)alkyl]NH. In some embodiments, R represents -[(Cl-C6)alkyl]COOH.
  • alkyl comprises an aliphatic hydrocarbon including straight chain and branched chain groups.
  • the alkyl group has 21 to 100 carbon atoms, and more preferably 21-50 carbon atoms.
  • the term "long alkyl” comprises an alkyl having at least 20 carbon atoms in its main chain (the longest path of continuous covalently attached atoms). A short alkyl therefore has 20 or less main-chain carbons.
  • an alkyl can be substituted or unsubstituted.
  • the term "alkyl”, as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl.
  • the term "alkenyl” describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond.
  • alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove.
  • alkynyl as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents.
  • cycloalkyl describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted.
  • aryl describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi- electron system.
  • an aryl group may be substituted or unsubstituted.
  • alkoxy describes both an -O-alkyl and an -O-cycloalkyl group.
  • aryloxy describes an -O-aryl.
  • alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule.
  • halide describes fluorine, chlorine, bromine or iodine.
  • haloalkyl describes an alkyl group as defined herein, further substituted by one or more halide(s).
  • haloalkoxy describes an alkoxy group as defined herein, further substituted by one or more halide(s).
  • hydroxyl or “hydroxy” describes a -OH group.
  • thiohydroxy or “thiol” describes a -SH group.
  • the term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group.
  • the term “thioaryloxy” describes both an -S-aryl and a -S-heteroaryl group.
  • the term “amine” describes a -NR’R” group, with R’ and R”.
  • the term “heteroaryl” describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups examples include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • heteroalicyclic or "heterocyclyl” describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. In one embodiment, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.
  • R' is as defined hereinabove.
  • the above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).
  • the term “nitro” group refers to a -N02 group.
  • the term “cyano” or “nitrile” group refers to a -CoN group.
  • the term azide refers to a -N3 group.
  • the term “phosphinyl” describes a -PR'R" group, with R' and R" as defined hereinabove.
  • alkaryl describes an alkyl, as defined herein, which substituted by an aryl, as described herein. In one embodiment, alkaryl is benzyl.
  • heteroaryl describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system.
  • heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine.
  • the heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.
  • halo and “halide”, which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine or iodine, also referred to herein as fluoride, chloride, bromide and iodide.
  • haloalkyl describes an alkyl group as defined above, further substituted by one or more halide(s).
  • the metal is a trivalent metal or a divalent metal.
  • the recombinant protein as described herein comprises both peroxidase activity and oxidative activity. In one embodiment, the recombinant protein bound to a porphyrin comprising a metal comprises both peroxidase activity and oxidative activity. In one embodiment, the recombinant protein bound to a porphyrin comprising a metal comprises both peroxidase activity and oxidase activity.
  • the recombinant protein is characterized by Michaelis-Menten constant K M : ' PP value which is at least 2, 3, 4 or 5, or more, higher compared to plain GDH, as measured under the same condition (e.g., glucose concentration) of glucose oxidation.
  • the recombinant protein is characterized by higher selectivity towards glucose oxidation as compared to other sugar's molecules.
  • the terms “peptide”, “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the peptides, polypeptides and proteins described herein have modifications rendering them more stable while in the body or more capable of penetrating into cells.
  • the terms “peptide”, “polypeptide” and “protein” apply to naturally occurring amino acid polymers.
  • the terms “peptide”, “polypeptide” and “protein” apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
  • the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring.
  • the term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector used to express the recombinant protein in a cell.
  • the recombinant protein is provided within a single composition or kit with the gamma subunit FAD-GDH. In one embodiment, the recombinant protein with the gamma subunit FAD-GDH are provided within a single composition or kit as two separate proteins (unbound).
  • the recombinant protein is a hybrid protein. In some embodiments, the recombinant protein is a chimeric protein. In some embodiments, the c-type cytochrome peptide is from a different protein than the alpha subunit. In some embodiments, the c-type cytochrome peptide is not from FAD-GDH. In some embodiments, the c-type cytochrome peptide is not from GDH. In some embodiments, the nucleic acid sequence encoding the alpha subunit and the sequence encoding the c- type cytochrome peptide are operably linked, such that a full-length protein is produced following translation and/or transcription.
  • operably linked is intended to mean that the two nucleotide sequences of interest are linked to each other in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vectors Another type of vector, wherein virally-derived DNA or RNA sequences are present in the virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses).
  • Viral vectors also include polynucleotides carried by a virus for transfecting into host cells.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively- linked. Such vectors are referred to herein as “expression vectors”.
  • Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid coding for the protein of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • additional elements such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
  • a vector or a plasmid is an artificial composite.
  • a vector or a plasmid as described herein is man made.
  • a vector or a plasmid as described herein is not a product of nature.
  • the vector may be a DNA plasmid delivered via non-viral methods or via viral methods.
  • the viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector.
  • the promoters may be active in mammalian cells.
  • the promoters may be a viral promoter.
  • the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et ah, Nature 327. 70- 73 (1987)), and/or the like.
  • electroporation e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • Heat shock e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • Heat shock e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)
  • a recombinant protein is produced by expressing the recombinant DNA in a cell and then purifying the protein.
  • the cells expressing the DNA are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide.
  • effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production.
  • an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention.
  • a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
  • cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates.
  • culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell.
  • culturing conditions are within the expertise of one of ordinary skill in the art.
  • Purification of a recombinant protein involves standard laboratory techniques for extracting a recombinant protein that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature. Purification can be carried out using a tag that is part of the recombinant protein or thought immuno-purification with antibodies directed to the recombinant protein. Kits are commercially available for such purifications and will be familiar to one skilled in the art.
  • a preparation of purified peptide contains the peptide in a highly-purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the amino acid sequence set forth below in SEQ ID NO: 1.
  • the amino acid sequence with at least 70% homology to SEQ ID NO: 1 is the amino acid sequence set forth in SEQ ID NO: 6.
  • Mutations and deletions in a recombinant protein are created by introducing the mutation or deletion into the recombinant DNA. Methods of site-directed mutagenesis, and routine DNA recombination can be found in such standard textbooks as are enumerated above. Mutagenesis of one amino acid to another may require mutation of 1, 2, or 3 of the bases that make up the codon corresponding to the amino acid to be changed.
  • coupled is directly bound.
  • first detectable range of light absorbance is detectable in visible light and the second range of light absorbance is non- detectable in visible light.
  • the reporter is 2,6-Dichloroindophenol.
  • the reporter is coupled to glucose.
  • 2,6-Dichloroindopheno is coupled to glucose.
  • a DNA molecule encoding the recombinant protein In one embodiment, provided herein is a DNA molecule comprising: a transcription regulatory element, a translation regulatory element or both; operably linked to a nucleotide sequence encoding the recombinant protein. In one embodiment, the invention provides a DNA molecule encoding both the recombinant protein and the gamma subunit of an FAD-GDH. In one embodiment, the invention provides a single DNA molecule encoding both the recombinant protein and the gamma subunit of an FAD-GDH as two separate proteins.
  • a DNA molecule comprising a nucleic acid sequence encoding the recombinant protein.
  • a DNA molecule comprising a nucleic acid sequence encoding the recombinant protein and the gamma subunit of an FAD-GDH.
  • a DNA molecule comprising the nucleic acid sequence selected from the group consisting SEQ ID NOs: 5-7.
  • the invention provides a plasmid or a vector (such as an expression vector) comprising a nucleic acid sequence encoding the recombinant protein.
  • a cell comprising a DNA molecule, a plasmid or a vector as described herein.
  • the cell is a prokaryotic cell.
  • the cell is a bacterial cell.
  • the alpha subunit of the FAD-GDH is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
  • the recombinant protein is encoded by a DNA molecule comprising a coding nucleotide sequence encoding the alpha subunit of the FAD-GDH.
  • the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1200 to 1700 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1200 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1300 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1400 to 1700 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1500 to 1700 nucleotides.
  • the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1500 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1550 to 1640 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1600 to 1650 nucleotides.
  • the alpha subunit of the FAD-GDH is a mutant of alpha FAD-GDH or a mutant of SEQ ID NO: 1.
  • Active mutants of alpha FAD-GDH or SEQ ID NO: 1 are readily available to one of skill in the art.
  • active mutant as used in conjunction with an FAD-GDH, is meant a mutated form of the naturally occurring FAD-GDH.
  • FAD-GDH mutant or variants will typically but not exclusively have at least 70%, e.g., 80%, 85%, 90% to 95% or more, and for example 98% or more amino acid sequence identity to the amino acid sequence of the reference FAD-GDH molecule.
  • the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 70% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD- GDH or a mutant of SEQ ID NO: 1 is at least 75% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 80% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 85% identical to SEQ ID NO: 1.
  • the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 90% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 95% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 97% identical to SEQ ID NO: 1.
  • the gamma subunit of the FAD-GDH is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
  • the gamma subunit of the FAD-GDH is translated into a discrete protein.
  • the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 400 to 700 nucleotides.
  • the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 450 to 650 nucleotides.
  • the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 450 to 550 nucleotides.
  • the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 480 to 530 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 500 to 540 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 500 to 530 nucleotides.
  • the gamma subunit of the FAD-GDH is a mutant of gamma FAD-GDH or a mutant of SEQ ID NO: 2.
  • active mutants of gamma FAD-GDH or SEQ ID NO: 2 are readily available to one of skill in the art.
  • the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 70% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD- GDH or a mutant of SEQ ID NO: 2 is at least 75% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 80% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 85% identical to SEQ ID NO: 2.
  • the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 90% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 95% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 97% identical to SEQ ID NO: 2.
  • the minimal cytochrome peptide is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
  • the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
  • the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
  • TTATCCAG SEQ ID NO: 6 with linker and without his and restriction sites.
  • the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence: CCATGG
  • the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1500 to 3000 nucleotides. In one embodiment, the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1600 to 2600 nucleotides. In one embodiment, the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1800 to 2500 nucleotides. In one embodiment, the recombinant protein is encoded by a DNA sequence of 2000 to 2400 nucleotides.
  • the recombinant protein is 350 to 700 amino acids long. In one embodiment, the recombinant protein is 220 to 600 amino acids long. In one embodiment, the recombinant protein is 250 to 550 amino acids long. In one embodiment, the recombinant protein is 450 to 850 amino acids long. In one embodiment, the recombinant protein is 470 to 750 amino acids long. In one embodiment, the recombinant protein is 500 to 700 amino acids long. In one embodiment, the recombinant protein is 710 to 780 amino acids long. In one embodiment, the recombinant protein is 500 to 600 amino acids long. In one embodiment, the recombinant protein is 520 to 580 amino acids long. In one embodiment, the recombinant protein is 350 to 550 amino acids long.
  • a DNA sequence encoding the recombinant protein and the gamma subunit protein as described herein of SEQ ID NOs: 5 and 6 further comprises a Methionine codon (the initiation codon nucleotide sequence) 5’ to SEQ ID NOs: 5 and/or 6.
  • a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises a Methionine codon (the initiation codon nucleotide sequence) 5’ to SEQ ID NOs: 5 and/or 6.
  • a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises at its 5’ end, a short DNA sequence comprising any 1-10 nucleotides sequence. In one embodiment, a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises at its 5’ end, a short DNA sequence comprising 1-10 nucleotides sequence. In one embodiment, the 1-10 nucleotides sequence comprises the Methionine codon.
  • a DNA sequence or molecule as described herein comprising a coding sequence encoding the recombinant protein, further encodes the gamma subunit protein as described herein (as a separate protein).
  • the DNA sequence encoding the recombinant protein of the invention is any DNA molecule encoding the amino acid sequence encoded by anyone of SEQ ID NOs: 5-7 or the amino acid sequence of anyone of SEQ ID NOs: 8-11.
  • the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 70% identical to anyone of SEQ ID NOs: 5-7.
  • the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 75% identical to anyone of SEQ ID NOs: 5-7.
  • the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 80% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 85% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 90% identical to anyone of SEQ ID NOs: 5-7.
  • the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 95% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 95% identical to anyone of SEQ ID NOs: 5-7.
  • a DNA molecule of the invention or a DNA sequence described herein comprises or consists any sequence encoding the recombinant protein including (but not limited to) the recombinant protein comprising or consisting anyone of SEQ ID NOs: 8-11.
  • a DNA molecule of the invention or a DNA sequence described herein comprises or consists any sequence encoding the recombinant protein and the gamma subunit, as described herein, including (but not limited to) the amino acid sequences set forth in anyone of SEQ ID NOs: 8-11.
  • the recombinant protein and/or the gamma subunit, as described herein is/are translated based on the DNA sequences provided herein.
  • the recombinant protein has an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 97% identical to: (a) a recombinant protein translated from the DNA sequences provided herein or (b) anyone of SEQ ID NOs: 8-11.
  • the protein of the invention comprises a tag.
  • the DNA encoding the proteins of the invention comprises sequence encoding the tag.
  • the tag is selected from an n-terminal tag, a c-terminal tag and an internal tag. A skilled artisan will appreciate that the tag should be positioned so as not to interfere with the function of the recombinant protein. Thus, the tag will not interfere with the redox activity or the DET activity.
  • the tag is a c-terminal tag.
  • the tag is a His tag.
  • the tag is a 6x His tag.
  • the His tag comprises or consists of the amino acid sequence HHHHHH.
  • the DNA encoding the His tag comprises the sequence CATCACCATCACCATCAC (SEQ ID NO: 24) (e.g., in addition to SEQ ID NO: 19-23).
  • SEQ ID NO: 24 any sequence which encodes the tag may be used.
  • Protein tags are well known in the art and include, but are not limited to, HA tags, His tags, GFP tags, Myc tags, biotin tags, FLAG tags, streptavidin tags, and many, many others. Tagging may be useful for purification of the protein, and the tag may be cleaved before the enzyme is used.
  • the tag is small.
  • the tag is equal to or smaller than 40, 35, 30, 25, 20, 15, 10, 7, or 5 amino acids. Each possibility represents a separate embodiment of the invention. A smaller tag may be advantageous in that it is less likely to interfere with DET.
  • the tag is connected to the recombinant protein by a linker.
  • the linker may be a linker such as has been described hereinabove.
  • the linker comprises or consists of the sequence GSGSG.
  • the DNA sequence that encodes the linker comprises or consists of the sequence GGCAGTGGTTCCGGC (SEQ ID NO: 25) (e.g., in addition to SEQ ID NO: 19-23).
  • SEQ ID NO: 25 e.g., in addition to SEQ ID NO: 19-23.
  • the linker is produced by the restriction site introduced into the DNA to produce the recombinant protein. Indeed, there may be a linker produced by restriction site insertion between any of the different parts of the recombinant protein.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”.
  • consisting of means “including and limited to”.
  • consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, and material arts.
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, of aesthetical symptoms of a condition.
  • FAD-GDH g subunit as well as its catalytic a subunit were cloned into pTrcHis6A2 vector between Ncol and Hindlll restriction sites.
  • the partial FAD-GDH gene was followed by a short flexible polypeptide linker (13 amino-acids long) and the MCR2 (minimal cytochrome domain - MCD) DNA sequence with 6xHis tag at the sequence’s C-terminal, as shown in Figure 6 in a map of the new fusion protein.
  • MCR2 minimal cytochrome domain - MCD
  • the Gamma subunit is expressed as a separate protein encoded from nucleotide 3 to nucleotide 512 of the DNA molecule described herein.
  • the Alpha subunit is from nucleotide 512 to nucleotide 2128.
  • the flexible linker is from nucleotide 2129 to nucleotide 2167.
  • the carboxy terminal MCD linker is from nucleotide 2168 to nucleotide 2238. 6 times His-Tag from nucleotide 2239 to nucleotide 2269.
  • the complete pTrcHis6A2-FAD-GDH-MCD plasmid was transformed into E-coli BL21 cells for the expression of the fusion protein (FAD-GDH-MCD).
  • Bacteria were cultured in an auto-induction medium (FormediumTM, Hunstanton, England) with 0.5% glycerol (Bio-Lab ltd., Jerusalem, Israel) and 10pg/mL carbenicillin (Apollo, Manchester, England) and grown in 37 °C, with shaking at 250 rpm, for 6 hours after which cells were transferred to 27 °C for 18 additional hours.
  • Tris-base buffer TB, Fisher scientific, Geel, Belgium
  • lysis buffer 300 mM KC1, 50 mM K
  • the fusion enzyme was purified using IMAC purification system (Bio-Rad, Profinia, Hercules, CA, USA) according to manufacturer instructions.
  • DMB dimethoxybenzidine
  • ImM hydrogen peroxide Sigma- Aldrich, Rehovot, Israel
  • peroxidase activity test was performed in the absence of hydrogen peroxide.
  • 0.17 mM DMB and 172 mM glucose were mixed with 8 pF of 0.1 mg/mF Horseradish peroxidase (HRP, Sigma-Aldrich, Rehovot, Israel) to generate a reaction mix.
  • Chronoamperometric measurements ( Figure 7) were performed under the same conditions with the application of 0 mV vs. Ag/AgCl with the addition of varying concentrations of glucose or potential interfering molecules.
  • Square wave voltammetry (SWV) measurements were performed under the same conditions.
  • FGM selectivity test FGM selectivity test
  • biocatalytic recombinant protein The biocatalytic recombinant protein
  • a fusion enzyme was designed in a combination of a biocatalytic function from a redox enzyme domain that was fused to a natural minimal ET domain via a short polypeptide linker as shown in Figure 1.
  • the catalytic domain the a subunit of an FAD-GDH from Burkholderia cepacia was used.
  • the c-type cytochrome domain MCR-2 from a MamP protein which originates from a magnetotactic bacteria magnetoovoid bacterium MO- 127 was chosen.
  • MamP is part of the magnetosome, a unique organelle that is found in magnetotactic bacteria that allows magneto taxis to occur in these bacteria.
  • MCR-2 is one of the shortest natural c-type cytochromes known today (23 amino-acids long), thus can be used to achieve DET.
  • FGM FAD-GDH-MCD
  • Fusion enzyme’s engineered DNA sequence was cloned into pTrcHis6A2 expression vector and was transformed into E-coli BL21.
  • FGM was overexpressed in the bacterial expression system and then purified by utilizing immobilized metal affinity chromatography (IMAC) purification system ( Figure 2A).
  • IMAC immobilized metal affinity chromatography
  • Figure 2B In-gel heme staining was performed to verify the presence of the heme compared to GDH and Anti his-tag Western blot analysis was performed in order to verify the full-length enzyme’s expression (Figure 2B, right panel).
  • IMAC immobilized metal affinity chromatography
  • Figure 2B right panel
  • both FGM and GDH enzymes were expressed and their respective bands appeared in the expected size - ca. 64 kDa and 62 kDa for FGM and GDH, respectively.
  • In-gel heme staining revealed a band for FGM only, indicating the presence of a porphyrin containing iron bound to
  • FGM catalytic redox activity and heme peroxidase activity were measured biochemically and compared to GDH as shown in Figure 3A.
  • FGM has oxidized D-glucose as was measured by FAD- GDH activity assay in 50 mM Tris-base (pH 7.0), 0.6 mM 2,6-Dichloroindophenol (DCIP) and 0.6 mM phenazine methyl sulfate (PMS) in 37 °C.
  • FGM has also shown peroxidase activity, measured by heme activity assay in 1 mM 3,3’- dimethoxybenzidine (DMB) and 1 mM of hydrogen peroxide. Absorbance in 455 nm was monitored while the DMB oxidation occurs by the MCD, resulted in an oxidized DMB (red color). Heme activity assay results indicate that FGM indeed binds a heme group while no heme molecules are bound by GDH. [0280] Absorbance measurements of protein sample spectrum revealed a peak in absorbance at 408 nm for FGM and no peak for GDH, indicating presence of heme c in FGM (Figure 3B).
  • 408 nm/A280 nm ratio was calculated to be 0.4 for FGM expressed in the presence of pEC86 plasmid, compared to 0.2 for FGM expressed in the absence of this plasmid, indicating more efficient heme maturation in the presence of the helper plasmid.
  • K M 3PP values were 157 ⁇ 5 m M and 174 ⁇ 9 m M for FGM and GDH, respectively, showing different affinity of the enzymes toward the substrate.
  • GDH GDH’s K M 3PP value is lower than reported values but yet in the same order of magnitude of some.
  • K M : ' PP value was ca. 3 times higher for FGM compared to GDH, indicating faster oxidation of D-glucose by FGM.
  • FGM also showed more than 3 times higher catalytic efficiency (k) compared to GDH (Table 1).
  • the electrochemical activity of the enzymes was measured to determine whether the addition of the minimal cytochrome domain improves enzyme-electrode ET.
  • a standard 3 electrode electrochemical cell with 0.9 mm graphite rod as the auxiliary electrode were used, 3M KC1 saturated Ag/AgCl reference electrode and 3 mm diameter glassy carbon electrode (GCE) as the working electrode.
  • 10 pL of ca. 30pg/mL FGM or GDH enzyme solution were dropped on the GCE surface and dried in 4 °C overnight.
  • the electrodes surface was then covered with 12-14 kDa dialysis membrane tightened to the surface with an O-ring to keep the enzyme close to the electrode surface during measurements and to avoid enzyme diffusion to the surrounding buffer.
  • Cyclic voltammetry (CV) measurements were performed to compare the enzyme-electrode communication of FGM to that of GDH.
  • FGM After the addition of glucose to a final concentration of 5 mM, FGM demonstrated a higher electrocatalytic current compared to that of GDH with an onset potential of ca. (-) 150 mV.
  • SWV square-wave voltammetry
  • the electrochemical K M : ' PP is 2.84 ⁇ 0.57 mM for GDH and 1.40 ⁇ 0.27 mM for FGM, indicating no significant difference in the affinity towards glucose.
  • the imax value is one order of magnitude higher for FGM compared to GDH - 2.04 ⁇ 0.45 pA cm 2 and 0.4 ⁇ 0.17 pA cm 2 , respectively.
  • the difference in the i max value is indicative that the DET efficiency is different between the two enzymes where FGM shows five to seven times higher current then GDH for the same glucose concentrations.
  • Burkholderia Cepacia is considered a good candidate for biosensing applications because of its stability in high temperatures and insensitivity to oxygen.
  • an improved DET was provided, showing higher catalytic currents compered to GDH with almost the same affinity to the substrate.
  • FGM showed much higher currents for the same substrate concentrations compared to GDH, which makes it more accurate for glucose biosensing with improved sensitivity than previously reported for GDH.
  • Non-canonical amino acids (ncAAs) incorporation into FGM for site-specific wiring to an electrode FGM electron transfer machineries investigation [0290]
  • ncAAs non-canonical amino acids
  • a few constructs containing the amber (TAG) mutation on pTrcHis6A2-FGM plasmid were prepared using standard site-directed mutagenesis PCR protocol. The mutations were chosen with a proximity to the protein different domains - FAD binding domain, MCD and one site that is distant from either FAD domain or MCD. For proximity to FAD binding domain, two sites were found to be possible for ncAA incorporation - R42X and S247X ( Figure 9 - red), both ca.
  • Mutated plasmids were transformed into super competent E. coli DH5a cells and were plated on selective LB-agar plates. Bacterial colonies were isolated and plasmids were purified using miniprep kit followed by sequencing.
  • Plasmids, with amber codon-containing FGM mutants sequences were co-transformed to E. coli BL21 strain containing the pEVOL plasmid expressing Pyrrolysyl orthogonal translation system to incorporate Propargyl-lysine (PrK) into the protein sequence.
  • PrK containing protein was expressed in 20 mL auto-induction medium (AIM) in the presence of ImM PrK, lysed using Bugbuster lysis solution and was isolated utilizing IMAC purification method.
  • PrK is an example to a clickable ncAA, all clickable biorthogonal chemical handles can be considered for the site-specific wiring of this enzyme.
  • a synthetic linker In order to wire FGM with site-specifically incorporated PrK to an electrode, a synthetic linker was used.
  • the synthetic linker contained a pyrene group in one pole and an azide group at the other. The two groups were connected by a tri-ethylene oxide, di-ethylene oxide or mono-ethylene oxide, to get three different lengths of 8.3, 6.4 and 4.3 A, respectively.
  • the pyrene group is a polycyclic aromatic hydrocarbon consisting of four fused benzene rings, results in a flat aromatic system. Due to overlapping of p-bonds between aromatic side chains, the pyrene group can be attached to glassy carbon electrodes surface through p-p stacking.
  • the azide group was used to attach the alkyne group of PrK using “click” chemistry. Exemplary pyrene-azide linker structures with different lengths are presented in Figures 10A-10B. Click reaction:
  • Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is based on the formation of 1,4- disubstituted 1,2,3-triazoles between a terminal alkyne and an aliphatic azide in the presence of copper.
  • the reaction is a facile, selective, high yielding with mild conditions and with few or no byproducts. It can be performed in room-temperature, what makes it relevant for use in proteins.
  • CuAAC was used to link FGM to a pyrene-azide synthetic linker.
  • TAMRA-azide is an azide-linked reporter tag that can be used for visualization of alkyne containing proteins.
  • purified FGM was clicked with the florescent marker TAMRA-azide.
  • Clicked protein sample was loaded on SDS-PAGE and the protein gel was checked for florescence using LAS4000 camera.
  • anti 6xHis-tag antibodies will be used for Western blot analysis.
  • MS -MS analysis was performed on isolated protein to give another validation for PrK incorporation.
  • R42X (SEQ ID NO: 14); S247X (SEQ ID NO: 15); D395X (SEQ ID NO: 16); T558X (SEQ ID NO: 17); P560X (SEQ ID NO: 18), wherein X is a non-canonical amino acid (ncAA).
  • FGM protein sequences (all sequences are of the FAD-GDH a-subunit+MCD, without the g-subunit): R42X (SEQ ID NO: 19); S247X (SEQ ID NO: 20); D395X (SEQ ID NO: 21); T558X (SEQ ID NO: 22); P560X (SEQ ID NO: 23), wherein X is a non-canonical amino acid (ncAA).
  • Dimethoxybenzidine was purchased from Alfa Aesar (Heysham, England), 2,6-Dichlorophenolindophenol was purchased from Sigma- Aldrich (Rehovot, Israel), phenazine methyl sulfate and PCA from Tokyo chemical industry (Tokyo, Japan).
  • TAMRA-Azide, THPTA and sodium ascorbate were purchased from Sigma-Aldrich (Rehovot, Israel).
  • PDAz linker was purchased from Lumiprobe (Hunt Valley, MD, USA).
  • Highly oriented pyrolytic graphite grade ZYB was purchased from Ted Pella inc. (Redding, CA, USA).
  • Sodium acetate was purchased from Avantor inc. (Radnor, PA, USA), acetic acid from Bio-Lab ltd. (Jerusalem, Israel) and glassy carbon electrodes, Ag ⁇ AgCl reference electrodes, alumina polishing pad and 0.05pm alumina slurry were purchased from ALS (Tokyo, Japan).
  • Urea, ascorbic acid and lactate were purchased from Sigma-Aldrich (Rehovot, Israel).
  • HOPG highly oriented pyrolytic graphite
  • HOPG surface was cleaned before each measurement by cleaving it and exposing a new layer using an adhesive tape. Measurements were performed using a Cypher-ES (Asylum research, Oxford instruments) on AC mode with a micro cantilever BL-AC40TS (Olympus, Japan).
  • E. coli BF21 bacterial expression system was used for the expression of the FGM enzyme with site-specifically incorporated UAA.
  • Pyrrolysyl orthogonal translation system (pylOTS) DNA sequence was amplified from the pEVOF plasmid using polymerase chain reaction (PCR) and cloned into the backbone of pec86 plasmid using Gibson’s assembly to generate a new plasmid called pec86-pylOTS ( Figure 15A).
  • pylOTS allows the continuous expression of an orthogonal translation system (orthogonal tRNA and aminoacyl tRNA synthetase) for incorporation of PrK (1) using the amber (TAG) stop codon suppression, while the pec86 plasmid is responsible for the continuous expression of E. coli cytochrome c maturation system.
  • the second plasmid, pETDuet-FGM ( Figure 15B) was used for the expression of FGM with a TAG mutation encoded in the desired incorporation site.
  • Four different TAG mutants were planned using site-directed mutagenesis, according to GDH crystal structure.
  • FGM-S247PrK expression was first verified using anti His-tag western-blot analyses (Figure 12A). Two expression cultures were grown, while only one of them was supplemented with 2 mM of (1), the cells were then lysed and analyzed. It can be seen that only with the supplementation of (1) to the expression culture we could observe a 67 kDa band indicating the successful expression of a full- length His-tagged protein. In the absence of (1) in the growth culture, we could not detect any protein band in the relevant size.
  • Figure 16 show the AFM measurements of HOPG surfaces after 10 minutes of incubation in acetate buffer ( Figure 16, left panel), FGM-S247 wired through (2) ( Figure 16, middle panel) and FGM-S247 wired through (3) ( Figure 16, right panel).
  • HOPG is atomically flat
  • the inventors could measure the height of the bound enzyme on the HOPG surface and compare it to an approximate enzyme foot -print and height calculated from the crystal structure of FAD-GDH (Figure 11A) taking into account the estimated lengths of the respective linkers.
  • Figure 11A only acetate buffer added to the surface, does not result in salt accumulation ( Figure 16, left panel).
  • Measurement of the FGM-S247 linked through (3) showed islands of bound molecules averaging around 70 nm in height, almost 10 times higher than the height expected from a monolayer of FGM. These islands may be a result of protein aggregation caused by the wiring of the enzyme through multiple sites as expected from non-specific wiring. Coupling to (3) may result in multiple pyrene molecules on each protein.
  • FGM- S247PCA shows two well-defined peaks at (-100) mV and (+75) mV
  • FGM-S247PDAz shows a minute peak at (-250) mV and a defined albeit wide peak at ca. 0 mV vs. Ag/AgCl reference electrode.
  • a control experiment where the DPV was performed on the conjugation reagents only (without adding protein samples) shows that the observed peaks indeed originate from FGM electroactive-sites (Figure 17) and not from the presence of other factors in the reaction mixture.
  • Figure 18E shows the bioelectrocatalytic currents of three site-specifically wired variants and one non-specifically wired enzyme in response to 5 mM glucose addition using CV with a slow potential scan rate of 10 mV/sec.
  • Figure 18C shows the catalytic currents from the different variants almost independently from ET rates (provided that those ET rates are faster than the potential scan rate).
  • the catalytic response of the site-specifically wired enzymes was much higher compared to the non-specifically wired enzyme (ca.
  • both GDH-S247PDAz and FGM-S247PDAz which are bound with proximity to the FAD domain, present almost the same catalytic current while the FGM-T558PDAz shows almost two-fold higher catalytic current compared to both of them.
  • the substrate has a direct access to the catalytic site and the electrons can be transferred directly to the electrode without any barriers.
  • ET can be deterred by proteins covering the electrode in different orientations that are not optimal for efficient ET.
  • the broader linear range of FGM-T558PDAz was found to be 0.01 - 2 mM glucose while FGM-S247PCA showed a linear range of 0.1 - 2 mM only (both with R 2 >0.92) ( Figures 19A-19B). Improving the surface coverage will allow more available reaction sites (in a similar manner as improving enzyme specific activity, this could be achieved by using porous electrodes for example) that can result in a broader dynamic range.
  • Non-specific wiring methods such as EDC-NHS coupling allows the covering of GCE with proteins but without the ability to control its orientation.
  • site-specific UAA incorporation the inventors have created a unique orthogonal “chemical handle” that allows the conjugation of a linker in only one anchoring point on the protein sequence, and by that allowed determination of a specific orientation towards the electrode.
  • the controlled orientation results in ca. 20 times higher catalytic currents in response to glucose, higher ET efficiency that was shown by the ability of the enzyme to transfer electrons in a scan rate as high as 500 mV/sec and a scattered mono-layer pattern on the surface as observed by AFM measurements. Wiring of proteins through different sites result in a significant effect on their ET characteristics and their ability to communicate with an electrode.
  • FGM expression gene is built from two sub-units, the alpha catalytic subunit, and the gamma subunit which is a helper protein that enables correct folding of the alpha subunit and responsible for the high stability of the whole protein complex. So far, the inventors have been working with an expression plasmid that has both genes under the same promoter (Trc promoter), leading to low expression levels of FGM. In order to improve its expression levels, the inventors have cloned the FGM gene into a pETDuet plasmid, where each of the subunits is expressed from “its own” T7 promoter. The use of pETDuet results in much higher expression levels for FGM ( Figure 20), indicating that using a different promoter for each subunit indeed increases expression efficiency.
  • the inventors have engineered the pec86 plasmid (cytochrome c maturation plasmid) by fusing into its backbone the orthogonal tRNA and aminoacyl tRNA synthetase DNA sequences from pEVOF-pylOTS plasmid (ncAA incorporation plasmid), creating a new plasmid - pEC86-pylOTS.
  • D395PrKFGM was barely active, probably due to an effect of the change on the enzyme active site.
  • the D395 site mutation was planned according to the SWISS-model homology and found to be different from the real protein structure (found in a surface exposed loop in the homology model, while the real protein structure showed that it is located in the middle of an important a-helix).

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medical Informatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Surgery (AREA)
  • Optics & Photonics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Emergency Medicine (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

A polypeptide comprising a residue substituted by a non-canonical amino acid (ncAA) is provided. Methods for: (a) transferring an electron to an electrode, by coupling the polypeptide to an electrode; and (b) quantifying the amount of an analyte e.g., glucose are also provided.

Description

RECOMBINANT FLAVIN-ADENINE DINUCLEOTIDE GLUCOSE
DEHYDROGENASE AND USES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[001] This application claims the benefit of priority of U.S. Patent Application No. 17/332,718, titled “RECOMBINANT FLAVIN-ADENINE DINUCLEOTIDE GLUCOSE DEHYDROGENASE AND USES THEREOF”, filed on May 27, 2021, the contents of which are incorporated herein by reference in their entirety.
FIELD OF INVENTION
[002] The present invention relates to recombinant proteins, methods of using the same such as for direct electron transfer in bio-electrochemical applications.
BACKGROUND
[003] Redox enzymes are proteins that participate in biocatalytic processes which involve electron transfer (ET). Depending on their redox potential, enzyme mediated redox reactions may be used in anodes and cathodes of biofuel cells as well as in biosensing applications. For the utilization of most redox enzymes in such devices, a mediator molecule should be used to mediate the ET between the enzyme and the electrode. The use of an external redox mediator results in a potential loss as well as in low power outputs.
[004] Redox mediator molecules introduce two major challenges to the system: the first is having a middle point potential value that affords an efficient electron transfer from an enzyme to the electrode, which results in insufficient energy production. The other, is the need for diffusion of the mediator molecule through solution towards the electrode which might cause an additional loss of energy.
SUMMARY OF THE INVENTION
[005] The present invention relates to a recombinant protein having superior FAD-GDH (flavin- adenine dinucleotide glucose dehydrogenase) activity and methods of using the same.
[006] According to one aspect, there is provided a recombinant protein, comprising (a) alpha subunit of an FAD-GDH; and (b) a minimal c-type cytochrome peptide, comprising an amino acid sequence comprising a non-canonical amino acid (ncAA), wherein the amino acid sequence is set forth in SEQ ID NO: 20 or SEQ ID NO: 22, and wherein the ncAA is covalently bound to a mediator molecule being represented by Formula I:
Figure imgf000004_0001
wherein n is an integer in a range from 1 to 5.
[007] According to another aspect, there is provided a polynucleotide comprising a nucleic acid sequence encoding the polypeptide disclosed herein.
[008] According to another aspect, there is provided an expression vector or a plasmid comprising the polynucleotide disclosed herein.
[009] According to another aspect, there is provided a transgenic or a transfected cell comprising any one of: (a) the recombinant protein disclosed herein; (b) the polynucleotide disclosed herein; (c) the expression vector or plasmid disclosed herein; or (d) both (a) and (b).
[010] According to another aspect, there is provided an extract obtained or derived from the transgenic or transfected cell disclosed herein.
[Oi l] According to another aspect, there is provided a composition comprising any one of: (a) the recombinant protein disclosed herein; (b) the polynucleotide disclosed herein; (c) the expression vector or plasmid disclosed herein; (d) the transgenic or transfected cell disclosed herein; (e) the extract disclosed herein; or (f) any one of (a) to (d), and an acceptable carrier.
[012] According to another aspect, there is provided an electrode coupled to the polypeptide disclosed herein, wherein coupled is by non-covalent interactions.
[013] According to another aspect, there is provided a device comprising the electrode disclosed herein. [014] According to another aspect, there is provided a method for determining presence, concentration, or both, of an analyte in a liquid medium, the analyte being capable of undergoing a biocatalytic oxidation or reduction reaction in the presence of an oxidizer or a reducer, respectively, the method comprising: (i) providing the device disclosed herein; (ii) contacting the device with the liquid medium; (iii) measuring the electric signal generated between a cathode and an anode, the electric signal being indicative of any one of the presence of the analyte, the concentration of the analyte, and both; and (iv) determining the presence, concentration, or both, of the analyte in the liquid medium based on the electric signal measured in step (c).
[015] In some embodiments, the ncAA comprises Propargyl-lysine (PrK).
[016] In some embodiments, n equals 2.
[017] In some embodiments, the mediator molecule is characterized by having a diethylene oxide group having a length of 6.6A at most.
[018] In some embodiments, the polynucleotide comprises a nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
[019] In some embodiments, the polynucleotide is operably linked to a regulatory element.
[020] In some embodiments, the regulatory element is a T7 promoter.
[021] In some embodiments, the expression vector or plasmid further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH.
[022] In some embodiments, the nucleic acid sequence encoding the gamma subunit of an FAD-GDH is operably linked to a regulatory element.
[023] In some embodiments, the transgenic or transfected cell is a prokaryotic cell.
[024] In some embodiments, the analyte comprises glucose.
[025] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.
BRIEF DESCRIPTION OF THE DRAWINGS
[026] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings and images. With specific reference now to the drawings and images in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings and images makes apparent to those skilled in the art how embodiments of the invention may be practiced. [027] Figure 1 includes a 3D model of Flavin-adenine dinucleotide dependent glucose dehydrogenase FAD-GDH-MCD (FGM) based on the structure of GDH, predicted by homology to formate oxidase using Swiss-model (3q9t) and on the structure of MCD from MamP crystal structure (4jj0). FAD-GDH from Burkholderia cepacia is presented with its FAD binding motif (orange, " 1 ") and the FAD co-factor MCD model (light orange, "2") and the linker (grey, "3") were cut from mamP known 3D structure and include the Heme binding motif (red, "4") and heme molecule (pink, "5"). Heme and FAD molecules were attached to the protein model manually using PyMOL;
[028] Figures 2A-2B include micrographs showing Coomassie stained sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) analysis of FGM and GDH elution fractions resulting from IMAC purification. FGM and GDH catalytic sub-units are shown between 60 to 75 kDa. Band at ca. 13 kDa in both lanes correlates to FAD-GDH g subunit (2A); and in-gel heme staining showing the presence of a heme molecule in FGM compared to its absence in GDH (2B, left panel) and anti 6xhis- tag western blot verifying the full-length protein expression;
[029] Figures 3A-3D include graphs showing 2,6-dichloroindophenol (DCIP) reduction assay comparing the oxidation of D-glucose by both FGM (■) and GDH (·) (3A) and Heme activity measurements verifying the presence of a porphyrin in FGM compared to GDH. Measurements were performed in 37°C, 50 mM Tris buffer, pH=7.0 (3B); Peroxidase activity interference test; FGM was compared to Glucose oxidase (GOx) which is oxygen sensitive; No hydrogen peroxide was detected upon glucose oxidation by FGM while GOx, as a positive control, showed hydrogen peroxide production and its subsequent reduction by HRP is visible (3C); and absorbance spectrum of FGM and GDH showing peak in absorbance at 408 nm for FGM and no peak for GDH (3D);
[030] Figures 4A-4C include graphs showing cyclic voltammograms of GCE/GDH and GCE/FGM with (+) and without (-) 5m M glucose. The measurements were performed in 150 mM phosphate-citrate buffer, pH=5.0 at room temperature vs Ag/AgCl as a reference electrode at a scan rate of 5mV s 1 (4A) and 100 mV s 1 (4B), and square-wave voltammetry (SWV)s of GCE/GDH and GCE/FGM. Measurements performed in 150 mM phosphate-citrate buffer, pH = 5.0 vs Ag/AgCl reference electrode with 5 mV steps, amplitude 10 mV and a frequency 5 Hz (4C). Background GCE current subtracted from the signal (GCE: glassy carbon electrode);
[031] Figures 5A-5B include graphs showing steady state currents from chronoamperometry measurements of GCE/GDH (·) and GCE/FGM (■) using different glucose concentrations (5A); and Linweaver-Burk plots of electrochemical activity for both, FGM and GDH. Linear trend line equations were calculated to be y=123x+42.3 for GDH and y=10.1x+7.1 for FGM (5B);
[032] Figure 6 includes a map of expressed FGM. For the GDH, MCD sequence has been removed; [033] Figures 7A-7C include graphs showing Michaelis-Menten (7A) and Lineweaver-Burk (7B) plots of biochemical FAD-GDH activity for both, FGM and GDH. Linear trend line equations were calculated to be y=1.2x+6.7 for GDH and y=1.2x+7.7 for FGM and chronoamperometric measurements of glucose catalytic oxidation by FGM and GDH at an applied potential of 0.0 mV (7C);
[034] Figures 8A-8C include graphs showing GCE/FGM selectivity test that was performed by adding 3.6 mM glucose followed by two sequential additions of sugars in their relevant physiological concentration - 1.67 and 3.3 mM galactose
Figure imgf000007_0001
0.3 and 0.6 mM lactose (···), 2.9 and 5.8 mM maltose (red — ) and 1.67 and 3.3 mM xylose (— ) (8A); other molecules interference was tested by adding 3.6 mM glucose followed by two additions of 0.17mM ascorbic acid and 0.2 mM acetaminophen at an applied potential of 0.0 mV (8B); and 300 mV vs. Ag/AgC (8C);
[035] Figure 9 includes a 3D model of FAD-GDH from B. cepacia (green) is presented with its FAD binding motif (orange, "1") and the FAD co-factor (blue, "2"). MCD model (cyan, "3") and the linker (grey, "4") were cut from MamP known 3D structure and include the heme molecule (pink, "5"). The fusion shown in this figure was manually generated using PyMOL software. ncAA possible incorporation sites were colored - proximity to FAD (red, "6"), to MCD (yellow, "7"), distant from both FAD and MCD (black, "8");
[036] Figures 10A-10B include non-limiting pyrene-azide linker structures with different lengths (10A) and 5-etramethylrhodamine (TAMRA)-azide chemical structure (10B);
[037] Figures 11A-11E include FGM structural model: a 3D model of FGM was modeled by fusing the crystal structure of FAD-GDH from Burkholderia cepacia (g-subunit in silver, a-subunit in gold, PDB ID: 6A2U) to the crystal structure of MCR2 domain from MamP crystal structure in dark green (PDB ID: 4JJ0) (11A). Dimensions of protein’s height and width were estimated using UCSF chimera software. Propargyl-L-lysine (PrK) at positions S247 and T558 are labeled in light blue, FAD co-factor is blue, heme is dark red; Chemical structures of PrK (1), PDAz (2) and PCA (3) (11B); distance measurements between S247PrK residue and the FAD co-factor (left panel) and between the T558PrK residue and the heme domain (right panel) (11C); schematic illustration of the glassy-carbon electrode modification procedure (11D); and schematic illustration of the expected orientation of FGM- S247PDAz, FGM-T558PDAz and FGM-S247PCA on the electrode surface (HE);
[038] Figures 12A-12C include protein expression verification gel micrographs and a graph: anti his- tag western blot of crude lysates from bacterial expression system with the supplementation (+) and without (-) of 2 mM PrK (12A); in-gel heme staining of FGM-S247PrK and FGM-T558PrK (12B); and a graph of DCIP FAD-GDH activity assay for glucose oxidation (12C). Curves showing the reduction of DCIP by FGM-S247PrK (square), GDH-S247PrK (triangle) and FGM-T558PrK (circle);
[039] Figure 13 includes a gel micrograph verification of PrK incorporation using TAMRA-Az. 6 m M FGM-S247PrK, GDH-S247PrK and FGM-T558PrK were conjugate to TAMRA-Az using click reaction. The conjugated protein was loaded into SDS-PAGE and a fluorescent image of the gel was taken. PM2700 was used as the protein size marker;
[040] Figure 14 includes graphs showing cyclic voltammograms in different scan-rates in the absence of glucose and peak current vs. scan rate plots. An arrow marks the relevant peak of each variant. The relation between the peak current and the scan rate was analyzed by simple linear regression and the R2 is presented;
[041] Figures 15A-15B include plasmid maps of pec86-pylOTS plasmid (15A) and pETDuet- FGMS247TAG expression plasmid (15B);
[042] Figure 16 includes images of atomic force microscopy (AFM) measurements of highly oriented pyrolytic graphite (HOPG) after incubation using different linkers. Measurement of the height of the HOPG surface after incubation with acetate buffer (left panel), FGM-S247PDAz (middle panel) and FGM-S247PCA (right panel). The square and line on the right panel indicate the area which its height was analyzed and is presented in the graphs below the respective image, arrows indicate a single protein on the HOPG surface. 200 nm scale bars are represented by white lines. Representative 3D images of the same modified surfaces are present. All images are of the same scale and measured areas of lpmxlpm;
[043] Figure 17 includes a graph of differential pulse voltammetry (DPV) curves of EDC-NHS+PCA coupling reaction mixture (1) and click reaction mixture (2) that lacks the protein sample. FGM- S247PCA (4) and FGM-S247PDAz (3) curves were added for the ease of comparison;
[044] Figures 18A-18H includes graphs of electrochemical characterization of wired FGM and GDH: DPV measurements of FGM-S247PDAz (2) and FGM-S247PCA (1) under argon in the absence of glucose (18A). Peaks are indicated by arrows; linearized multistep amperometry current decay plot of FGM-S247PDAz (circle), FGM-T558PDAz (diamond), GDH-S247PDAz (triangle) and FGM- S247PCA (square) (18B); CVs of FGM-S247PDAz, FGM-T558PDAz, GDH-S247PDAz and FGM- S247PCA in high scan rates before (1) and after (4, 5, 3 and 2, respectively) the addition of 5 mM glucose (18C); CV of heme -binding domain attached to the electrode surface before (1) and after (2) the addition of 5 mM of glucose (18D); CVs in scan rate of 10 mV/sec for FGM-S247PDAz, FGM-T558PDAz, GDH-S247PDAz and FGM-S247PCA before the addition of 5 mM glucose (1), and after the addition of 5 mM glucose (3, 5, 4 and 2, respectively) (Figure 18E); Glucose-current calibration curves of FGM- S247PCA (circle), FGM-S247PDAz (square), FGM-T558PDAz (triangle) and GDH-S247PDAz (inverted triangle) measured using chronoamperometric detection upon application of a potential of 300 mV vs. Ag/AgCl reference electrode. The current from each glucose concentration (10 m M - 10 mM) was measured in triplicates; curves of the mean currents are presented with standard deviations. Inset presents the current response of FGM-T558PDAz towards glucose in physiologically relevant glucose concentrations of sweat and tears (18F); FGM-T558PDAz response to glucose in ALU interference solution that was measured upon application of 150 mV vs. Ag/AgCl reference electrode. Black arrows indicate 0.1 mM glucose additions (18G); and the current response of FGM-T558PDAz in physiologically relevant range of glucose in the presence of interfering molecules. The current was measured in triplicates and the mean current values were used for the linear regression and are presented with error bars (18H);
[045] Figures 19A-19B include graphs showing linear range of FGM-T558PDAz (19A) and FGM- S247PCA (19B) current response towards glucose;
[046] Figure 20 includes anti-His tag western blot analysis of FGM expressed from pTrc plasmid (1) vs. the use of a pETDuet plasmid (2);
[047] Figure 21 includes FGM 3D model structure with arrows indicating the ncAA incorporation sites;
[048] Figures 22A-22C include gel micrographs and a graph showing ncAA incorporated FGM variants characterization: FGM variants after ‘click’ with a fluorescent marker (22A), in-gel heme staining of protein concentrated elution samples (22B) and a graph of FAD-GDH glucose oxidation activity assay (22C);
[049] Figures 23A-23C include graphs and a chemical structure elucidation showing site-specific ‘wiring’ verification: Cyclic voltammetry (CV) of electrodes after incubation with a ‘clicked’ FGM and S247PrKFGM using a pyrene-azide linker (23A); FGM - blue and red curves, S247PrKFGM - green and purple curves; CV of ‘clicked’ S247PrKFGM vs. S247PrKFGM entrapped under a dialysis membrane. Entrapped enzymes: blue and red curves, ‘clicked’ enzymes: green and purple curves (23B); and pyrene-di-ethylene oxide-azide linker chemical structure (23C);
[050] Figures 24A-24B include graphs showing click reaction effect on S247PrKFGM: CV of S247PrKFGM + pDAz no glucose (green), S247PrKFGM + pDAz 5 mM glucose (purple), S247PrKFGM without pDAz no glucose (blue), S247PrKFGM without pDAz 5 mM glucose (red); and chronoamperometry of S247PrKFGM without pDAz (blue), S247PrKFGM + pDAz (red) (24A) and S247PrKFGMpDAz specificity test (24B); and
[051] Figure 25 includes a graph showing CV of S247PrKFGM response to 5 mM glucose in different scan rates.
DETAILED DESCRIPTION
[052] Provided herein is a recombinant flavin-adenine dinucleotide glucose dehydrogenase (FAD- GDH) enzyme, polynucleotides sequences encoding same, useful for direct electron transfer such as in bio-electrochemical applications, including, but not limited to, glucose monitoring.
[053] The present invention is directed to a polypeptide comprising a non-canonical amino acid (ncAA). According to some embodiments, the present invention provides an electrode coupled to the polypeptide of the present invention.
[054] The present invention is based, in part, on the surprising finding that using a site-specific incorporation of the ncAA, allows for a of a specific orientation towards the electrode. By controlling the incorporation of the ncAA and orientation, the catalytic current can be improved in response to glucose.
[055] In some embodiments, the wiring of the polypeptides through different sites result in a significant effect on their electron transfer (ET) characteristics and their ability to communicate with an electrode. In some embodiments, the site-specific wiring of the polypeptides allows higher catalytic currents and lower onset potential, indicating highly efficient ET that is gained due to the site-specific wiring.
[056] As provided in some embodiments of the present invention, fusing the enzyme with a minimal cytochrome domain (MCD), instead of large cytochrome, allows to shorten the enzyme-electrode distance and improve the direct electron transfer (DET) capabilities of the enzyme. As demonstrated hereinbelow under a non-limiting example, the fusion enzyme communicated with an electrode directly, without the use of a mediator molecule. Direct electron transfer between the redox enzyme and an electrode resulted in enhancement of chemical detection.
[057] As demonstrated hereinbelow, the disclosed recombinant enzyme showed a substantially reduced redox potential, e.g., from +400 mV to 0 mV, thereby improving enzyme selectivity in various electrochemical applications including glucose sensing and monitoring as well as an electromotive force in a bio-electrochemical power device. [058] Before explaining further embodiments of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.
Polypeptides
[059] According to some embodiments, there is provided a recombinant polypeptide comprising the amino acid sequence:
MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTD FMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEEFGVWGPGPEEDFY SPRKEPYPMPPFPFSFNEQTIKS A FNGYDPKFHVVTEPV ARNSRPYDGRPTCCGNNNCMPICPIGAMYN GIVHVEKAEQAGAKFID XAVVYKFETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKIFFMSANRDFPNGVAN SSDM V GRNFMDHPGTGVSFY ANEKFWPGRGPQEMTSFIGFRDGPFRANEAAKKIHFSNMSRI N QETQKIFKGGKFMKPEEFD AQIRDRS ARF V QFDCFHEIFPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVFGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC R AFDHPNFFIS S S STMPT V GTVN VTFTI A AF AFRMSDTFKKEVEFGS G Y GS GPPGPIR AG ATMP HRDRGPCGACHAIIQ (SEQ ID NO: 20), wherein X is a non-canonical amino acid (ncAA).
[060] According to some embodiments, there is provided a recombinant polypeptide comprising the amino acid sequence:
MADTDTQKADVVVVGSGVAGAIVAHQFAMAGKSVIFFEAGPRMPRWEIVERFRNQVDKTD FMAPYPSSAWAPHPEYGPPNDYFIFKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEEFGVWGPGPEEDFY SPRKEPYPMPPFPFSFNEQTIKS A LNGYDPKFHVVTEPV ARNSRPYDGRPTCCGNNNCMPICPIGAMYN GIVHVEKAEQAGAKLID SAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVAN SSDM V GRNLMDHPGTGVSFY ANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRI N QETQKIFKGGKLMKPEELD AQIRDRS ARF V QFDCFHEILPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC R AFDHPNLFIS S S STMPTV GT VN VTLTI A AL ALRMS DTLKKE VEF GS GY GS GPPGPIR AG AXMP HRDRGPCGACHAIIQ (SEQ ID NO: 20), wherein X is a non-canonical amino acid (ncAA).
[061] In some embodiments, the ncAA comprises Propargyl-lysine (PrK). [062] In some embodiments, the ncAA is covalently bound to a mediator molecule comprising polycyclic aromatic system.
[063] In some embodiments, the ncAA is covalently bound to a mediator molecule, wherein the mediator molecule is represented by Formula I:
Figure imgf000012_0001
(I), wherein n is an integer in a range from 1 to 5.
[064] In some embodiments, n is an integer in a range from 1 to 4, 1 to 3, 2 to 5, 2 to 4, or 2 to 3, including any range therebetween. Each possibility represents a separate embodiment of the invention. [065] In some embodiments, n equals 3. In some embodiments, n equals 2.
[066] As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably to refer to two or more amino acids linked together. The terms “polypeptide”, “peptide”, “protein”, and “amino acid sequence” as used herein refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the term “peptide”, “oligopeptide”, “polypeptide”, or “protein”. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, multiple antigenic peptide (MAP) forms, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. [067] In some embodiments, the mediator molecule comprises a diethylene oxide arm or group. In some embodiments, the mediator molecule is a linker. In some embodiments, the terms” mediator molecule” and “linker: are used herein interchangeably.
[068] In some embodiments, the diethylene oxide arm or group has a length of 2-8 A, 3-8 A, 4-8 A, 5- 8 A, 6-8 A, 6-7 A, 4-6.5 A, 5.5-7.5 A, or 5.8-6.6 A. each possibility represents a separate embodiment of the invention. [069] In some embodiments, the diethylene oxide arm or group has a length of not more than 5 A, 5.2 A, 5.4 A, 5.6 A, 5.8 A, 6.0 A, 6.1 A, 6.2 A, 6.3 A, 6.4 A, 6.6 A, 6.8 A, 7.0 A, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.
[070] In some embodiments, “diethylene oxide arm or group length” as used herein, comprises total length, average length, or maximal length, of the diethylene oxide arm or group.
Polynucleotides
[071] According to some embodiments, the present invention provides a polynucleotide comprising a nucleic acid sequence encoding the polypeptide described herein.
[072] In some embodiments, the polynucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
[073] In some embodiments, the polynucleotide comprises the nucleic acid sequence:
ATGGCGGATACGGATACCCAGAAAGCGGACGTGGTCGTGGTTGGATCCGGCGTGGCAGG
CGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAAAAGCGTGATCCTGCTGGAAGCTGG
TCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTTCCGCAATCAAGTCGATAAAACCGA
CTTTATGGCACCGTATCCGAGCAGCGCATGGGCACCGCATCCGGAATATGGTCCGCCGAA
TGATTACCTGATCCTGAAAGGCGAACACAAATTTAACTCACAGTACATTCGTGCAGTGGG
CGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCGCTTCATCCCGAACGATTTTAAAAT
GAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATTCAGTACGATGACATCGAACATTA
TTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGGTCCGGGCCCGGAAGAAGACCTGT
ATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCTGCCGCTGAGTTTCAATGAACAAA
CCATTAAATCCGCTCTGAACGGCTATGATCCGAAATTTCACGTGGTTACGGAACCGGTGG
CCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTGCTGTGGCAACAATAACTGCATGC
CGATTTGTCCGATCGGTGCAATGTATAACGGCATCGTCCATGTGGAAAAAGCTGAACAGG
CAGGTGCTAAACTGATTGATTAGGCGGTCGTGTACAAACTGGAAACGGGCCCGGACAAA
CGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTGCGGACCATCGCGTCGAAGGCAAA
TACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGAAAATTCTGCTGATGAGCGCGAAC
CGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATATGGTTGGCCGCAATCTGATGGAC
CATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAAAACTGTGGCCGGGTCGTGGTCCG
CAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCCCGTTTCGCGCGAATGAAGCAGCG
AAGAAAATTCATCTGTCAAATATGTCGCGTATCAACCAGGAAACCCAAAAAATCTTTAAA
GGCGGTAAACTGATGAAACCGGAAGAACTGGATGCGCAGATCCGTGACCGCAGTGCCCG CTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGCAGCCGGAAAATCGTATTGTCCCG
TCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTCCGGAAATTACGTATGCGATCGAT
GACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAGTTTATGCTACCGCGGCCAAAGTG
CTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTGCGCCGAATAACCACATCACCGGT
GCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGGTTGATAAAGACTGTCGCGCCTTC
GATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCATGCCGACGGTTGGCACCGTTAAC
GTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCTGATACGCTGAAAAAAGAAGTC
GAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCGATTCGTGCAGGTGCTACCATGC
CGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTATTATCCAGGGCAGTGGTTCCG
GCCATCACCATCACCATCACTAA (SEQ ID NO: 15).
[074] In some embodiments, the polynucleotide comprises the nucleic acid sequence:
ATGGCGGATACGGATACCCAGAAAGCGGACGTGGTCGTGGTTGGATCCGGCGTGGCAGG
CGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAAAAGCGTGATCCTGCTGGAAGCTGG
TCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTTCCGCAATCAAGTCGATAAAACCGA
CTTTATGGCACCGTATCCGAGCAGCGCATGGGCACCGCATCCGGAATATGGTCCGCCGAA
TGATTACCTGATCCTGAAAGGCGAACACAAATTTAACTCACAGTACATTCGTGCAGTGGG
CGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCGCTTCATCCCGAACGATTTTAAAAT
GAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATTCAGTACGATGACATCGAACATTA
TTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGGTCCGGGCCCGGAAGAAGACCTGT
ATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCTGCCGCTGAGTTTCAATGAACAAA
CCATTAAATCCGCTCTGAACGGCTATGATCCGAAATTTCACGTGGTTACGGAACCGGTGG
CCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTGCTGTGGCAACAATAACTGCATGC
CGATTTGTCCGATCGGTGCAATGTATAACGGCATCGTCCATGTGGAAAAAGCTGAACAGG
CAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAAACTGGAAACGGGCCCGGACAAA
CGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTGCGGACCATCGCGTCGAAGGCAAA
TACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGAAAATTCTGCTGATGAGCGCGAAC
CGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATATGGTTGGCCGCAATCTGATGGAC
CATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAAAACTGTGGCCGGGTCGTGGTCCG
CAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCCCGTTTCGCGCGAATGAAGCAGCG
AAGAAAATTCATCTGTCAAATATGTCGCGTATCAACCAGGAAACCCAAAAAATCTTTAAA
GGCGGTAAACTGATGAAACCGGAAGAACTGGATGCGCAGATCCGTGACCGCAGTGCCCG CTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGCAGCCGGAAAATCGTATTGTCCCG
TCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTCCGGAAATTACGTATGCGATCGAT
GACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAGTTTATGCTACCGCGGCCAAAGTG
CTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTGCGCCGAATAACCACATCACCGGT
GCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGGTTGATAAAGACTGTCGCGCCTTC
GATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCATGCCGACGGTTGGCACCGTTAAC
GTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCTGATACGCTGAAAAAAGAAGTC
GAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCGATTCGTGCAGGTGCTTAGATGC
CGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTATTATCCAGGGCAGTGGTTCCG
GCCATCACCATCACCATCACTAA(SEQ ID NO: 17).
[075] In some embodiments, the polynucleotide comprises the nucleic acid sequence:
TAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTT
TGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCCATGGCTCACAATGACAACACCCC
GCACTCCCGCCGTACCGGCGATGCGGCCGTGACCGGTATTACGCGTCGCCAGTGGCTGCA
AGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGGGTTCCCTGGCCCTGCGCGCACTGGC
TGATGATCCGGGCACCGCACCGCTGGATACCTTTATGACGCTGAGCGAAGCTCTGACGGG
CAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTTTCTGCAAGCGCTGCAAAAAGGTTC
ATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGGCGCCCTGGCAAGCGGTTCTCTGAA
CCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGAAGCATGGTATCTGGGCATTGTTGA
TAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTTAGTGTCGTGTCCGACACGCTGGTC
ATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGGGCCGAAAAACCGATCGAACGTCAG
GCATAATGGCAGCCATCACCATCATCACCACAGCCAGGATCCGAATTCGAGCTCGGCGCG
CCTGCAGGTCGACAAGCTTGCGGCCGCATAATGCTTAAGTCGAACAGAAAGTAATCGTAT
TGTACACGGCCGCATAATCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATA
ACAATTCCCCATCTTAGTATATTAGTTAAGTATAAGAAGGAGATATACATATGGCAGATC
TCAATTGATGGCGGATACGGATACCCAGAAAGCGGACGTGGTCGTGGTTGGATCCGGCGT
GGCAGGCGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAAAAGCGTGATCCTGCTGGA
AGCTGGTCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTTCCGCAATCAAGTCGATAA
AACCGACTTTATGGCACCGTATCCGAGCAGCGCATGGGCACCGCATCCGGAATATGGTCC
GCCGAATGATTACCTGATCCTGAAAGGCGAACACAAATTTAACTCACAGTACATTCGTGC
AGTGGGCGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCGCTTCATCCCGAACGATTT TAAAATGAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATTCAGTACGATGACATCGA
ACATTATTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGGTCCGGGCCCGGAAGAAG
ACCTGTATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCTGCCGCTGAGTTTCAATG
AACAAACCATTAAATCCGCTCTGAACGGCTATGATCCGAAATTTCACGTGGTTACGGAAC
CGGTGGCCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTGCTGTGGCAACAATAACT
GCATGCCGATTTGTCCGATCGGTGCAATGTATAACGGCATCGTCCATGTGGAAAAAGCTG
AACAGGCAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAAACTGGAAACGGGCCCG
GACAAACGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTGCGGACCATCGCGTCGAA
GGCAAATACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGAAAATTCTGCTGATGAGC
GCGAACCGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATATGGTTGGCCGCAATCTG
ATGGACCATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAAAACTGTGGCCGGGTCGT
GGTCCGCAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCCCGTTTCGCGCGAATGAA
GCAGCGAAGAAAATTCATCTGTCAAATATGTCGCGTATCAACCAGGAAACCCAAAAAATC
TTTAAAGGCGGTAAACTGATGAAACCGGAAGAACTGGATGCGCAGATCCGTGACCGCAG
TGCCCGCTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGCAGCCGGAAAATCGTATT
GTCCCGTCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTCCGGAAATTACGTATGCG
ATCGATGACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAGTTTATGCTACCGCGGCC
AAAGTGCTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTGCGCCGAATAACCACATC
ACCGGTGCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGGTTGATAAAGACTGTCGC
GCCTTCGATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCATGCCGACGGTTGGCACC
GTTAACGTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCTGATACGCTGAAAAAA
GAAGTCGAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCGATTCGTGCAGGTGCTA
CCATGCCGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTATTATCCAGGGCAGTG
GTTCCGGCCATCACCATCACCATCACTAAAAGCGATATCGGCCGGCCACGCGATCGCTGA
CGTCGGTACCCTCGAGTCTGGTAAAGAAACCGCTGCTGCGAAATTTGAACGCCAGCACAT
GGACTCGTCTACTAGCGCAGCTTAATTAACCTAGGCTGCTGCCACCGCTGAGCAATAACT
AGCATAACCCCTTGGGGCCTCTAAACGGGTCTTG (SEQ ID NO: 7).
[076] The terms “polynucleotide” and “nucleic acid” as used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA, or any combination thereof.
[077] Polynucleotides encoding polypeptides may be obtained from any source including, but not limited to, a cDNA library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level. Accordingly, polynucleotides encoding a polypeptide can be conveniently obtained from a cDNA library prepared from human tissue. The polypeptide-encoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).
[078] In some embodiments, the polynucleotide is codon optimized to facilitate or increase translation efficiency in a host cell. In some embodiments, the polynucleotide is codon optimized for expression in a microorganism cell. Methods for codon optimization are common and would be apparent to one of ordinary skill in the art, as well as codon preference of various types of cells, e.g., of E. coli.
[079] In some embodiments, there is provided a polynucleotide comprising: (a) a first regulatory element operably linked to the alpha catalytic subunit of the FGM expression gene; and (b) a second regulatory element operably linked to the gamma catalytic subunit of the FGM expression gene.
[080] In some embodiments, the regulatory element is a promoter.
[081] In some embodiments, the first regulatory element and the second regulatory element are identical. In some embodiments, the first regulatory element and the second regulatory element is a bacteriophage. In some embodiments, the first regulatory element and the second regulatory element is a T7 phage.
[082] In some embodiments, the polynucleotide is a DNA molecule.
[083] According to some embodiments, there is provided a vector or a plasmid comprising the polynucleotide of the invention.
[084] In some embodiments, the expression vector or plasmid further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH.
[085] In some embodiments, each of the polynucleotide encoding the polypeptide of the invention, and the nucleic acid sequence encoding the gamma subunit of an FAD-GDH are operably linked to a separate regulatory element.
[086] In some embodiments, the expression vector further comprises a nucleic acid sequence encoding a gamma subunit of an FAD-GDH, wherein each of the polynucleotide encoding the polypeptide of the present invention, and the nucleic acid sequence encoding the gamma subunit of an FAD-GDH are operably linked to a separate regulatory element. In some embodiments, the regulatory element is a promoter. In some embodiments, the promoter is a T7 promoter. [087] According to some embodiments, there is provided a transgenic or a transfected cell comprising: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; or d) any combination of (a) to (c).
[088] In some embodiments, the cell is a prokaryotic cell. In one embodiment, the cell is a bacterial cell.
[089] According to some embodiments, there is provided an extract obtained or derived from the cell disclosed herein. In some embodiments, the extract comprises: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; d) any combination of (a) to (c).
[090] According to some embodiments, there is provided a composition comprising: a) the polypeptide disclosed herein; b) the polynucleotide disclosed herein; c) the expression vector or the plasmid disclosed herein; d) the cell disclosed herein; e) the extract disclosed herein; f) any combination of (a) to (e); and g) and an acceptable carrier.
[091] According to some embodiments, there is provided an electrode coupled to the polypeptide described herein. In some embodiments, coupled is by non-covalent interactions. In some embodiments, non-covalent interactions refer to pi-pi stacking interactions between the pyrene groups of the mediator molecule represented by Formula I and the electrode surface. As used herein “pi-pi stacking” refers to attractive, non-covalent interactions between aromatic rings.
[092] According to some embodiments, there is provided a device comprising the electrode described hereinabove.
[093] According to some embodiments, there is provided a method for determining an analyte in a liquid medium, the analyte being capable to undergo a biocatalytic oxidation or reduction reaction in the presence of an oxidizer or a reducer, respectively, the method comprising: (i) providing the device described hereinabove; (ii) contacting the device with the liquid medium; (iii) measuring the electric signal generated between the cathode and the anode, the electric signal being indicative of the presence and/or the concentration of the analyte; and (iv) determining the analyte based on the electric signal. [094] In some embodiments, the analyte comprises glucose.
[095] According to some embodiments, there is provided a method for transferring an electron to an electrode, comprising coupling the polypeptide described herein to an electrode, thereby transferring an electron to the electrode. [096] According to some embodiments, there is provided a method for quantifying the amount of a reporter in a sample having a first detectable range of light absorbance in an oxidized state a second range of light absorbance in a non-oxidized state, comprising: (a) contacting polypeptide described herein with the reporter in a non-oxidized state; and (b) measuring the amount of the reporter in an oxidized state, thereby quantifying the amount of a reporter in a sample.
[097] In some embodiments, the first detectable range of light absorbance is detectable in visible light and the second range of light absorbance is non-detectable in visible light. In some embodiments, the reporter is 2,6-Dichloroindophenol. In some embodiments, the 2,6-Dichloroindophenol is coupled to glucose.
[098] According to some embodiments, there is provided a method for expression of the polypeptide described herein, comprising expressing a first subunit from a first regulatory element and expressing a second subunit from a second regulatory element, thereby expressing the polypeptide described herein. [099] In some embodiments, a first subunit is an alpha catalytic subunit of the FGM. In some embodiments, a second subunit is a gamma catalytic subunit of the FGM.
[0100] In some embodiments, the regulatory element is a promoter.
[0101] In some embodiments, the first regulatory element and the second regulatory element are identical. In some embodiments, the first regulatory element and the second regulatory element is a bacteriophage. In some embodiments, the first regulatory element and the second regulatory element is a T7 phage.
[0102] The present invention provides, in some embodiments, a recombinant protein comprising: (a) an alpha subunit of an FAD-GDH; and (b) a minimal cytochrome peptide.
[0103] In one embodiment, the alpha subunit of the FAD-GDH is derived or recovered from a prokaryotic cell. In one embodiment, the alpha subunit of the FAD-GDH is derived or recovered from a bacterial cell. In one embodiment, the alpha subunit of the FAD-GDH is Burkholderia cepacian alpha subunit of FAD-GDH. In one embodiment, the alpha subunit of the FAD-GDH of the present invention is derived from a thermostable enzyme, an oxygen independent enzyme, or both.
[0104] The term “thermostable enzyme” refers to an enzyme that is relatively stable to heat. The thermostable enzymes can withstand the high temperature incubation used to remove the modifier groups, typically, but not exclusively, greater than 50° C, without suffering an irreversible loss of activity. [0105] In one embodiment, the recombinant protein further comprises the gamma subunit of an FAD- GDH. In one embodiment, the invention provides a composition comprising or consisting the recombinant protein with or without the gamma subunit of an FAD-GDH. In one embodiment, the invention provides a composition comprising or consisting the recombinant protein and the gamma subunit of an FAD-GDH. In one embodiment, the invention provides a composition comprising or consisting at least two different proteins: (a) the recombinant protein; and (b) the gamma subunit of an FAD-GDH. In one embodiment, the at least two different proteins are unbound. In some embodiments, the gamma subunit is from the same FAD-GDH as the alpha subunit. In some embodiments, the gamma subunit is from a different FAD-GDH as the alpha subunit. In some embodiments, the gamma subunit is from the same or different FAD-GDH as the alpha subunit.
[0106] In one embodiment, the recombinant protein further comprises a minimal cytochrome peptide. In one embodiment, the minimal cytochrome peptide is a natural peptide. In one embodiment, the minimal cytochrome peptide comprises a non-natural peptide.
[0107] In one embodiment, the recombinant protein is devoid of the gamma subunit of the FAD-GDH. In one embodiment, the minimal cytochrome peptide comprises a c-type cytochrome domain. In one embodiment, the minimal cytochrome peptide does not comprise a b-type cytochrome domain. In one embodiment, the minimal cytochrome peptide comprises a c-type cytochrome domain MCR-2 from a MamP protein. In one embodiment, the minimal cytochrome peptide comprises a magnetotactic bacterius minimal cytochrome peptide. In one embodiment, the minimal cytochrome peptide is a magnetotactic bacterius minimal cytochrome peptide.
[0108] In one embodiment, the minimal cytochrome peptide is a peptide comprising or consisting of 11 to 30 amino acids. In one embodiment, the minimal cytochrome peptide is a peptide comprising or consisting of 11 to 24 amino acids. In some embodiments, the minimal domain is a naturally occurring cytochrome. In some embodiments, the minimal domain is a synthetic cytochrome.
[0109] In one embodiment, the minimal cytochrome peptide is a cytochrome peptide (e.g., c-type cytochrome) comprising or consisting of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, including any range therebetween. In some embodiments, the peptide comprises cytochrome functionality. In some embodiments, the peptide comprises ET functionality. [0110] In one embodiment, the minimal cytochrome peptide is linked to the amino terminus of the alpha subunit of an FAD-GDH. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus of the alpha subunit of an FAD-GDH. In one embodiment, the minimal cytochrome peptide is linked to the amino terminus of the gamma subunit of an FAD-GDH. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus of the gamma subunit of an FAD-GDH.
[0111] In one embodiment, the minimal cytochrome peptide is linked to the subunit of an FAD-GDH directly or indirectly. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus of the subunit of an FAD-GDH directly or indirectly.
[0112] In one embodiment, the recombinant protein, further comprises a linker. In one embodiment, the recombinant protein, further comprises a peptide linker. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus or the amino terminus of the alpha subunit of an FAD-GDH via a peptide linker. In one embodiment, the minimal cytochrome peptide is linked to the carboxy terminus or the amino terminus of the gamma subunit of an FAD-GDH via a peptide linker.
[0113] In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 5 to 20 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 8 to 18 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 12 to 15 amino acids. In one embodiment, the minimal cytochrome peptide is linked to the amino or carboxy terminus of the subunit via a linker comprising or consisting 5 to 15 amino acids. In some embodiments, the linker is between 1 and 10, 1 and 9, 1 and 8, 1 and 7, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 3 and 10, 3 and 9, 2 and 8 or 2 and 7 amino acids in length.
[0114] In some embodiments, the linker comprises 30% to 60% glycine. In some embodiments, the linker comprises 30% to 60% serine. In some embodiments, the linker is hydrophilic. In some embodiments, the linker does not cause steric hinderance. In some embodiments, the linker is a flexible linker. In some embodiments, the linker does not interfere with maturation of the porphyrin binding MCD. In some embodiments, the linker does not interfere with enzymatic activity of the other subunits. In some embodiments, the linker is not so short that the other subunit interferes with maturation of the porphyrin binding MCD. In some embodiments, the linker retains the subunits in close enough proximity to allow electron transfer. In some embodiments, the linker has a length of up to 20 A, up to 25 A, or up to 30 A. In some embodiments, the linker has a length of greater than or equal to 5 A, 10 A, 12 A, 15 A or 17 A. Non-limiting exemplary linker is a peptide comprising GSGYGSG. In some embodiments, the linker comprises or consists of SEQ ID NO: 24. In some embodiments, the linker comprises or consists a non-peptide backbone. [0115] In one embodiment, the linker is encoded by a DNA sequence comprising or consisting the nucleotide sequence: GAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCG (SEQ ID NO: 4). It will be understood by a skilled artisan that synonymous substitutions may be made to this sequence. [0116] In one embodiment, the linker is encoded by a DNA sequence comprising or consisting of a nucleotide sequence synonymous with SEQ ID NO: 4).
[0117] Without being bound by any particular mechanism it is assumed that a short linker containing glycine renders the linker with a desired flexibility. Further, and without being bound by any particular mechanism it is assumed that a short linker containing serine renders the linker with a desired hydrophilicity.
[0118] Further, a shorter linker (e.g. shorter than to 5 A) could prevent proper maturation of the porphyrin binding MCD (due to its close proximity to GDH); on the other hand, a longer linker (e.g., longer than 30 A) could prevent efficient ET between the two domains.
[0119] In one embodiment, the recombinant protein further comprises a short tag peptide (3 to 20 amino acids long). In one embodiment, the short tag peptide is his tag. In some embodiments, the tag is a 6x his tag. Protein tags are well known in the art and any tag that does not interfere with the function (redox and ET) of the recombinant protein may be used. In some embodiments, the short tag is between 1 and 30, 1 and 25, 1 and 20, 1 and 15, 1 and 10, 2 and 30, 2 and 25, 2 and 20, 2 and 25, 2 and 10, 3 and 30, 3 and 25, 3 and 20, 3 and 15 or 3 and 10 amino acids in length. Each possibility represents a separate embodiment of the invention.
[0120] In one embodiment, the recombinant protein has a molecular weight in the range of 58 to 75 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 60 to 70 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 63 to 65 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 62 to 68 kDa. In one embodiment, the recombinant protein has a molecular weight in the range of 63 to 65 kDa.
[0121] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTD FMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEELGVWGPGPEEDLY SPRKEPYPMPPLPLSFNEQTIKS A LNGYDPKFHVVTEPV ARNSRPYDGRPTCCGNNNCMPICPIGAMYN GIVHVEKAEQAGAKLID SAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVAN SSDM V GRNLMDHPGTGVSFY ANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRI N QETQKIFKGGKLMKPEELD AQIRDRS ARF V QFDCFHEILPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC R AFDHPNLFIS S S STMPTV GT VN VTLTI A AL (SEQ ID NO: 8, shortened).
[0122] Herein throughout, in some embodiments, by "shortened" or "partial" it is meant to refer to without e.g., tag peptide, and/or a linker.
[0123] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTD FMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEELGVWGPGPEEDLY SPRKEPYPMPPLPLSFNEQTIKS A LNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLID SAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVAN SSDM V GRNLMDHPGTGVSFY ANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRI N QETQKIFKGGKLMKPEELD AQIRDRS ARF V QFDCFHEILPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC RAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDTLKKEVIRAGATMPHRDRGPCGACHA IIQ (SEQ ID NO: 9, full).
[0124] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
M ADTDTQKAD V V V V GS G V AG AI V AHQL AM AGKS VILLE AGPRMPRWEIVERFRN Q VD KTD FMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEELGVWGPGPEEDLY SPRKEPYPMPPLPLSFNEQTIKS A LNGYDPKFHVVTEPV ARNSRPYDGRPTCCGNNNCMPICPIGAMYN GIVHVEKAEQAGAKLID SAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGVAN SSDM V GRNLMDHPGTGVSFY ANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRI N QETQKIFKGGKLMKPEELD AQIRDRS ARF V QFDCFHEILPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC RAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDTLKKEVEFGSGYGSGPPGPIRAGATMP HRDRGPCGACHAIIQGSGSGHHHHHH (SEQ ID NO: 10, full). [0125] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWEIVERFRNQVDKTD FMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKMK TVY GV GRD WPIQYDDIEHYY QRAEEEFGVWGPGPEEDFY SPRKEPYPMPPFPFSFNEQTIKS A FNGYDPKFHVVTEPV ARNSRPYDGRPTCCGNNNCMPICPIGAMYN GIVHVEKAEQAGAKFID SAVVYKFETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKIFFMSANRDFPNGVAN SSDMVGRNFMDHPGTGVSFYANEKFWPGRGPQEMTSFIGFRDGPFRANEAAKKIHFSNMSRI N QETQKIFKGGKFMKPEEFD AQIRDRS ARF V QFDCFHEIFPQPENRI VPS KTATD A V GIPRPEIT YAIDDYVKRGAVHTREVYATAAKVFGGTEVVFNDEFAPNNHITGATIMGADARDSVVDKDC RAFDHPNFFISSSSTMPTVGTVNVTFTIAAFAFRMSDTFKKEVEFGSGYGSGPPGPIRAGATMP HRDRGPCGACHAIIQ (SEQ ID NO: 11, partial).
[0126] An electrode
[0127] In some embodiments, there is provided an electrode carrying or coupled to a recombinant protein comprising A, B, C, and D, wherein: A is a cofactor of a redox enzyme; B is a redox enzyme; C is a linker moiety; and D is an electron transfer (ET) domain that is configured to transfer electrons between the electrode and A. In some embodiment the ET comprises a cytochrome.
[0128] In one embodiment, the A, B, C, and D are linked to each other under the following order: A-B- C-D.
[0129] In some embodiments, the cofactor, when not bound to a linker moiety, comprises at least one pair of hydroxyl groups.
[0130] In one embodiment, there is provided a device comprising the electrode.
[0131] In one embodiment, the electrode comprises a material selected from, without being limited thereto, graphite and glassy carbon electrode (GCE).
[0132] In one embodiment, the electrode is made of or coated by an electrically conducting substance, such as, without being limited thereto gold, platinum, silver, conducting glass such as indium tin oxide (ITO).
[0133] In one embodiment, by "chemically attach to" it is meant to refer to being attached via a covalent bond. As used herein, the term “coupled” refers to a physical attachment, such that the two are bonded together. In some embodiments, the bond is a covalent bond. In some embodiments, the bond is a synthetic bond. In some embodiments, the bond is a chemical bond. [0134] In another embodiment, by "chemically attach to" it is meant to refer to being attached to ("wiring") the electrode via a synthetic linker (also referred "electrode linker" or "mediator molecule" or "mediator") being configured to link the recombinant protein to an electrode.
[0135] In one embodiment, the wiring can be obtained by a non-specific process, by using a chemical modification and conductive matrices such as, without limitation, graphene oxide and multi -walled carbon nanotubes.
[0136] In one embodiment, the wiring is a site-specific wiring, performed e.g., by inserting at least one non-canonical amino acid in a desired site of one of the groups (A to D) that, optionally, covalently links to a moiety that binds to an electrode as described herein.
[0137] Without being bound by any particular mechanism, it is assumed that the recombinant protein disclosed herein (e.g., in the form of A-B-C-D) allows direct electron transfer (DET) via a domain that affords DET, resulting in a “built in” redox mediator.
[0138] In some embodiments, in order to achieve an efficient DET, the distance between the enzyme’s active site and the electrode is as short as a few Angstroms (e.g., 1 to 20 A). In some embodiments, the distance is between 0-20, 0-19, 0-18, 0-17, 0-16, 0-15, 0-14, 0-13, 0-12, 0-11, 0-10, 1-20, 1-19, 1-18, 1- 17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 3-10, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, or 3-10 A. Each possibility represents a separate embodiment of the invention.
[0139] In some embodiments, when linked to the enzyme, the ET domain is minimal so as not to introduce additional insulation to the system by a complex proteinaceous matrix. In some embodiments, the ET domain is linked by a flexible linker. As described herein, the flexibility allows avoiding interruption of the catalytic redox activity. In some embodiments, the minimal domain is not more than 10, 12, 15, 17, 20, 22, 25, 27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 amino acids. Each possibility represents a separate embodiment of the invention.
[0140] Embodiments of "minimal domain" are described hereinabove e.g., for the c-type cytochrome. [0141] In some embodiments, the cofactor is selected from, without being limited thereto, FAD, NAD+, and NADP+.
[0142] In one embodiment, the redox enzyme refers to an enzyme that can catalyze a redox reaction. [0143] In one embodiment, the redox enzyme may be selected from, without being limited thereto, oxidase, dehydrogenase, and malic enzyme (e.g., malate dehydrogenase). In some embodiments, the redox enzyme is selected from an oxidase, a dehydrogenase, a reductase, a peroxidase, a glyoxalase, a hydroxylase and a malic enzyme. In some embodiments, the redox enzyme is sugar dehydrogenase. In some embodiments, the sugar is glucose. In some embodiments, enzyme is alcohol dehydrogenase. [0144] In one embodiment, the redox enzyme in the immobilized group is characterized by a redox potential of less than 50 mV. In one embodiment, the redox enzyme in the immobilized group is characterized by a redox potential of 50 mV, 40 mV, 30 mV, 20 mV, 10 mV, 0 V, -10 mV, -20 mV, -30 mV, -40 mV, -50 mV, -60 mV, -70 mV, -80 mV, -90 mV, -100 mV, -110 mV, -120 mV, -130 mV, -140 mV, -150 mV, -160 mV, -170 mV, -180 mV, -190 mV, or -200 mV (induced potential vs. Ag/AgCl) including any value and range therebetween.
[0145] In one embodiment, the dehydrogenase is selected from, without being limited thereto, alcohol dehydrogenase, glutamic acid dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, and glycerol dehydrogenase.
[0146] In one embodiment, the linker moiety comprises a peptide. In one embodiment, the peptide comprises serine. In one embodiment, the linker moiety comprises a short peptide e.g. having 5 to 20 amino acids, or 5 to 15 amino acids, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, including any range therebetween. In some embodiments, the linker is selected from any embodiments of a linker described hereinabove. In some embodiments, the linker moiety is a flexible linker. In some embodiments, the linker moiety is hydrophilic. In some embodiments, the linker moiety is synthetic. In some embodiments, the linker moiety is not from cellobiose dehydrogenase. In some embodiments, the linker moiety is not from pyranose dehydrogenase. In some embodiments, the linker moiety does not interfere with enzyme function and/or DET.
[0147] A non-limiting example of a linker is a peptide comprising GSGYGSG.
[0148] In one embodiment, the linker is characterized by a length of: 5 to 40, or 20 to 30 A, e.g., 5, 10, 15, 20, 25, 30, 35, or 40A, including any value and range therebetween.
[0149] In one embodiment, the electron transfer domain comprises a cytochrome, e.g., MCD.
[0150] Embodiments of the cytochrome are described hereinabove. In some embodiments, the electron transfer domain comprises a cytochrome c. In some embodiments, the electron transfer domain does not comprise a cytochrome b. In some embodiments, the recombinant protein is not a naturally occurring protein.
[0151] In one embodiment, the device is a biosensor. [0152] As used herein and in the art, biosensors are analytical devices that combine a biological material (e.g., tissues, microorganisms, enzymes, antibodies, nucleic acids etc.) or a biologically-derived material with a physicochemical transducer or transducing microsystem.
[0153] In one embodiment, the device comprises or is configured to attach to an electronic circuitry for energizing the electrode and measuring the response.
[0154] In some embodiments, the biosensor is for measuring the concentration of a sugar in a medium. [0155] In some embodiments, the biosensor is for measuring the concentration of glucose in a medium. In some embodiments, the biosensor is for measuring the concentration of alcohol in a medium.
[0156] In some embodiments the medium is a bodily fluid. In some embodiments, the bodily fluid is selected from at least one of: blood, serum, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, urine, breast milk, interstitial fluid, and stool. In some embodiments, the biosensor is capable of measuring whole blood, serum or plasma glucose without dilution or sample processing, which can evaluate the current status of glucose or an individual suffering from diabetes. In some embodiments, the bodily fluid is undiluted. In some embodiments, the bodily fluid is diluted with a buffer.
[0157] In some embodiments, the biosensor comprises an anode compartment and a cathode compartment, the compartments being in fluid communication, wherein the anode compartment comprises an anode electrode and a substrate; and wherein the cathode compartment comprises a cathode electrode, the anode electrode and cathode electrode in electrical communication.
[0158] In some embodiments, the electrode disclosed herein is the anode.
[0159] According to an aspect, the present invention provides a method for determining an analyte in a liquid medium, the analyte being capable to undergo a biocatalytic oxidation or reduction reaction in the presence of an oxidizer or a reducer, respectively, the method comprising:
[0160] (i) providing the disclosed device in an embodiment thereof;
[0161] (ii) contacting the device with the liquid medium;
[0162] (iii) measuring the electric signal generated between the cathode and the anode, the electric signal being indicative of the presence and/or the concentration of the analyte; and [0163] (iv) determining the analyte based on the electric signal.
[0164] In some embodiments, the method is for determining the presence of the analyte in the medium. In some embodiments, the method is for determining the concentration of the analyte in the medium. When the liquid medium is, for example, a body fluid e.g. blood, lymph fluid or cerebro-spinal fluid, and the method may be carried out in an invasive manner, the method comprises inserting the biosensor into the body and bringing it into contact with the body fluid and determining the analyte in the body fluid within the body. Alternatively, body fluids or any other analyte may be tested non-invasively, and in such cases the method may comprise adding a buffer to the fluid. In some embodiments, the buffer has pH 4 to 8. In some embodiments, the buffer has pH 5 (±1).
[0165] In some embodiments, the contacting is in vivo. In some embodiments, the contacting is ex vivo. In some embodiments, a detected electrical signal indicates the analyte is present. In some embodiments, the greater the electrical signal the greater the concentration of analyte. In some embodiments, the electrical signal is compared to a predetermined standard that indicates the concentration of the analyte based on the electrical signal.
[0166] Non-limiting examples of analytes are sugar molecules e.g., galactose, lactose, maltose and xylose glucose, fructose, maltose; lactate; bilirubin; alcohols or amino acids.
[0167] In some embodiments, provided herein is a method for transferring an electron to an electrode, comprising coupling the disclosed recombinant protein in an embodiment thereof to an electrode, thereby transferring an electron to an electrode. In one embodiment, coupling is in the absence of a mediator molecule.
[0168] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
M ADTDTQKAD V V V V GS G V AG AI V AHQLAM AGKS VILLE AGPX 1 MPRWEIVERFRNQVDKT DFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKM KTVY GV GRD WPIQYDDIEHYY QRAEEEFG VWGPGPEEDFY SPRKEPYPMPPFPFSFNEQTIKS ALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLI DX2AVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGV ANSSDMVGRNLMDHPGTGVSFYANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLSNM SRINQETQKIFKGGKLMKPEELDAQIRX3RSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPR PEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVD KDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDTLKKEVEFGSGYGSGPPGPIRAGA X4MX5HRDRGPCGACHAIIQGSGSGHHHHHH (SEQ ID NO: 12, full).
[0169] In some embodiments, the amino acid sequence of the recombinant protein comprises or consists of the following sequence:
M ADTDTQKAD VVVV GS G V AG AI V AHQLAM AGKS VILLE AGPX 1 MPRWEIVERFRNQVDKT DFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYIRAVGGTTWHWAASAWRFIPNDFKM KTVY GV GRD WPIQYDDIEHYY QRAEEELG VWGPGPEEDLY SPRKEPYPMPPLPLSFNEQTIKS ALNGYDPKFHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAGAKLI DX2AVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIETPKILLMSANRDFPNGV ANSSDM V GRNLMDHPGTGVSFY ANEKLWPGRGPQEMTSLIGFRDGPFRANEAAKKIHLS NM SRINQETQKIFKGGKLMKPEELDAQIRX3RSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPR PEITYAIDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGADARDSVVD KDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDTLKKEVEFGSGYGSGPPGPIRAGA X4MX5HRDRGPCGACHAIIQ (SEQ ID NO: 13, partial).
[0170] In some embodiments, XI is R. In some embodiments, X2 is S. In some embodiments, X3 is D. In some embodiments, X4 is T. In some embodiments, X5 is P.
[0171] In some embodiments, one or more amino acids selected from XI to X5 comprise at least one non-canonical amino acid (ncAA) residue.
[0172] Exemplary embodiments of SEQ ID NO: 12-13 are presented in the Examples section below (e.g., SEQ ID NOs: 19-23).
[0173] The term "non-canonical amino acid residue" refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, b-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains.
[0174] Non-canonical amino acid residues may be incorporated into a peptide within the scope of the invention by employing known techniques of protein engineering that use recombinantly expressing cells.
[0175] In some embodiments one or more from XI to X5 are present in proximity to the protein domain selected from, without being limited thereto: FAD binding domain, or MCD. In some embodiments one or more from XI to X5 are present in a site that is distant from either FAD domain or MCD.
[0176] In some embodiments, by "proximity" it is meant to refer to a distance of 10 to 25 A, e.g., 10, 15, 20, or 25 A, including any value and range therebetween.
[0177] In some embodiments, by "distant" it is meant to refer to a distance of 30 to 100 A, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 A.
[0178] In some embodiments, the ncAA is a clickable ncAA.
[0179] By "clickable ncAA" it is meant to refer to ncAA attachable to another group or moiety by biorthogonal chemical mechanism. [0180] In exemplary embodiments, the ncAA comprises Propargyl -lysine (PrK).
[0181] In some embodiments, the ncAA has attached thereto an electrode linker (mediator) configured to couple the recombinant protein to an electrode.
[0182] In some embodiments, the linker comprises an aromatic group.
[0183] In some embodiments, the aromatic group comprises polycyclic aromatic hydrocarbon system. [0184] In some embodiments, by "polycyclic aromatic hydrocarbon system" it is meant to refer to a system comprising e.g., 3, 4, 5, or 6, fused benzene rings, which, in some embodiments, is in the form in a flat aromatic system.
[0185] In some embodiments, the aromatic group is selected from, without being limited thereto, pyrene, perylene, benzopyrene, oxoperylene, rubrene, perylene bisimide, styrene, anthracene, tetracene, pentacene, or any derivative thereof.
[0186] In exemplary embodiments, the aromatic group comprises pyrene or a derivative thereof.
[0187] In some embodiments, the linker further comprises an azide group.
[0188] In some embodiments, the azide group allows to bind to the PrK, for example, via an alkyne group, e.g., by “click” chemistry.
[0189] In some embodiments, the aromatic system (e.g., pyrene group) is present in one pole of the linker, and the azide group is present at the other pole of the mediator.
[0190] In some embodiments, the two groups (e.g., azide group and the aromatic group) are connected to each other by an alkyl oxide, for example, and without being limited thereto, tri-ethylene oxide, di ethylene oxide or mono -ethylene oxide. In some embodiments, the mediator is characterized by a length of 3 to 9 or 4 to 8 A, for example 3, 4, 5, 6, 7, 8, or 9 A, including any value and range therebetween. [0191] The Porphyrin
[0192] In one embodiment, the recombinant protein (e.g., the cytochrome domain) is bound to a porphyrin comprising a metal. In one embodiment, the recombinant protein is bound to a compound of formula I:
Figure imgf000031_0001
wherein R is any electron donor, or a compound as further described herein, wherein the compound of formula I is bound to a metal. In one embodiment, the recombinant protein but not the gamma subunit is bound to a porphyrin as described herein.
[0193] In one embodiment, R represents, independently and in each occurrence, a substituent selected from the group consisting of: alkyl, cycloalkyl, aryl, heteroalicyclic, heteroaryl, alkoxy, hydroxy, phosphonate, thiohydroxy, thioalkoxy, aryloxy, thioaryloxy, amino, nitro, halo, trihalomethyl, cyano, amide, amine, alkanoamine, carboxy, sulfonyl, sulfoxy, sulfinyl, and sulfonamide, or is absent. In one embodiment, R represents, independently and in each occurrence, hydrogen.
[0194] In one embodiment, R represents -(Ci-C6)alkyl. In some embodiments, R represents -(Ci- C6)alkoxy. In some embodiments, R represents -(Ci-C6)alkylthio. In some embodiments, R represents - (Ci-C6)alkylsulfinyl. In some embodiments, R represents -(Ci-C6)alkylsulfonyl. In some embodiments, R represents -[(Cl-C6)alkyl]NH. In some embodiments, R represents -[(Cl-C6)alkyl]COOH.
[0195] In one embodiment, the term "alkyl" comprises an aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 21 to 100 carbon atoms, and more preferably 21-50 carbon atoms. Whenever a numerical range; e.g., “21-100”, is stated herein, it implies that the group, in this case the alkyl group, may contain 21 carbon atoms, 22 carbon atoms, 23 carbon atoms, etc., up to and including 100 carbon atoms.
[0196] In one embodiment, the term "long alkyl" comprises an alkyl having at least 20 carbon atoms in its main chain (the longest path of continuous covalently attached atoms). A short alkyl therefore has 20 or less main-chain carbons. In one embodiment, an alkyl can be substituted or unsubstituted. In one embodiment, the term "alkyl", as used herein, also encompasses saturated or unsaturated hydrocarbon, hence this term further encompasses alkenyl and alkynyl. [0197] In one embodiment, the term "alkenyl" describes an unsaturated alkyl, as defined herein, having at least two carbon atoms and at least one carbon-carbon double bond. The alkenyl may be substituted or unsubstituted by one or more substituents, as described hereinabove. In one embodiment, the term "alkynyl", as defined herein, is an unsaturated alkyl having at least two carbon atoms and at least one carbon-carbon triple bond. The alkynyl may be substituted or unsubstituted by one or more substituents. [0198] In one embodiment, the term "cycloalkyl" describes an all-carbon monocyclic or fused ring (i.e., rings which share an adjacent pair of carbon atoms) group where one or more of the rings does not have a completely conjugated pi-electron system. The cycloalkyl group may be substituted or unsubstituted. [0199] In one embodiment, the term "aryl" describes an all-carbon monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups having a completely conjugated pi- electron system. In one embodiment, an aryl group may be substituted or unsubstituted.
[0200] In one embodiment, the term alkoxy" describes both an -O-alkyl and an -O-cycloalkyl group. In one embodiment, the term "aryloxy" describes an -O-aryl. In one embodiment, the term alkyl, cycloalkyl and aryl groups in the general formulas herein may be substituted by one or more substituents, whereby each substituent group can independently be, for example, halide, alkyl, alkoxy, cycloalkyl, alkoxy, nitro, amine, hydroxyl, thiol, thioalkoxy, thiohydroxy, carboxy, amide, aryl and aryloxy, depending on the substituted group and its position in the molecule.
[0201] In one embodiment, "halide", "halogen" or “halo” describes fluorine, chlorine, bromine or iodine. In one embodiment, “haloalkyl” describes an alkyl group as defined herein, further substituted by one or more halide(s). In one embodiment, “haloalkoxy” describes an alkoxy group as defined herein, further substituted by one or more halide(s). In one embodiment, the term “hydroxyl” or "hydroxy" describes a -OH group. In one embodiment, the term "thiohydroxy" or “thiol” describes a -SH group. In one embodiment, the term "thioalkoxy" describes both an -S-alkyl group, and a -S-cycloalkyl group. In one embodiment, the term "thioaryloxy" describes both an -S-aryl and a -S-heteroaryl group. In one embodiment, the term “amine” describes a -NR’R” group, with R’ and R”. In one embodiment, the term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. [0202] In one embodiment, the term "heteroalicyclic" or "heterocyclyl" describes a monocyclic or fused ring group having in the ring(s) one or more atoms such as nitrogen, oxygen and sulfur. The rings may also have one or more double bonds. In one embodiment, the rings do not have a completely conjugated pi-electron system. Representative examples are piperidine, piperazine, tetrahydrofurane, tetrahydropyrane, morpholino and the like.
[0203] In one embodiment, the term "carboxy" or "carboxylate" describes a -C(=0)-OR' group, where R' is hydrogen, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl (bonded through a ring carbon) or heteroalicyclic (bonded through a ring carbon).
[0204] In one embodiment, the term “carbonyl” describes a -C(=0)-R' group, where R' is as defined hereinabove. In one embodiment, the above-terms also encompass thio-derivatives thereof (thiocarboxy and thiocarbonyl).
[0205] In one embodiment, the term “thiocarbonyl” describes a -C(=S)-R' group, where R' is as defined hereinabove. In one embodiment, the term "thiocarboxy" group describes a -C(=S)-OR' group, where R' is as defined herein. In one embodiment, the term sulfinyl" group describes an -S(=0)-R' group, where R' is as defined herein. In one embodiment, the term sulfonyl" or “sulfonate” group describes an - S(=0)2-R' group, where R' is as defined herein. In one embodiment, the term "carbamyl" or “carbamate” group describes an -OC(=0)-NR'R" group, where R' is as defined herein and R" is as defined for R'. [0206] In one embodiment, the term "nitro" group refers to a -N02 group. In one embodiment, the term "cyano" or "nitrile" group refers to a -CºN group. In one embodiment, the term azide” refers to a -N3 group. In one embodiment, the term “sulfonamide” refers to a -S(=0)2-NR'R" group, with R' and R" as defined herein.
[0207] In one embodiment, the term “phosphonyl” or “phosphonate” describes an -0-P(=0)(0R')2 group, with R' as defined hereinabove. In one embodiment, the term “phosphinyl” describes a -PR'R" group, with R' and R" as defined hereinabove.
[0208] In one embodiment, the term “alkaryl” describes an alkyl, as defined herein, which substituted by an aryl, as described herein. In one embodiment, alkaryl is benzyl.
[0209] In one embodiment, the term "heteroaryl" describes a monocyclic or fused ring (i.e., rings which share an adjacent pair of atoms) group having in the ring(s) one or more atoms, such as, for example, nitrogen, oxygen and sulfur and, in addition, having a completely conjugated pi-electron system. Examples, without limitation, of heteroaryl groups include pyrrole, furane, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline and purine. The heteroaryl group may be substituted or unsubstituted by one or more substituents, as described hereinabove. Representative examples are thiadiazole, pyridine, pyrrole, oxazole, indole, purine and the like.
[0210] In one embodiment, the terms "halo" and "halide", which are referred to herein interchangeably, describe an atom of a halogen, that is fluorine, chlorine, bromine or iodine, also referred to herein as fluoride, chloride, bromide and iodide. In one embodiment, the term “haloalkyl” describes an alkyl group as defined above, further substituted by one or more halide(s).
[0211] In one embodiment, the metal is a trivalent metal or a divalent metal.
[0212] In one embodiment, the recombinant protein as described herein comprises both peroxidase activity and oxidative activity. In one embodiment, the recombinant protein bound to a porphyrin comprising a metal comprises both peroxidase activity and oxidative activity. In one embodiment, the recombinant protein bound to a porphyrin comprising a metal comprises both peroxidase activity and oxidase activity.
[0213] In one embodiment, the recombinant protein is characterized by Michaelis-Menten constant KM :'PP value which is at least 2, 3, 4 or 5, or more, higher compared to plain GDH, as measured under the same condition (e.g., glucose concentration) of glucose oxidation.
[0214] In one embodiment, the recombinant protein is characterized by higher selectivity towards glucose oxidation as compared to other sugar's molecules. In one embodiment, the means that the rate of glucose oxidation is higher by at least 10%, by at least 20%, by at least 30%, by at least 40%, or by at least 50% than a other sugar molecule.
[0215] As used herein, the terms “peptide”, "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. In some embodiment, the peptides, polypeptides and proteins described herein have modifications rendering them more stable while in the body or more capable of penetrating into cells. In some embodiment, the terms “peptide”, "polypeptide" and "protein" apply to naturally occurring amino acid polymers. In another embodiment, the terms “peptide”, "polypeptide" and "protein" apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid.
[0216] As used herein, the term “recombinant protein” refers to a protein which is coded for by a recombinant DNA and is thus not naturally occurring. The term “recombinant DNA” refers to DNA molecules formed by laboratory methods of genetic recombination. Generally, this recombinant DNA is in the form of a vector used to express the recombinant protein in a cell. In one embodiment, the recombinant protein is provided within a single composition or kit with the gamma subunit FAD-GDH. In one embodiment, the recombinant protein with the gamma subunit FAD-GDH are provided within a single composition or kit as two separate proteins (unbound).
[0217] In some embodiments, the recombinant protein is a hybrid protein. In some embodiments, the recombinant protein is a chimeric protein. In some embodiments, the c-type cytochrome peptide is from a different protein than the alpha subunit. In some embodiments, the c-type cytochrome peptide is not from FAD-GDH. In some embodiments, the c-type cytochrome peptide is not from GDH. In some embodiments, the nucleic acid sequence encoding the alpha subunit and the sequence encoding the c- type cytochrome peptide are operably linked, such that a full-length protein is produced following translation and/or transcription. The term “operably linked” is intended to mean that the two nucleotide sequences of interest are linked to each other in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0218] In general, and throughout this specification, the term “vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a “plasmid” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector, wherein virally-derived DNA or RNA sequences are present in the virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfecting into host cells. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively- linked. Such vectors are referred to herein as “expression vectors”. Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
[0219] Recombinant expression vectors can comprise a nucleic acid coding for the protein of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
[0220] A vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), selectable marker (e.g., antibiotic resistance), poly-Adenine sequence.
[0221] A vector or a plasmid is an artificial composite. A vector or a plasmid as described herein is man made. A vector or a plasmid as described herein is not a product of nature.
[0222] The vector may be a DNA plasmid delivered via non-viral methods or via viral methods. The viral vector may be a retroviral vector, a herpesviral vector, an adenoviral vector, an adeno-associated viral vector or a poxviral vector. The promoters may be active in mammalian cells. The promoters may be a viral promoter.
[0223] In some embodiments, the vector is introduced into the cell by standard methods including electroporation (e.g., as described in From et ah, Proc. Natl. Acad. Sci. USA 82, 5824 (1985)), Heat shock, infection by viral vectors, high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface (Klein et ah, Nature 327. 70- 73 (1987)), and/or the like.
[0224] General methods in molecular and cellular biochemistry, such as may be useful for carrying out DNA and protein recombination, as well as other techniques described herein, can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et ah, HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et ah, John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998).
[0225] It should be well understood to one skilled in the art that a recombinant protein is produced by expressing the recombinant DNA in a cell and then purifying the protein. The cells expressing the DNA are cultured under effective conditions, which allow for the expression of high amounts of recombinant polypeptide. Such effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce the recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.
[0226] Purification of a recombinant protein involves standard laboratory techniques for extracting a recombinant protein that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the peptide in nature. Purification can be carried out using a tag that is part of the recombinant protein or thought immuno-purification with antibodies directed to the recombinant protein. Kits are commercially available for such purifications and will be familiar to one skilled in the art. Typically, a preparation of purified peptide contains the peptide in a highly-purified form, i.e., at least about 80% pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
[0227] In some embodiments, the protein comprises an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% homology to the amino acid sequence set forth below in SEQ ID NO: 1. In some embodiments, the amino acid sequence with at least 70% homology to SEQ ID NO: 1 is the amino acid sequence set forth in SEQ ID NO: 6.
[0228] Mutations and deletions in a recombinant protein are created by introducing the mutation or deletion into the recombinant DNA. Methods of site-directed mutagenesis, and routine DNA recombination can be found in such standard textbooks as are enumerated above. Mutagenesis of one amino acid to another may require mutation of 1, 2, or 3 of the bases that make up the codon corresponding to the amino acid to be changed.
[0229] In some embodiments, provided herein a method for quantifying the amount of a reporter in a sample having a first detectable range of light absorbance in an oxidized state a second range of light absorbance in a non-oxidized state and, comprising: contacting the recombinant protein with the reporter in a non-oxidized state; and measuring the amount of the reporter in an oxidized state, thereby quantifying the amount of a reporter in a sample. In some embodiments, coupled is directly bound. In another embodiment, first detectable range of light absorbance is detectable in visible light and the second range of light absorbance is non- detectable in visible light. In some embodiments, the reporter is 2,6-Dichloroindophenol. In some embodiments, the reporter is coupled to glucose. In some embodiments, 2,6-Dichloroindopheno is coupled to glucose.
DNA Sequences
[0230] In one embodiment, provided herein is a DNA molecule encoding the recombinant protein. In one embodiment, provided herein is a DNA molecule comprising: a transcription regulatory element, a translation regulatory element or both; operably linked to a nucleotide sequence encoding the recombinant protein. In one embodiment, the invention provides a DNA molecule encoding both the recombinant protein and the gamma subunit of an FAD-GDH. In one embodiment, the invention provides a single DNA molecule encoding both the recombinant protein and the gamma subunit of an FAD-GDH as two separate proteins.
[0231] In one embodiment, provided herein is a DNA molecule comprising a nucleic acid sequence encoding the recombinant protein. In one embodiment, provided herein is a DNA molecule comprising a nucleic acid sequence encoding the recombinant protein and the gamma subunit of an FAD-GDH. In one embodiment, provided herein is a DNA molecule comprising the nucleic acid sequence selected from the group consisting SEQ ID NOs: 5-7. In one embodiment, the invention provides a plasmid or a vector (such as an expression vector) comprising a nucleic acid sequence encoding the recombinant protein. In one embodiment, provided herein is a cell comprising a DNA molecule, a plasmid or a vector as described herein. In one embodiment, the cell is a prokaryotic cell. In one embodiment, the cell is a bacterial cell.
[0232] In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
ATGGCGGATACGGATACCCAGAAAGCGGACGTGGTCGTGGTTGGATCCGGCGTGGCAGG
CGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAAAAGCGTGATCCTGCTGGAAGCTGG
TCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTTCCGCAATCAAGTCGATAAAACCGA
CTTTATGGCACCGTATCCGAGCAGCGCATGGGCACCGCATCCGGAATATGGTCCGCCGAA
TGATTACCTGATCCTGAAAGGCGAACACAAATTTAACTCACAGTACATTCGTGCAGTGGG CGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCGCTTCATCCCGAACGATTTTAAAAT
GAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATTCAGTACGATGACATCGAACATTA
TTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGGTCCGGGCCCGGAAGAAGACCTGT
ATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCTGCCGCTGAGTTTCAATGAACAAA
CCATTAAATCCGCTCTGAACGGCTATGATCCGAAATTTCACGTGGTTACGGAACCGGTGG
CCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTGCTGTGGCAACAATAACTGCATGC
CGATTTGTCCGATCGGTGCAATGTATAACGGCATCGTCCATGTGGAAAAAGCTGAACAGG
CAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAAACTGGAAACGGGCCCGGACAAA
CGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTGCGGACCATCGCGTCGAAGGCAAA
TACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGAAAATTCTGCTGATGAGCGCGAAC
CGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATATGGTTGGCCGCAATCTGATGGAC
CATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAAAACTGTGGCCGGGTCGTGGTCCG
CAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCCCGTTTCGCGCGAATGAAGCAGCG
AAGAAAATTCATCTGTCAAATATGTCGCGTATCAACCAGGAAACCCAAAAAATCTTTAAA
GGCGGTAAACTGATGAAACCGGAAGAACTGGATGCGCAGATCCGTGACCGCAGTGCCCG
CTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGCAGCCGGAAAATCGTATTGTCCCG
TCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTCCGGAAATTACGTATGCGATCGAT
GACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAGTTTATGCTACCGCGGCCAAAGTG
CTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTGCGCCGAATAACCACATCACCGGT
GCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGGTTGATAAAGACTGTCGCGCCTTC
GATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCATGCCGACGGTTGGCACCGTTAAC
GTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCTGATACGCTGAAAAAAGAAGTC
(SEQ ID NO: 1). In one embodiment, the recombinant protein is encoded by a DNA molecule comprising a coding nucleotide sequence encoding the alpha subunit of the FAD-GDH.
[0233] In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1200 to 1700 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1200 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1300 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1400 to 1700 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1500 to 1700 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1500 to 1650 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1550 to 1640 nucleotides. In one embodiment, the alpha subunit of the FAD-GDH is encoded by a DNA sequence of 1600 to 1650 nucleotides.
[0234] In one embodiment, the alpha subunit of the FAD-GDH is a mutant of alpha FAD-GDH or a mutant of SEQ ID NO: 1. Active mutants of alpha FAD-GDH or SEQ ID NO: 1 are readily available to one of skill in the art. By the term active mutant, as used in conjunction with an FAD-GDH, is meant a mutated form of the naturally occurring FAD-GDH. FAD-GDH mutant or variants will typically but not exclusively have at least 70%, e.g., 80%, 85%, 90% to 95% or more, and for example 98% or more amino acid sequence identity to the amino acid sequence of the reference FAD-GDH molecule.
[0235] In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 70% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD- GDH or a mutant of SEQ ID NO: 1 is at least 75% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 80% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 85% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 90% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 95% identical to SEQ ID NO: 1. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 1 is at least 97% identical to SEQ ID NO: 1.
[0236] In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
ATGGCTCACAATGACAACACCCCGCACTCCCGCCGTACCGGCGATGCGGCCGTGACCGGT
ATTACGCGTCGCCAGTGGCTGCAAGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGGGT
TCCCTGGCCCTGCGCGCACTGGCTGATGATCCGGGCACCGCACCGCTGGATACCTTTATG
ACGCTGAGCGAAGCTCTGACGGGCAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTTT
CTGCAAGCGCTGCAAAAAGGTTCATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGGC
GCCCTGGCAAGCGGTTCTCTGAACCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGAA
GCATGGTATCTGGGCATTGTTGATAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTTA
GTGTCGTGTCCGACACGCTGGTCATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGGG
CCGAAAAACCGATCGAACGTCAGGCATAA (SEQ ID NO: 2). [0237] In one embodiment, the gamma subunit of the FAD-GDH is translated into a discrete protein. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 400 to 700 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 450 to 650 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 450 to 550 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 480 to 530 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 500 to 540 nucleotides. In one embodiment, the gamma subunit of the FAD-GDH is encoded by a DNA sequence of 500 to 530 nucleotides.
[0238] In one embodiment, the gamma subunit of the FAD-GDH is a mutant of gamma FAD-GDH or a mutant of SEQ ID NO: 2. In one embodiment, active mutants of gamma FAD-GDH or SEQ ID NO: 2 are readily available to one of skill in the art.
[0239] In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 70% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD- GDH or a mutant of SEQ ID NO: 2 is at least 75% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 80% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 85% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 90% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 95% identical to SEQ ID NO: 2. In one embodiment, the DNA sequence of a mutant of FAD-GDH or a mutant of SEQ ID NO: 2 is at least 97% identical to SEQ ID NO: 2.
[0240] In one embodiment, the minimal cytochrome peptide is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
ATTCGTGCAGGTGCTACCATGCCGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTA TTATCCAG (SEQ ID NO: 3).
[0241] In one embodiment, the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
CTCACAATGACAACACCCCGCACTCCCGCCGTACCGGCGATGCGGCCGTGACCGGTATTA
CGCGTCGCCAGTGGCTGCAAGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGGGTTCCC
TGGCCCTGCGCGCACTGGCTGATGATCCGGGCACCGCACCGCTGGATACCTTTATGACGC
TGAGCGAAGCTCTGACGGGCAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTTTCTGC AAGCGCTGCAAAAAGGTTCATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGGCGCCC
TGGCAAGCGGTTCTCTGAACCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGAAGCAT
GGTATCTGGGCATTGTTGATAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTTAGTGT
CGTGTCCGACACGCTGGTCATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGGGCCGA
AAAACCGATCGAACGTCAGGCATAATGGCGGATACGGATACCCAGAAAGCGGACGTGGT
CGTGGTTGGATCCGGCGTGGCAGGCGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAA
AAGCGTGATCCTGCTGGAAGCTGGTCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTT
CCGCAATCAAGTCGATAAAACCGACTTTATGGCACCGTATCCGAGCAGCGCATGGGCACC
GCATCCGGAATATGGTCCGCCGAATGATTACCTGATCCTGAAAGGCGAACACAAATTTAA
CTCACAGTACATTCGTGCAGTGGGCGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCG
CTTCATCCCGAACGATTTTAAAATGAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATT
CAGTACGATGACATCGAACATTATTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGG
TCCGGGCCCGGAAGAAGACCTGTATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCT
GCCGCTGAGTTTCAATGAACAAACCATTAAATCCGCTCTGAACGGCTATGATCCGAAATT
TCACGTGGTTACGGAACCGGTGGCCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTG
CTGTGGCAACAATAACTGCATGCCGATTTGTCCGATCGGTGCAATGTATAACGGCATCGT
CCATGTGGAAAAAGCTGAACAGGCAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAA
ACTGGAAACGGGCCCGGACAAACGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTG
CGGACCATCGCGTCGAAGGCAAATACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGA
AAATTCTGCTGATGAGCGCGAACCGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATA
TGGTTGGCCGCAATCTGATGGACCATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAA
AACTGTGGCCGGGTCGTGGTCCGCAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCC
CGTTTCGCGCGAATGAAGCAGCGAAGAAAATTCATCTGTCAAATATGTCGCGTATCAACC
AGGAAACCCAAAAAATCTTTAAAGGCGGTAAACTGATGAAACCGGAAGAACTGGATGCG
CAGATCCGTGACCGCAGTGCCCGCTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGC
AGCCGGAAAATCGTATTGTCCCGTCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTC
CGGAAATTACGTATGCGATCGATGACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAG
TTTATGCTACCGCGGCCAAAGTGCTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTG
CGCCGAATAACCACATCACCGGTGCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGG
TTGATAAAGACTGTCGCGCCTTCGATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCAT
GCCGACGGTTGGCACCGTTAACGTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCT GATACGCTGAAAAAAGAAGTCATTCGTGCAGGTGCTACCATGCCGCATCGTGATCG TGGTCCGTGCGGTGCATGTCACGCTATTATCCAG (SEQ ID NO: 5 without linker, His and restriction sites).
[0242] In one embodiment, the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence:
CTCACAATGACAACACCCCGCACTCCCGCCGTACCGGCGATGCGGCCGTGACCGGTATTA
CGCGTCGCCAGTGGCTGCAAGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGGGTTCCC
TGGCCCTGCGCGCACTGGCTGATGATCCGGGCACCGCACCGCTGGATACCTTTATGACGC
TGAGCGAAGCTCTGACGGGCAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTTTCTGC
AAGCGCTGCAAAAAGGTTCATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGGCGCCC
TGGCAAGCGGTTCTCTGAACCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGAAGCAT
GGTATCTGGGCATTGTTGATAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTTAGTGT
CGTGTCCGACACGCTGGTCATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGGGCCGA
AAAACCGATCGAACGTCAGGCATAATGGCGGATACGGATACCCAGAAAGCGGACGTGGT
CGTGGTTGGATCCGGCGTGGCAGGCGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAA
AAGCGTGATCCTGCTGGAAGCTGGTCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTT
CCGCAATCAAGTCGATAAAACCGACTTTATGGCACCGTATCCGAGCAGCGCATGGGCACC
GCATCCGGAATATGGTCCGCCGAATGATTACCTGATCCTGAAAGGCGAACACAAATTTAA
CTCACAGTACATTCGTGCAGTGGGCGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCG
CTTCATCCCGAACGATTTTAAAATGAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATT
CAGTACGATGACATCGAACATTATTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGG
TCCGGGCCCGGAAGAAGACCTGTATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCT
GCCGCTGAGTTTCAATGAACAAACCATTAAATCCGCTCTGAACGGCTATGATCCGAAATT
TCACGTGGTTACGGAACCGGTGGCCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTG
CTGTGGCAACAATAACTGCATGCCGATTTGTCCGATCGGTGCAATGTATAACGGCATCGT
CCATGTGGAAAAAGCTGAACAGGCAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAA
ACTGGAAACGGGCCCGGACAAACGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTG
CGGACCATCGCGTCGAAGGCAAATACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGA
AAATTCTGCTGATGAGCGCGAACCGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATA
TGGTTGGCCGCAATCTGATGGACCATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAA
AACTGTGGCCGGGTCGTGGTCCGCAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCC CGTTTCGCGCGAATGAAGCAGCGAAGAAAATTCATCTGTCAAATATGTCGCGTATCAACC
AGGAAACCCAAAAAATCTTTAAAGGCGGTAAACTGATGAAACCGGAAGAACTGGATGCG
CAGATCCGTGACCGCAGTGCCCGCTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGC
AGCCGGAAAATCGTATTGTCCCGTCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTC
CGGAAATTACGTATGCGATCGATGACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAG
TTTATGCTACCGCGGCCAAAGTGCTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTG
CGCCGAATAACCACATCACCGGTGCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGG
TTGATAAAGACTGTCGCGCCTTCGATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCAT
GCCGACGGTTGGCACCGTTAACGTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCT
GATACGCTGAAAAAAGAAGTCGAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCG
ATTCGTGCAGGTGCTACCATGCCGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTA
TTATCCAG (SEQ ID NO: 6 with linker and without his and restriction sites).
[0243] In one embodiment, the recombinant protein is encoded by a DNA sequence comprising or consisting of the nucleotide sequence: CCATGG
CTCACAATGACAACACCCCGCACTCCCGCCGTACCGGCGATGCGGCCGTGACCGGTATTA
CGCGTCGCCAGTGGCTGCAAGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGGGTTCCC
TGGCCCTGCGCGCACTGGCTGATGATCCGGGCACCGCACCGCTGGATACCTTTATGACGC
TGAGCGAAGCTCTGACGGGCAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTTTCTGC
AAGCGCTGCAAAAAGGTTCATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGGCGCCC
TGGCAAGCGGTTCTCTGAACCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGAAGCAT
GGTATCTGGGCATTGTTGATAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTTAGTGT
CGTGTCCGACACGCTGGTCATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGGGCCGA
AAAACCGATCGAACGTCAGGCATAATGGCGGATACGGATACCCAGAAAGCGGACGTGGT
CGTGGTTGGATCCGGCGTGGCAGGCGCAATCGTGGCTCATCAACTGGCAATGGCAGGTAA
AAGCGTGATCCTGCTGGAAGCTGGTCCGCGTATGCCGCGTTGGGAAATTGTTGAACGTTT
CCGCAATCAAGTCGATAAAACCGACTTTATGGCACCGTATCCGAGCAGCGCATGGGCACC
GCATCCGGAATATGGTCCGCCGAATGATTACCTGATCCTGAAAGGCGAACACAAATTTAA
CTCACAGTACATTCGTGCAGTGGGCGGCACCACGTGGCATTGGGCAGCCTCGGCATGGCG
CTTCATCCCGAACGATTTTAAAATGAAAACCGTGTATGGCGTTGGTCGTGACTGGCCGATT
CAGTACGATGACATCGAACATTATTACCAACGCGCGGAAGAAGAACTGGGCGTGTGGGG
TCCGGGCCCGGAAGAAGACCTGTATTCACCGCGTAAAGAACCGTACCCGATGCCGCCGCT GCCGCTGAGTTTCAATGAACAAACCATTAAATCCGCTCTGAACGGCTATGATCCGAAATT
TCACGTGGTTACGGAACCGGTGGCCCGTAATTCGCGCCCGTACGACGGTCGCCCGACCTG
CTGTGGCAACAATAACTGCATGCCGATTTGTCCGATCGGTGCAATGTATAACGGCATCGT
CCATGTGGAAAAAGCTGAACAGGCAGGTGCTAAACTGATTGATAGTGCGGTCGTGTACAA
ACTGGAAACGGGCCCGGACAAACGTATTACCGCAGCTGTTTATAAAGATAAAACGGGTG
CGGACCATCGCGTCGAAGGCAAATACTTCGTGATTGCGGCCAATGGTATCGAAACCCCGA
AAATTCTGCTGATGAGCGCGAACCGTGATTTTCCGAATGGTGTGGCCAACAGTTCCGATA
TGGTTGGCCGCAATCTGATGGACCATCCGGGCACCGGCGTGAGCTTTTATGCAAACGAAA
AACTGTGGCCGGGTCGTGGTCCGCAGGAAATGACCTCTCTGATCGGTTTCCGTGATGGCC
CGTTTCGCGCGAATGAAGCAGCGAAGAAAATTCATCTGTCAAATATGTCGCGTATCAACC
AGGAAACCCAAAAAATCTTTAAAGGCGGTAAACTGATGAAACCGGAAGAACTGGATGCG
CAGATCCGTGACCGCAGTGCCCGCTTTGTTCAATTCGATTGCTTTCACGAAATCCTGCCGC
AGCCGGAAAATCGTATTGTCCCGTCCAAAACCGCAACGGACGCAGTGGGTATTCCGCGTC
CGGAAATTACGTATGCGATCGATGACTACGTCAAACGTGGCGCAGTGCATACGCGCGAAG
TTTATGCTACCGCGGCCAAAGTGCTGGGCGGCACCGAAGTGGTCTTCAACGATGAATTTG
CGCCGAATAACCACATCACCGGTGCCACGATTATGGGCGCGGATGCCCGTGACTCAGTGG
TTGATAAAGACTGTCGCGCCTTCGATCATCCGAACCTGTTTATTAGCAGCAGCAGCACCAT
GCCGACGGTTGGCACCGTTAACGTCACCCTGACGATTGCAGCTCTGGCACTGCGTATGTCT
GATACGCTGAAAAAAGAAGTCGAATTCGGTTCTGGTTATGGCTCTGGTCCGCCGGGTCCG
ATTCGTGCAGGTGCTACCATGCCGCATCGTGATCGTGGTCCGTGCGGTGCATGTCACGCTA
TTATCCAGGGCAGTGGTTCCGGCCATCACCATCACCATCACTAAAAGCTT (SEQ ID NO: 7 with linker, His and restriction sites).
[0244] In one embodiment, the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1500 to 3000 nucleotides. In one embodiment, the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1600 to 2600 nucleotides. In one embodiment, the recombinant protein with or without the gamma subunit as described herein is encoded by a DNA sequence of 1800 to 2500 nucleotides. In one embodiment, the recombinant protein is encoded by a DNA sequence of 2000 to 2400 nucleotides.
[0245] In one embodiment, the recombinant protein is 350 to 700 amino acids long. In one embodiment, the recombinant protein is 220 to 600 amino acids long. In one embodiment, the recombinant protein is 250 to 550 amino acids long. In one embodiment, the recombinant protein is 450 to 850 amino acids long. In one embodiment, the recombinant protein is 470 to 750 amino acids long. In one embodiment, the recombinant protein is 500 to 700 amino acids long. In one embodiment, the recombinant protein is 710 to 780 amino acids long. In one embodiment, the recombinant protein is 500 to 600 amino acids long. In one embodiment, the recombinant protein is 520 to 580 amino acids long. In one embodiment, the recombinant protein is 350 to 550 amino acids long.
[0246] In one embodiment, a DNA sequence encoding the recombinant protein and the gamma subunit protein as described herein of SEQ ID NOs: 5 and 6 further comprises a Methionine codon (the initiation codon nucleotide sequence) 5’ to SEQ ID NOs: 5 and/or 6. In one embodiment, a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises a Methionine codon (the initiation codon nucleotide sequence) 5’ to SEQ ID NOs: 5 and/or 6. In one embodiment, a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises at its 5’ end, a short DNA sequence comprising any 1-10 nucleotides sequence. In one embodiment, a DNA sequence encoding the recombinant protein of SEQ ID NOs: 5 and 6 further comprises at its 5’ end, a short DNA sequence comprising 1-10 nucleotides sequence. In one embodiment, the 1-10 nucleotides sequence comprises the Methionine codon.
[0247] In one embodiment, a DNA sequence or molecule as described herein comprising a coding sequence encoding the recombinant protein, further encodes the gamma subunit protein as described herein (as a separate protein).
[0248] In one embodiment, the DNA sequence encoding the recombinant protein of the invention is any DNA molecule encoding the amino acid sequence encoded by anyone of SEQ ID NOs: 5-7 or the amino acid sequence of anyone of SEQ ID NOs: 8-11. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 70% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 75% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 80% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 85% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 90% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 95% identical to anyone of SEQ ID NOs: 5-7. In one embodiment, the DNA sequence encoding the recombinant protein with or without the gamma subunit as described herein is at least 95% identical to anyone of SEQ ID NOs: 5-7.
[0249] In some embodiments, a DNA molecule of the invention or a DNA sequence described herein comprises or consists any sequence encoding the recombinant protein including (but not limited to) the recombinant protein comprising or consisting anyone of SEQ ID NOs: 8-11. In some embodiments, a DNA molecule of the invention or a DNA sequence described herein comprises or consists any sequence encoding the recombinant protein and the gamma subunit, as described herein, including (but not limited to) the amino acid sequences set forth in anyone of SEQ ID NOs: 8-11. In one embodiment, the recombinant protein and/or the gamma subunit, as described herein is/are translated based on the DNA sequences provided herein. In one embodiment, the recombinant protein has an amino acid sequence that is at least 70%, 80%, 90%, 95%, or 97% identical to: (a) a recombinant protein translated from the DNA sequences provided herein or (b) anyone of SEQ ID NOs: 8-11.
[0250] In some embodiments, the protein of the invention comprises a tag. In some embodiments, the DNA encoding the proteins of the invention comprises sequence encoding the tag. In some embodiments, the tag is selected from an n-terminal tag, a c-terminal tag and an internal tag. A skilled artisan will appreciate that the tag should be positioned so as not to interfere with the function of the recombinant protein. Thus, the tag will not interfere with the redox activity or the DET activity. In some embodiments, the tag is a c-terminal tag. In some embodiments, the tag is a His tag. In some embodiments, the tag is a 6x His tag. In some embodiments, the His tag comprises or consists of the amino acid sequence HHHHHH. In some embodiments, the DNA encoding the His tag comprises the sequence CATCACCATCACCATCAC (SEQ ID NO: 24) (e.g., in addition to SEQ ID NO: 19-23). A skilled artisan will appreciate that any sequence which encodes the tag may be used. Protein tags are well known in the art and include, but are not limited to, HA tags, His tags, GFP tags, Myc tags, biotin tags, FLAG tags, streptavidin tags, and many, many others. Tagging may be useful for purification of the protein, and the tag may be cleaved before the enzyme is used. In some embodiment, the tag is small. In some embodiments, the tag is equal to or smaller than 40, 35, 30, 25, 20, 15, 10, 7, or 5 amino acids. Each possibility represents a separate embodiment of the invention. A smaller tag may be advantageous in that it is less likely to interfere with DET.
[0251] In some embodiments, the tag is connected to the recombinant protein by a linker. The linker may be a linker such as has been described hereinabove. In some embodiments, the linker comprises or consists of the sequence GSGSG. In some embodiments, the DNA sequence that encodes the linker comprises or consists of the sequence GGCAGTGGTTCCGGC (SEQ ID NO: 25) (e.g., in addition to SEQ ID NO: 19-23). A skilled artisan will appreciate that any sequence which encodes the linker may be used. In some embodiments, the linker is produced by the restriction site introduced into the DNA to produce the recombinant protein. Indeed, there may be a linker produced by restriction site insertion between any of the different parts of the recombinant protein.
General
[0252] As used herein the term “about” refers to ± 10 %.
[0253] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of’ means “including and limited to”. The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
[0254] The word “exemplary” is used herein to mean “serving as an example, instance or illustration”. Any embodiment described as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments and/or to exclude the incorporation of features from other embodiments.
[0255] The word “optionally” is used herein to mean “is provided in some embodiments and not provided in other embodiments”. Any particular embodiment of the invention may include a plurality of “optional” features unless such features conflict.
[0256] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
[0257] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0258] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
[0259] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, and material arts.
[0260] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, of aesthetical symptoms of a condition.
[0261] In those instances where a convention analogous to "at least one of A, B, and C, etc." is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."
[0262] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
[0263] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES
[0264] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting fashion.
[0265] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes T-TTT Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.
EXAMPLE 1
Plasmid construction
[0266] FAD-GDH g subunit as well as its catalytic a subunit were cloned into pTrcHis6A2 vector between Ncol and Hindlll restriction sites. The partial FAD-GDH gene was followed by a short flexible polypeptide linker (13 amino-acids long) and the MCR2 (minimal cytochrome domain - MCD) DNA sequence with 6xHis tag at the sequence’s C-terminal, as shown in Figure 6 in a map of the new fusion protein. For the WT GDH the same construct was used, but without the MCD sequence. Full DNA construct:
CCATGGCTCACAATGACAACACCCCGCACTCCCGCCGTACCGGCGATGCGGCCGTGACCG
GTATTACGCGTCGCCAGTGGCTGCAAGGCGCGCTGGCCCTGACCGCAGCTGGCCTGACGG
GTTCCCTGGCCCTGCGCGCACTGGCTGATGATCCGGGCACCGCACCGCTGGATACCTTTAT GACGCTGAGCGAAGCTCTGACGGGCAAAAAAGGTCTGTCTCGTGTTCTGGGCCAGCGTTT
TCTGCAAGCGCTGCAAAAAGGTTCATTCAAAACCGCGGATTCGCTGCCGCAGCTGGCGGG
CGCCCTGGCAAGCGGTTCTCTGAACCCGGACCAAGAAGCTCTGGCGCTGAAAATCCTGGA
AGCATGGTATCTGGGCATTGTTGATAATGTGGTTATCACCTACGAAGAAGCCCTGATGTTT
AGTGTCGTGTCCGACACGCTGGTCATTCCGAGCTATTGCCCGAACAAACCGGGTTTCTGG
GCCGAAAAACCGATCGAACGTCAGGCATAATGGCGGATACGGATACCCAGAAAGCGGAC
GTGGTCGTGGTTGGATCCGGCGTGGCAGGCGCAATCGTGGCTCATCAACTGGCAATGGCA
GGTAAAAGCGTGATCCTGCTGGAAGCTGGTCCGCGTATGCCGCGTTGGGAAATTGTTGAA
CGTTTCCGCAATCAAGTCGATAAAACCGACTTTATGGCACCGTATCCGAGCAGCGCATGG
GCACCGCATCCGGAATATGGTCCGCCGAATGATTACCTGATCCTGAAAGGCGAACACAAA
TTTAACTCACAGTACATTCGTGCAGTGGGCGGCACCACGTGGCATTGGGCAGCCTCGGCA
TGGCGCTTCATCCCGAACGATTTTAAAATGAAAACCGTGTATGGCGTTGGTCGTGACTGG
CCGATTCAGTACGATGACATCGAACATTATTACCAACGCGCGGAAGAAGAACTGGGCGTG
TGGGGTCCGGGCCCGGAAGAAGACCTGTATTCACCGCGTAAAGAACCGTACCCGATGCCG
CCGCTGCCGCTGAGTTTCAATGAACAAACCATTAAATCCGCTCTGAACGGCTATGATCCG
AAATTTCACGTGGTTACGGAACCGGTGGCCCGTAATTCGCGCCCGTACGACGGTCGCCCG
ACCTGCTGTGGCAACAATAACTGCATGCCGATTTGTCCGATCGGTGCAATGTATAACGGC
ATCGTCCATGTGGAAAAAGCTGAACAGGCAGGTGCTAAACTGATTGATAGTGCGGTCGTG
TACAAACTGGAAACGGGCCCGGACAAACGTATTACCGCAGCTGTTTATAAAGATAAAAC
GGGTGCGGACCATCGCGTCGAAGGCAAATACTTCGTGATTGCGGCCAATGGTATCGAAAC
CCCGAAAATTCTGCTGATGAGCGCGAACCGTGATTTTCCGAATGGTGTGGCCAACAGTTC
CGATATGGTTGGCCGCAATCTGATGGACCATCCGGGCACCGGCGTGAGCTTTTATGCAAA
CGAAAAACTGTGGCCGGGTCGTGGTCCGCAGGAAATGACCTCTCTGATCGGTTTCCGTGA
TGGCCCGTTTCGCGCGAATGAAGCAGCGAAGAAAATTCATCTGTCAAATATGTCGCGTAT
CAACCAGGAAACCCAAAAAATCTTTAAAGGCGGTAAACTGATGAAACCGGAAGAACTGG
ATGCGCAGATCCGTGACCGCAGTGCCCGCTTTGTTCAATTCGATTGCTTTCACGAAATCCT
GCCGCAGCCGGAAAATCGTATTGTCCCGTCCAAAACCGCAACGGACGCAGTGGGTATTCC
GCGTCCGGAAATTACGTATGCGATCGATGACTACGTCAAACGTGGCGCAGTGCATACGCG
CGAAGTTTATGCTACCGCGGCCAAAGTGCTGGGCGGCACCGAAGTGGTCTTCAACGATGA
ATTTGCGCCGAATAACCACATCACCGGTGCCACGATTATGGGCGCGGATGCCCGTGACTC
AGTGGTTGATAAAGACTGTCGCGCCTTCGATCATCCGAACCTGTTTATTAGCAGCAGCAG CACCATGCCGACGGTTGGCACCGTTAACGTCACCCTGACGATTGCAGCTCTGGCACTGCG TATGTCTGATACGCTGAAAAAAGAAGTCGAATTCGGTTCTGGTTATGGCTCTGGTCCGCC GGGTCCGATTCGTGCAGGTGCTACCATGCCGCATCGTGATCGTGGTCCGTGCGGTGCATG TCACGCTATTATCCAGGGCAGTGGTTCCGGCCATCACCATCACCATCACTAAAAGCTT (SEQ ID NO: 26). The first 6 nucleotides and the last 6 nucleotides are Ncol and Hindlll restriction sites. The Gamma subunit is expressed as a separate protein encoded from nucleotide 3 to nucleotide 512 of the DNA molecule described herein. The Alpha subunit is from nucleotide 512 to nucleotide 2128. The flexible linker is from nucleotide 2129 to nucleotide 2167. The carboxy terminal MCD linker is from nucleotide 2168 to nucleotide 2238. 6 times His-Tag from nucleotide 2239 to nucleotide 2269. Enzyme expression and purification
[0267] The complete pTrcHis6A2-FAD-GDH-MCD plasmid was transformed into E-coli BL21 cells for the expression of the fusion protein (FAD-GDH-MCD). Bacteria were cultured in an auto-induction medium (FormediumTM, Hunstanton, England) with 0.5% glycerol (Bio-Lab ltd., Jerusalem, Israel) and 10pg/mL carbenicillin (Apollo, Manchester, England) and grown in 37 °C, with shaking at 250 rpm, for 6 hours after which cells were transferred to 27 °C for 18 additional hours.
[0268] The cells were then centrifuged at 6,000 rpm for 10 minutes, the pallet was resuspended using 20 mL 50 mM Tris-base buffer (TB, Fisher scientific, Geel, Belgium) pH=7.0, centrifuged, weighted, and resuspended using lysis buffer (300 mM KC1, 50 mM KH2P04, 10 mM imidazole pH=7.0) in a 1:3 (weighkbuffer volume) ratio. Protease inhibitors, lysozyme and super nuclease were added to the suspension in 1:500, 1:400, and 1:5000 ratios, respectively. The cells were lysed using sonication needle followed by 15 minutes of 50 °C incubation and centrifugation (11,500 rpm for 25 minutes). The supernatant was filtered using 0.22 pm filter.
[0269] The fusion enzyme was purified using IMAC purification system (Bio-Rad, Profinia, Hercules, CA, USA) according to manufacturer instructions.
FAD-GDH activity assay
[0270] To assess FGM’s D-glucose oxidation activity, glucose oxidation was measured in the presence of 0.6 mM 2,6-Dichloroindophenol (DCIP, Sigma-Aldrich, Rehovot, Israel), 0.6 mM phenazine methyl sulfate (PMS, Tokyo chemical industry, Tokyo, Japan), different concentrations of D-glucose and 0.2 mM FGM, all dissolved in 50 mM TB pH=7.0. Assay was performed in 37 °C using a 96-well plate reader (BioTek instruments, Winoosky, VT, USA) with shaking between measurements, monitoring the decrease in the DCIP absorbance l=610 nm every 15 seconds over 25 min of activity. Heme activity measurements
[0271] To verify the attachment of a porphyrin to the MCD domain, heme activity was measured using 1 mM dimethoxybenzidine (DMB, Alfa Aesar, Heysham, England), ImM hydrogen peroxide (Sigma- Aldrich, Rehovot, Israel), and 0.2 mM of the enzyme. Measurements were performed at 37 °C using a 96-wells plate reader, monitoring the increase in DMB absorbance l =455 nm every 15 seconds over 30 min of activity.
Peroxidase activity interference measurements
[0272] To verify that FGM does not transfer electrons to oxygen, producing hydrogen peroxide, that interferes with the FAD-GDH activity assay, peroxidase activity test was performed in the absence of hydrogen peroxide. 0.17 mM DMB and 172 mM glucose were mixed with 8 pF of 0.1 mg/mF Horseradish peroxidase (HRP, Sigma-Aldrich, Rehovot, Israel) to generate a reaction mix. 8 pF of concentrated FGM or glucose oxidase (GOx) were added to the reaction mix right before measurement. Measurements were performed at 37 °C using a plate reader, monitoring the increase in DMB absorbance l=500 nm every 15 seconds over 25 min of activity (Figure 3C).
Electrode preparation
[0273] Glassy carbon electrodes (GCE, 3mm in diameter; AFS, Tokyo, Japan) were polished using 0.05 pm alumina slurry on polishing pad for two minutes, then transferred to a 20 mF glass with 10 mF double-distilled water (DDW) and sonicated for five minutes in a sonication bath. The electrodes were then dried under nitrogen gas. 10 pF of enzyme solution in wanted concentrations was dropped on the electrode surface. The electrodes were incubated in 4 °C overnight to generate an enzyme film on the GCE. Electrode’s surfaces were then covered with 12-14 kDa dialysis membrane (Membrane Filtration Products, Seguin, TX, USA) and tightened using an O-ring rubber to prevent diffusion of the enzyme to the solution.
Electrochemical measurements
[0274] Cyclic voltametric measurements were performed using a PalmSense2 potentiostat (Palm Instruments, Houten, The Netherlands) using a standard three electrodes system with 0.9 mm graphite rod as an auxiliary electrode, 3 M KC1 saturated Ag/AgCl reference electrode (AFS, Tokyo, Japan) and GCE as the working electrode in 0.15 M phosphate-citrate buffer pH=5.0. Chronoamperometric measurements (Figure 7) were performed under the same conditions with the application of 0 mV vs. Ag/AgCl with the addition of varying concentrations of glucose or potential interfering molecules. Square wave voltammetry (SWV) measurements were performed under the same conditions. FGM selectivity test
[0275] The interference of different sugars and molecules on glucose biosensing by GCE/FGM was measured using chronoamperometry (see selectivity Figures 8A-8C). Measurements were performed in 0.15 M phosphate-citrate buffer pH=5.0 with the application of 0 mV vs. Ag/AgCl reference electrode. FGM selectivity was tested by adding 3.6 mM of glucose followed by two sequential additions of one of the different sugars or molecules in their relevant physiological concentrations. The sugars used for this test were D-galactose, lactose, D-maltose and D-xylose and the molecules were ascorbic acid and acetaminophen (all from Sigma- Aldrich, Rehovot, Israel). Selectivity test have revealed a small interference caused by galactose while other sugars did not interfere with the measurement. This result indicates high selectivity towards glucose and low/no reaction with other sugars that can be found in human blood samples, which is very important for biosensing accuracy. GCE\FGM showed low sensitivity to ascorbic acid and no sensitivity to acetaminophen.
The biocatalytic recombinant protein
[0276] In the present study, a fusion enzyme was designed in a combination of a biocatalytic function from a redox enzyme domain that was fused to a natural minimal ET domain via a short polypeptide linker as shown in Figure 1. As the catalytic domain, the a subunit of an FAD-GDH from Burkholderia cepacia was used. As a minimal ET unit, the c-type cytochrome domain MCR-2 from a MamP protein which originates from a magnetotactic bacteria magnetoovoid bacterium MO- 127 was chosen. MamP is part of the magnetosome, a unique organelle that is found in magnetotactic bacteria that allows magneto taxis to occur in these bacteria. MCR-2 is one of the shortest natural c-type cytochromes known today (23 amino-acids long), thus can be used to achieve DET.
[0277] In order for FAD-GDH-MCD (FGM) fused enzyme to mature properly in the host cell, FAD- GDH a subunit should correctly fold. The Enzyme’s g subunit aids in the maturation of the a subunit and locates it in the periplasm. Within the bacterial periplasmatic environment, the maturation of c-type cytochromes (heme binding cytochromes) occurs with the help of a specific gene cluster called ccmA- H29. In that manner, the holo-enzyme is being transferred to the periplasm and there the MCD’s maturation process occurs.
[0278] Fusion enzyme’s engineered DNA sequence was cloned into pTrcHis6A2 expression vector and was transformed into E-coli BL21. FGM was overexpressed in the bacterial expression system and then purified by utilizing immobilized metal affinity chromatography (IMAC) purification system (Figure 2A). In-gel heme staining was performed to verify the presence of the heme compared to GDH and Anti his-tag Western blot analysis was performed in order to verify the full-length enzyme’s expression (Figure 2B, right panel). As shown in Figure 2B, both FGM and GDH enzymes were expressed and their respective bands appeared in the expected size - ca. 64 kDa and 62 kDa for FGM and GDH, respectively. In-gel heme staining revealed a band for FGM only, indicating the presence of a porphyrin containing iron bound to FGM.
[0279] FGM catalytic redox activity and heme peroxidase activity were measured biochemically and compared to GDH as shown in Figure 3A. FGM has oxidized D-glucose as was measured by FAD- GDH activity assay in 50 mM Tris-base (pH 7.0), 0.6 mM 2,6-Dichloroindophenol (DCIP) and 0.6 mM phenazine methyl sulfate (PMS) in 37 °C. Absorbance (lambda=610nm) was monitored using a plate reader while the oxidized DCIP (blue color) was being reduced by FGM to its reduced form (colorless). FGM has also shown peroxidase activity, measured by heme activity assay in 1 mM 3,3’- dimethoxybenzidine (DMB) and 1 mM of hydrogen peroxide. Absorbance in 455 nm was monitored while the DMB oxidation occurs by the MCD, resulted in an oxidized DMB (red color). Heme activity assay results indicate that FGM indeed binds a heme group while no heme molecules are bound by GDH. [0280] Absorbance measurements of protein sample spectrum revealed a peak in absorbance at 408 nm for FGM and no peak for GDH, indicating presence of heme c in FGM (Figure 3B). 408 nm/A280 nm ratio was calculated to be 0.4 for FGM expressed in the presence of pEC86 plasmid, compared to 0.2 for FGM expressed in the absence of this plasmid, indicating more efficient heme maturation in the presence of the helper plasmid.
[0281] The apparent kinetic and thermodynamic parameters of FGM were calculated using FAD-GDH biochemical activity assay in 37 °C (Table 1). FGM and GDH concentrations were first determined spectrophotometrically using a standard Bradford assay. Michaelis-Menten curves were transformed to Lineweaver-Burk curves in order to determine the kinetic and thermodynamic parameters of the enzyme. One enzyme activity unit was defined as amount of enzyme oxidizing 1 mM of substrate per minute. The molar absorption coefficient of DCIP was calculated to be 4.7 cm^mM 1. By using the biochemical activity assay and DCIP molar absorption coefficient, FGM and GDH specific activity were calculated to be 16 mU.
[0282] Lineweaver-Burk plots (Figures 7A-7B) were used to calculate kinetic and thermodynamic parameters of the enzyme. KM 3PP values were 157±5 m M and 174 ± 9 m M for FGM and GDH, respectively, showing different affinity of the enzymes toward the substrate. GDH’s KM 3PP value is lower than reported values but yet in the same order of magnitude of some. KM :'PP value was ca. 3 times higher for FGM compared to GDH, indicating faster oxidation of D-glucose by FGM. FGM also showed more than 3 times higher catalytic efficiency (k) compared to GDH (Table 1). Next, the electrochemical activity of the enzymes was measured to determine whether the addition of the minimal cytochrome domain improves enzyme-electrode ET.
[0283] Chronoamperometric measurements (Figure 7C) were performed to determine the apparent kinetic parameters of FGM compared to GDH. Using a standard 3 electrode electrochemical cell, the current was measured vs. successive glucose additions. A potential of 0V was applied during the measurements. The current for each glucose concentration was determined and is presented in Figure 5A. As described above, using the linear part of the transient curves and Linewaver-Burk transformation - the electrochemical kinetic constants were calculated (Figure 5B).
Table 1. Apparent biochemical kinetic parameters of FGM and GDH
Figure imgf000056_0001
[0284] For the electrochemical measurements, a standard 3 electrode electrochemical cell with 0.9 mm graphite rod as the auxiliary electrode were used, 3M KC1 saturated Ag/AgCl reference electrode and 3 mm diameter glassy carbon electrode (GCE) as the working electrode. 10 pL of ca. 30pg/mL FGM or GDH enzyme solution were dropped on the GCE surface and dried in 4 °C overnight. The electrodes surface was then covered with 12-14 kDa dialysis membrane tightened to the surface with an O-ring to keep the enzyme close to the electrode surface during measurements and to avoid enzyme diffusion to the surrounding buffer. Cyclic voltammetry (CV) measurements were performed to compare the enzyme-electrode communication of FGM to that of GDH.
[0285] Optimal pH value for electrochemical measurements was tested by measuring the catalytic current in pH values of 3.6-7.0 and found to be 5.0.
[0286] Measurements were performed in phosphate-citrate buffer pH=5.0 at room temperature and a scan rate of 5mV/sec for both enzymes with and without the addition of 5 mM glucose. It can be seen in Figures 4A-B that the CVs of both enzymes before the addition of glucose are almost identical. No clear anodic or cathodic peaks were identified for both enzymes. After the addition of glucose to a final concentration of 5mM, FGM has demonstrated 10 times higher electrocatalytic current compared to that of GDH with an onset potential of ca. -100 mV for both enzymes (indicating no apparent shift in potentials due to the fusion of MCD). That difference in the ET efficiency is probably due to the addition of the minimal cytochrome domain that mediates the ET from the buried FAD co-factor to GCE.
[0287] After the addition of glucose to a final concentration of 5 mM, FGM demonstrated a higher electrocatalytic current compared to that of GDH with an onset potential of ca. (-) 150 mV. The fact that no catalytic current was observed at this high scan rate using GDH, but a significant catalytic current evolved using FGM, is an indication of fast ET rates of FGM. That observed ET efficiency is probably due to addition of the minimal cytochrome domain that mediates ET from the buried FAD cofactor to GCE. To identify a peak originating from the MCD domain, square-wave voltammetry (SWV) was performed. As shown in Figures 4C, the voltammogram of an electrode with FGM revealed a peak around (-) 230 mV, whereas GDH had no observable peak at this potential.
[0288] As shown in Table 2, the electrochemical KM :'PP is 2.84±0.57 mM for GDH and 1.40±0.27 mM for FGM, indicating no significant difference in the affinity towards glucose. The imax value is one order of magnitude higher for FGM compared to GDH - 2.04±0.45 pA cm 2 and 0.4±0.17 pA cm 2, respectively. The difference in the imax value is indicative that the DET efficiency is different between the two enzymes where FGM shows five to seven times higher current then GDH for the same glucose concentrations.
Table 2. Apparent electrochemical kinetic and thermodynamic parameters of FGM and GDH
Figure imgf000057_0001
[0289] Burkholderia Cepacia is considered a good candidate for biosensing applications because of its stability in high temperatures and insensitivity to oxygen. By adding a minimal cytochrome domain to FAD-GDH c-terminus an improved DET was provided, showing higher catalytic currents compered to GDH with almost the same affinity to the substrate. When tested electrochemically with 0 V induced potential vs Ag/AgCl, FGM showed much higher currents for the same substrate concentrations compared to GDH, which makes it more accurate for glucose biosensing with improved sensitivity than previously reported for GDH.
Non-canonical amino acids (ncAAs) incorporation into FGM for site-specific wiring to an electrode: FGM electron transfer machineries investigation [0290] To incorporate non-canonical amino acids (ncAAs) into FGM, a few constructs containing the amber (TAG) mutation on pTrcHis6A2-FGM plasmid were prepared using standard site-directed mutagenesis PCR protocol. The mutations were chosen with a proximity to the protein different domains - FAD binding domain, MCD and one site that is distant from either FAD domain or MCD. For proximity to FAD binding domain, two sites were found to be possible for ncAA incorporation - R42X and S247X (Figure 9 - red), both ca. 11 A from FAD binding domain. For proximity to MCD, T558X and P560X (yellow) are possible sites for incorporation, with 10 and 7 A from MCD, respectively. D395X (black) was found to be far from both FAD and MCD as it is about 37 A from FAD and 83 A from MCD.
[0291] Mutated plasmids were transformed into super competent E. coli DH5a cells and were plated on selective LB-agar plates. Bacterial colonies were isolated and plasmids were purified using miniprep kit followed by sequencing.
Non-canonical amino acids ( ncAA ) incorporation into FGM
[0292] Plasmids, with amber codon-containing FGM mutants sequences were co-transformed to E. coli BL21 strain containing the pEVOL plasmid expressing Pyrrolysyl orthogonal translation system to incorporate Propargyl-lysine (PrK) into the protein sequence. PrK containing protein was expressed in 20 mL auto-induction medium (AIM) in the presence of ImM PrK, lysed using Bugbuster lysis solution and was isolated utilizing IMAC purification method. PrK is an example to a clickable ncAA, all clickable biorthogonal chemical handles can be considered for the site-specific wiring of this enzyme. Pyrene-azide linkers for enzyme wiring to electrode
[0293] In order to wire FGM with site-specifically incorporated PrK to an electrode, a synthetic linker was used. The synthetic linker contained a pyrene group in one pole and an azide group at the other. The two groups were connected by a tri-ethylene oxide, di-ethylene oxide or mono-ethylene oxide, to get three different lengths of 8.3, 6.4 and 4.3 A, respectively. The pyrene group is a polycyclic aromatic hydrocarbon consisting of four fused benzene rings, results in a flat aromatic system. Due to overlapping of p-bonds between aromatic side chains, the pyrene group can be attached to glassy carbon electrodes surface through p-p stacking. The azide group was used to attach the alkyne group of PrK using “click” chemistry. Exemplary pyrene-azide linker structures with different lengths are presented in Figures 10A-10B. Click reaction:
[0294] Copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction is based on the formation of 1,4- disubstituted 1,2,3-triazoles between a terminal alkyne and an aliphatic azide in the presence of copper. The reaction is a facile, selective, high yielding with mild conditions and with few or no byproducts. It can be performed in room-temperature, what makes it relevant for use in proteins. CuAAC was used to link FGM to a pyrene-azide synthetic linker. Click reaction mix contained tris-KCl buffer (pH=8.2), pyrene-azide linker, Cu+, Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), FGM with incorporated PrK and sodium-ascorbate (Na-Asc). Reaction mix was incubated in RT for 30-60 min with moderate mixing, centrifuged for 10 min, and the supernatant was collected for further examinations. ncAA incorporation validation
[0295] TAMRA-azide is an azide-linked reporter tag that can be used for visualization of alkyne containing proteins. To validate incorporation of PrK into FGM sequence, purified FGM was clicked with the florescent marker TAMRA-azide. Clicked protein sample was loaded on SDS-PAGE and the protein gel was checked for florescence using LAS4000 camera. In addition, anti 6xHis-tag antibodies will be used for Western blot analysis. MS -MS analysis was performed on isolated protein to give another validation for PrK incorporation.
Enzyme site-specific wiring to GCE
[0296] Ten pL of pyrene-azide clicked to PrK containing FGM was dropped on clean GCE and incubate in RT for 15min. The pyrene groups from the linker adhere to GCE surface through p-p stacking. GCE was washed using DW to avoid unbound protein showing signal in electrochemical measurements. Relevant FGM DNA sequences (all sequences encode the FAD-GDH q-subunit+MCD without the y- subunif):
[0297] R42X (SEQ ID NO: 14); S247X (SEQ ID NO: 15); D395X (SEQ ID NO: 16); T558X (SEQ ID NO: 17); P560X (SEQ ID NO: 18), wherein X is a non-canonical amino acid (ncAA).
[0298] Relevant FGM protein sequences (all sequences are of the FAD-GDH a-subunit+MCD, without the g-subunit): R42X (SEQ ID NO: 19); S247X (SEQ ID NO: 20); D395X (SEQ ID NO: 21); T558X (SEQ ID NO: 22); P560X (SEQ ID NO: 23), wherein X is a non-canonical amino acid (ncAA). EXAMPLE 2
Reagents and materials
[0299] All DNA oligos used for the plasmid construction were purchased from Syntezza Bioscience Ltd. (Jerusalem, Israel). For the bacterial expression cultures, Auto-induction medium was purchased from Formedium™ (Hunstanton, England), glycerol was purchased from Bio-Lab ltd. (Jerusalem, Israel), chloramphenicol was purchased from Chem-impex int’l (Wood Dale, IL, USA), carbenicillin was purchased from Apollo (Manchester, England) and Propargyl-l-lysine was purchased from Synchem (Elk Grove Village, IL, USA). Tris buffer was purchased from Fisher scientific (Geel, Belgium), sodium chloride from Bio-Lab ltd. (Jerusalem, Israel), Imidzole from Glentham Life Sciences Ltd (Corsham, England), and Bradford reagent and Ni-NTA beads from Merck (Rehovot, Israel). Dimethoxybenzidine was purchased from Alfa Aesar (Heysham, England), 2,6-Dichlorophenolindophenol was purchased from Sigma- Aldrich (Rehovot, Israel), phenazine methyl sulfate and PCA from Tokyo chemical industry (Tokyo, Japan). TAMRA-Azide, THPTA and sodium ascorbate were purchased from Sigma-Aldrich (Rehovot, Israel). PDAz linker was purchased from Lumiprobe (Hunt Valley, MD, USA). Highly oriented pyrolytic graphite grade ZYB was purchased from Ted Pella inc. (Redding, CA, USA). Sodium acetate was purchased from Avantor inc. (Radnor, PA, USA), acetic acid from Bio-Lab ltd. (Jerusalem, Israel) and glassy carbon electrodes, Ag\AgCl reference electrodes, alumina polishing pad and 0.05pm alumina slurry were purchased from ALS (Tokyo, Japan). Urea, ascorbic acid and lactate were purchased from Sigma-Aldrich (Rehovot, Israel).
Protein expression and verification
[0300] FGM-S247PrK (Figure 11A) was expressed in 100 mL Auto-induction media supplemented with 1:100 overnight cultured bacteria, 50 pg/mL chloramphenicol, 100 pg/mL carbenicillin, 0.5% glycerol and 2 mM of UAA Propargyl-l-lysine (PrK) (Figure 11B (1)) for 48 h in 20 °C. The cells were then collected using centrifugation in 8000 rpm for 10 minutes followed by two washes with 15 mL lysis buffer (20 mM tris pH=8.0, 20 mM Imidazole, 500 mM NaCl). Cells were lysed using sonication needle and lysates were centrifuged for 30 min at 11 ,000 rpm to get a clear lysate. The lysate was then purified using immobilized metal affinity chromatography purification beads. The eluted protein concentration was then estimated using a Bradford assay to be 1 mg/mL and verified using anti his-tag Western blot analysis (Figure 12A), in-gel heme staining (Figure 12 B) and 2,6-Dichlorophenolindophenol (DCIP) glucose oxidation activity assay (Figure 12C). Verification of UAA incorporation into the protein sequence
[0301] To verify UAA incorporation in response to the TAG mutation, a Cu(I)-catalyzed azide-alkyne cycloaddition (click reaction) was used to bind the alkyne residue of (1) to the azide residue of the fluorescent marker 5-Carboxytetramethylrhodamine-azide (TAMRA-Az). Six (6) m M purified protein was mixed with 50 mM TAMRA-Az, 100m phosphate buffer (PB) pH=7.0, 200 mM CuCb, 1.2 mM Tris(3-hydroxypropyltriazolylmethyl) amine (THPTA) and 2.5 mM sodium ascorbate (NaAsc), and incubated for 1 h in the dark at RT with shaking. The fluorescence of the conjugated protein-TAMRA- Az was then analyzed by SDS-PAGE imaging using ImageQuant LAS4000 imager on a Cy3 mode (GE Healthcare, Little Chalfont, UK) (Figure 13).
In-gel heme staining
[0302] Polyacrylamide gel after SDS-PAGE was first washed with DDW to remove the electrophoresis buffer. The gel then incubated in 20 mL DDW, and 5 mL of dimethoxybenzidine (DMB) solution were added to a final concentration of 0.8 mg/mL. After 10 minutes incubation with shake, hydrogen peroxide was added to a final concentration of 0.7%v/v and gel was incubated with shake until bands of the oxidized DMB have appeared. Gel image was taken using ImageQuant LAS4000 imager.
FAD-GDH glucose oxidation activity assay using DCIP
[0303] D-glucose oxidation was measured biochemically in the presence of 0.6 mM phenazine methyl sulfate (PMS), 0.6 mM 2,6-Dichlorophenolindophenol (DCIP), 100 mM D-glucose and 4.5 mM FGM- S247PrK solution in total volume of 80 pL. All dissolved in tris buffer pH=7.0. The 80 pL mix absorbance in 610 nm was measured using plate reader (BioTek instruments, Winoosky, VT) in 37 °C with shacking. Decrease in the DCIP absorbance indicates glucose oxidation. Enzyme activity unit was determined to be the amount of enzyme oxidizing one pmol of substrate per minute. The inventors have calculated the extinction coefficient of DCIP to be 4.7 cm 1 mM 1.
AFM analysis
[0304] To assess the protein wiring and its distribution on the electrode surface, a highly oriented pyrolytic graphite (HOPG) grade ZYB was used as the substrate for AFM measurements. 5 pL drop of 3 mM pyrene-conjugated protein sample was incubated on a freshly cleaved HOPG surface for 10 minutes followed by a wash with double distilled water (DDW), to remove unbound molecules, then drying in air before a measurement in 100 pL of 100 mM acetate buffer pH=5.0. HOPG surface was cleaned before each measurement by cleaving it and exposing a new layer using an adhesive tape. Measurements were performed using a Cypher-ES (Asylum research, Oxford instruments) on AC mode with a micro cantilever BL-AC40TS (Olympus, Japan).
Verification of protein wiring on the electrode
[0305] To verify that both, FGM-S247PDAz and FGM-S247PCA are wired on the electrode surface, we first identified peaks using CVs (at 100 mV and 145 mV vs. Ag/AgCl, respectively). By using different scan rates, we have plotted the anodic peak current vs. the scan rates (Figure 14). It can be seen that both samples showed high linearity of peak currents with the scan rate and not with its square root (R2>0.99) which indicates wired specie on the electrode surface.
UAA incorporation into FGM
[0306] E. coli BF21 bacterial expression system was used for the expression of the FGM enzyme with site-specifically incorporated UAA. Pyrrolysyl orthogonal translation system (pylOTS) DNA sequence was amplified from the pEVOF plasmid using polymerase chain reaction (PCR) and cloned into the backbone of pec86 plasmid using Gibson’s assembly to generate a new plasmid called pec86-pylOTS (Figure 15A). pylOTS allows the continuous expression of an orthogonal translation system (orthogonal tRNA and aminoacyl tRNA synthetase) for incorporation of PrK (1) using the amber (TAG) stop codon suppression, while the pec86 plasmid is responsible for the continuous expression of E. coli cytochrome c maturation system. The second plasmid, pETDuet-FGM (Figure 15B) was used for the expression of FGM with a TAG mutation encoded in the desired incorporation site. Four different TAG mutants were planned using site-directed mutagenesis, according to GDH crystal structure. Our guiding rational for mutation sites was to control the enzyme orientation in a manner that will allow proximity of 14 A or less between the FAD or heme site and the electrode, without interference with the protein structure and correct folding. The sites that were tested were R42TAG and S247TAG, which are in close proximity to the FAD binding-site, while T558TAG (Figure 11C, right panel) is in a close proximity to the heme domain. For the FAD proximity mutation site, we had the highest expression levels and activity with the S247TAG mutant (Figure 11C, left panel). Hence, from here on we have focused our studies on S247TAG and T558TAG mutants. As a control that will allow us to find out whether the MCD affects ET properties of a site-specifically wired enzyme, we have also generated an S247TAG mutant GDH that lacks its MCD domain.
Protein conjugation with pyrene containing linkers
[0307] For the site-specific wiring of FGM-S247PrK, FGM-T558PrK and GDH-S247PrK the inventors have used a pyrene-diethyleneglycol-azide (PDAz) linker (Figure 11B, 2). The azide in (2) was clicked to the alkyne residue of (Figure 11B, 1). Three (3) mM of protein sample was mixed with 400 mM (2), 2 mM tris pH=8.2, 10 mM KC1, 1 mM Cu(I), 1.2 mM THPTA, 2.5 mM NaAsc and 10% Dimethyl sulfoxide (DMSO). The reaction was incubated for 1 h in the dark at room temperature with shaking, then transferred to 4 °C for two days before use. For the non-specific wiring of FGM-S247PrK we have used a standard EDC-N-Hydroxysuccinimide (NHS) coupling with a PCA linker (Figure 11B, 3). 3 mM of protein sample was mixed with 200 mM EDC, 400 mM NHS, 25 mM 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES) buffer pH=7.0 and 100 mM (3) and incubated for 12 h at RT with shaking.
Electrode preparations and electrochemical measurements
[0308] Glassy carbon electrode was first polished on 0.05 pm alumina polishing pad followed by five minutes sonication in DDW and drying using pure argon gas. Protein wiring to electrode was performed by dropping 5 pL of 3 pM pyrene-conjugated protein sample on GCE surface for 10 minutes incubation at RT (Figure ID) to allow the wiring of the protein through pi-pi stacking interactions between the pyrene groups and the GCE surface. After 10 minutes, the electrode was rinsed with DDW to remove unbound molecules and measured using a standard 3 -electrodes electrochemical system with Ag/AgCl reference electrode and a pencil rod as the counter electrode. The expected orientations of all FGM variants towards the electrode are presented in Figure HE. GDH-S247PDAz is not presented since it is expected to be oriented like FGM-S247PDAz, lacking MCD. All measurements were performed using Palmsense 3 potentiostat (Palm Instruments, Houten, The Netherlands) at RT in 100 mM acetate buffer pH=5.0 under argon atmosphere and at variable scan rates. DPV measurements were performed with potential pulse of 100 mV for 0.1 sec, with scan rate of 25 mV/sec. In the multistep amperometry measurements the electrode’s potential was biased from (-200) mV to 0 mV, framing the onset potentials of all modified electrodes, measuring the current decay for 10 sec for each step. In case of negative current values, a constant value was added to allow current decay linearization using natural logarithm (In) transformation. To test the enzyme -electrode performance in the presence of interfering molecules, we have used acetate buffer supplemented with 50 mM ascorbic acid, 10 mM lactate and 10 mM urea (ALU) and measured the current response upon application of 150 mV vs. Ag/AgCl reference electrode. Results and discussion Enzyme characterization
[0309] FGM-S247PrK expression was first verified using anti His-tag western-blot analyses (Figure 12A). Two expression cultures were grown, while only one of them was supplemented with 2 mM of (1), the cells were then lysed and analyzed. It can be seen that only with the supplementation of (1) to the expression culture we could observe a 67 kDa band indicating the successful expression of a full- length His-tagged protein. In the absence of (1) in the growth culture, we could not detect any protein band in the relevant size. The incorporation of (1) into the protein was verified by “clicking” a tetramethylrhodamine-Azide (TAMRA-Az) to the FGM-S247PrK, GDH-S247PrK and FGM-T558PrK protein variants. Fluorescent gel image (Figure 13) has demonstrated that the mutants of FGM and GDH indeed had (1) incorporated into their amino-acid sequence. One band in the expected size indicated that the FGM/GDH mutants are the only proteins in the sample that contain the alkyne residue of (1). Next, the inventors have used in-gel heme staining to verify a proper minimal cytochrome c maturation process (Figure 12B), where a band of around 67 kDa was observed, indicating a successful heme maturation. The enzyme’s catalytic activity towards glucose was tested biochemically using 2,6- Dichlorophenolindophenol (DCIP) colorimetric assay for glucose oxidation (Figure 12C). The colorimetric activity assay indicated that FGM-S247PrK, GDH-S247PrK and FGM-T558PrK are all capable of oxidizing glucose with a similar specific activity.
AFM measurements
[0310] Understanding protein orientation on carbon surface can be realized by the combination of the knowledge the inventors have from the protein crystal structure (Figure 11A) with AFM measurements. The inventors have analyzed the topography of an atomically flat carbon surface after wiring FGM-S247 in different methods and compare it to the known expected size of the protein. Figure 16 show the AFM measurements of HOPG surfaces after 10 minutes of incubation in acetate buffer (Figure 16, left panel), FGM-S247 wired through (2) (Figure 16, middle panel) and FGM-S247 wired through (3) (Figure 16, right panel). Since HOPG is atomically flat, the inventors could measure the height of the bound enzyme on the HOPG surface and compare it to an approximate enzyme foot -print and height calculated from the crystal structure of FAD-GDH (Figure 11A) taking into account the estimated lengths of the respective linkers. As a control, only acetate buffer added to the surface, does not result in salt accumulation (Figure 16, left panel). Measurement of the FGM-S247 linked through (3) showed islands of bound molecules averaging around 70 nm in height, almost 10 times higher than the height expected from a monolayer of FGM. These islands may be a result of protein aggregation caused by the wiring of the enzyme through multiple sites as expected from non-specific wiring. Coupling to (3) may result in multiple pyrene molecules on each protein. The adhesion of the protein to the surface through multiple pyrene residues can expose the hydrophobic core of the protein, which may result in the aggregation observed in the AFM measurements. However, measurement of the FGM-S247 clicked to (2) showed scattered particles on the surface with an average height of ca. 6 nm. When taking into consideration the anchoring point of the protein (S247) and the length of (2) (which is estimated to be 0.6 nm in its stretched form), this observation is very close to the estimated height of a single layer of a wired FGM in its expected orientation (Figure 11A) on the HOPG surface. These results suggest that using site- specific wiring affords a relatively good control over the protein orientation towards the surface as well as better surface coverage of active and correctly folded enzyme with a mono-layer pattern, although not a densely packed one, rather than multi layered and partially unfolded aggregates.
Electrochemical characterization
[0311] For the electrochemical characterization of the wired enzyme, three different enzyme variants were compared using a site-specific wiring approach with the PDAz linker. In addition, the site-specific wiring approach was compared to a non-specific wiring approach using EDC coupling of one of the mutants to the electrode (FGM-S247PCA). In order to see whether the electroactive peaks can be detected and differentiated between the wiring approaches, the inventors have conducted DPV measurements that are more sensitive and less prone to background from non-Faradaic currents. Figure 18A shows DPVs of FGM-S247PDAz and FGM-S247PCA in the absence of glucose. While FGM- S247PCA shows two well-defined peaks at (-100) mV and (+75) mV, FGM-S247PDAz shows a minute peak at (-250) mV and a defined albeit wide peak at ca. 0 mV vs. Ag/AgCl reference electrode. A control experiment where the DPV was performed on the conjugation reagents only (without adding protein samples) shows that the observed peaks indeed originate from FGM electroactive-sites (Figure 17) and not from the presence of other factors in the reaction mixture.
[0312] In order to elucidate the source of the two defined peaks in the non-specifically bound enzyme compared with the broad peak in the site-specifically bound enzyme, the inventors have calculated the apparent ET coefficient ( k^p ) using multistep amperometry as was previously described by the inventors. In Figure 18B it can be seen that the linearized current decay of the site-specifically wired enzyme has a mono-exponential decay characteristic (R2>0.93) while the non-specifically wired enzyme has a bi-exponential decay dependence, which indicates ET from one or two redox active-sites. It cannot be excluded that the observed mono-exponential characteristic signal is an average from both, FAD and heme domain, but it can be deduced from this result that only one orientation is dominant on the electrode surface. Hence, the inventors could extract k^p values from the slope of the linearized current decay curve as summarized in Table 3. Table 3. Apparent electrochemical constants of the different tested variants
Figure imgf000066_0001
[0313] Since the inventors have observed a mono-exponential current decay from the site-specifically bound enzymes variants and a bi-exponential current decay from the non-specifically bound one, it could be an indication of the source of the two peaks: whereas one peak origin is a direct communication between the heme and the electrode and the other is a mediated ET from the FAD to the heme and then to the electrode. The inventors hypothesize that the porphyrin, in the case of non-specific attachment is adsorbed directly on the electrode due to two lysine residues in a close proximity to its binding site. Whereas an internal ET (between the FAD and the heme) can be hardly observed when fast scan rates are being used as no electrocatalytic current is apparent in the presence of glucose under higher scan rates regime and only the two separate peaks are visible and are well defined (which is an indication of Faradaic processes but not catalytic ones) (Figure 18C). This result is in agreement with a control experiment that have shown that no electrocatalytic current is observed from attaching the heme binding domain only to the electrode in the presence of glucose as it cannot oxidize glucose in the absence of the FAD binding domain (Figure 18D).
[0314] Figure 18E shows the bioelectrocatalytic currents of three site-specifically wired variants and one non-specifically wired enzyme in response to 5 mM glucose addition using CV with a slow potential scan rate of 10 mV/sec. As opposed to a high-potential-scan rate measurements (Figure 18C), at a low scan rate, the inventors observed the catalytic currents from the different variants almost independently from ET rates (provided that those ET rates are faster than the potential scan rate). The catalytic response of the site-specifically wired enzymes was much higher compared to the non-specifically wired enzyme (ca. 10 folds higher increase in maximal currents) and its onset potential is lower (-100 mV for all the site-specifically wired enzymes compared to -45 mV for FGM-S247PCA). This result indicates better accessibility of the protein’s electroactive site towards the electrode. Within the site-specifically wired enzymes, both GDH-S247PDAz and FGM-S247PDAz, which are bound with proximity to the FAD domain, present almost the same catalytic current while the FGM-T558PDAz shows almost two-fold higher catalytic current compared to both of them. In the site-specifically wired enzyme the substrate has a direct access to the catalytic site and the electrons can be transferred directly to the electrode without any barriers. By comparing the two points of attachment it was evident that higher catalytic currents are observed when the enzyme was wired close to its cytochrome domain (FGMT558TAG) rather than through sites that are close to its FAD binding-site (FGMS274TAG). While with the non- specifically wired enzyme, ET can be deterred by proteins covering the electrode in different orientations that are not optimal for efficient ET.
[0315] For a given glucose concentration, the highest catalytic current was observed for FGM- T558PDAz variant, followed by FGM-S247PDAz and the lower catalytic current was observed with the GDH-S247PDAz electrode. Using high potential scan rates can help us understand whether the MCD has a role in the electron transfer to the electrode. While using low potential-scan rates resulted in almost the same catalytic current for both GDH and FGM, which are wired through the S247 site, using high scan rates resulted in higher currents for FGM. This observation suggests that the MCD participates in the ET process to the electrode, rendering it more efficient.
[0316] Apparent electron transfer rate constants were determined for the different variants, the highest was observed for FGM-T558PDAz wired to the electrode close to its MCD, next was the FGM- S247PDAz wired close to its FAD binding-site, the lowest value was that of GDH-S247PDAz which is wired in a close proximity to its FAD however in the absence of MCD and the lowest value was that of the non-specifically wired FGM-S247PCA. Glucose concentration vs. current calibration curves of all variants is presented in Figure 18F. It can be seen that the site-specifically wired enzymes show much higher currents than the non-specific wired ones. The apparent electrochemical Michaelis-Menten constants were determined based on a non-linear fit of the calibration curves shown in Figure 18F (Table 3). K^pp of all samples did not differ significantly between variants within the error of the measurements in the range of 1.55-1.89 mM which is an indication that the mutagenesis and the site of UAA incorporation did not modify the enzyme affinity to glucose. However, imax values were calculated to be more than 15 times higher for FGM-S247PDAz with 8.88 ± 0.40 m A cm2 vs. 0.55 ± 0.02 m A cm 2 for FGM-S247PCA. The site of attachment has also an effect on the imax values. Wiring through the T558TAG site with a proximity to the heme domain resulted in the highest current value, a slightly lower value was that of the S247TAG variant and the lowest value was that of the GDH-S247TAG. This result is another indication that the MCD has an effect on the ET abilities of the protein and are in correlation with the calculated kg PP values. In comparison with imax value previously reported by the present inventors for non-wired FGM, which was 2.0 ± 0.5 mA cm 2, the site-specific wiring demonstrates more than four times higher imax value which is a significant improvement. Those high currents allow much higher resolution for glucose biosensing with higher sensitivity, which could be very useful and physiologically relevant for samples with low glucose concentrations such as in sweat, subcutaneous plasma or tears samples. The limit of detection (LOD) was found to be 10 mM glucose for the site- specifically wired enzymes compared to 100 mM for the non-specifically wired enzyme, with a signal to noise ratio that is larger than 3. Since FGM-T558PDAz showed the highest currents in response to glucose, the inventors have presented its current response to the physiologically relevant concentrations in tears and sweat in the inset of fig. 2(F). FGM-T558PDAz present a resolution of 10 mM glucose and high linearity (R2=0.988) of the current measured in the 0.01 to 0.6 mM of glucose. The broader linear range of FGM-T558PDAz was found to be 0.01 - 2 mM glucose while FGM-S247PCA showed a linear range of 0.1 - 2 mM only (both with R2>0.92) (Figures 19A-19B). Improving the surface coverage will allow more available reaction sites (in a similar manner as improving enzyme specific activity, this could be achieved by using porous electrodes for example) that can result in a broader dynamic range. To test the herein disclosed wired enzyme response in the presence of interfering molecules, the inventors have used known interfering molecules in tears samples as previously described. The current response of FGM-T558PDAz was tested in ALU solution upon the addition of 0.1 mM glucose increments (Figure 18G). The current response in the physiologically relevant glucose concentrations in tears (0.1 -0.6 mM) was found linear under these conditions as shown in Figure 18H, no interference was observed under these conditions. Being able to detect glucose in the relevant concentrations without being interfered by the ALU solution makes our system suitable for glucose detection in bodily fluids other than blood. [0317] In conclusion, the inventors have presented the importance of a redox enzyme orientation towards an electrode. Non-specific wiring methods such as EDC-NHS coupling allows the covering of GCE with proteins but without the ability to control its orientation. By using site-specific UAA incorporation, the inventors have created a unique orthogonal “chemical handle” that allows the conjugation of a linker in only one anchoring point on the protein sequence, and by that allowed determination of a specific orientation towards the electrode. The controlled orientation results in ca. 20 times higher catalytic currents in response to glucose, higher ET efficiency that was shown by the ability of the enzyme to transfer electrons in a scan rate as high as 500 mV/sec and a scattered mono-layer pattern on the surface as observed by AFM measurements. Wiring of proteins through different sites result in a significant effect on their ET characteristics and their ability to communicate with an electrode. Using this method allows high flexibility in the UAA incorporation site which makes it a powerful tool for advanced protein engineering for enzyme -based biosensors. When site-specifically orienting a glucose oxidizing enzyme on the surface of an electrode, the inventors could observe a gain in sensitivity (down to a concentration of 10 m M glucose), however this gain in sensitivity comes with a cost, in gaining sensitivity the inventors have lost the dynamic range that allows for higher glucose concentrations sensing. Testing FGM enzyme-electrode in ALU solution demonstrated no interference when using known interfering molecules present in tears. This makes the enzyme more suitable for non- invasive biosensors for bodily fluids other than blood such as sweat, tears and urine.
EXAMPLE 3
FGM expression improvement
[0318] FGM expression gene is built from two sub-units, the alpha catalytic subunit, and the gamma subunit which is a helper protein that enables correct folding of the alpha subunit and responsible for the high stability of the whole protein complex. So far, the inventors have been working with an expression plasmid that has both genes under the same promoter (Trc promoter), leading to low expression levels of FGM. In order to improve its expression levels, the inventors have cloned the FGM gene into a pETDuet plasmid, where each of the subunits is expressed from “its own” T7 promoter. The use of pETDuet results in much higher expression levels for FGM (Figure 20), indicating that using a different promoter for each subunit indeed increases expression efficiency.
Site-specific wiring of FGM to an electrode
[0319] The structure of FAD-GDH from Burkholderia cepacia was published recently and enabled the inventors to better understand the structure of the protein and better plan ncAA incorporation sites for site-specific wiring to an electrode. S247 is estimated to be 9.9 A from the FAD site and T558 is estimated to be 10 A from the heme site (Figure 21). Using those variants allow site-specific ‘wiring’ to an electrode with high proximity to allow direct electron transfer. To allow the incorporation of ncAA along with heme maturation, the inventors have engineered the pec86 plasmid (cytochrome c maturation plasmid) by fusing into its backbone the orthogonal tRNA and aminoacyl tRNA synthetase DNA sequences from pEVOF-pylOTS plasmid (ncAA incorporation plasmid), creating a new plasmid - pEC86-pylOTS.
[0320] Using the new ncAA incorporation and heme maturation plasmid (pec86-pylOTS) the inventors have managed to express the FGM ncAA variants with a mature c-type cytochrome. The incorporation of propargyl-lysine (PrK) was verified using a click reaction to a fluorescent marker (TAMRA-Azide) and the heme maturation was verified using in-gel heme staining. The glucose oxidation activity of the different variants was tested using a standard FAD-GDH activity assay (Figures 22A-22C).
[0321] The ‘click’ reaction with TAMRA-Azide has proved that the inventors system successfully expressed FGM with PrK incorporation (Figure 22A). In-gel heme staining approved that our variants have a matured c-type cytochrome and that our pec89-pylOTS plasmid can do both, ncAA incorporation and heme maturation (Figure 22B). Using the FAD-GDH activity assay proved that the inventors variants are active and can oxidize glucose (Figure 22C). The difference in the activity rate between S247PrKFGM and T558PrKFGM is probably due to the difference in protein concentrations. Different ncAA locations have been shown to affect the protein expression efficiency and it correlates with the bends’ intensities in the protein gels (Figure 22A, 22B).
[0322] D395PrKFGM was barely active, probably due to an effect of the change on the enzyme active site. The D395 site mutation was planned according to the SWISS-model homology and found to be different from the real protein structure (found in a surface exposed loop in the homology model, while the real protein structure showed that it is located in the middle of an important a-helix).
[0323] In the next step the inventors ‘clicked’ the different variants to a pyrene-azide linker (Figure 23C) to test its site-specific ‘wiring’ to a glassy carbon electrode (GCE).
[0324] The inventors performed a click reaction on S247PrK FGM and on WT FGM to verify their site specific wiring. The inventors expected that wiring FGM will not work since it doesn’t have an incorporated PrK, while S247PrKFGM will be wired to the electrode. From the results (Figure 23A) it could be seen that S247PrKFGM was successfully wired to the electrode and allowed high catalytic currents in response to glucose addition while WT FGM showed no bioelectrocatalytic current. As opposed to S247PrKFGM, WT FGM couldn’t stay on the electrode surface and was washed away in the measurements’ buffer. When comparing wired and entrapped S247PrKFGM (Figure 23B), it can be seen that the wiring of the protein allows higher catalytic currents and lower onset potential, indicating highly efficient electron transfer (ET) that is gained due to the site-specific wiring. Same tests were performed on T558PrKFGM variant, and it has been proved to be wired as well.
[0325] To eliminate the possibility that the click reaction reagents have affected the 247PrKFGM structure in a way that will allow higher catalytic response, the inventors have performed the click reaction without the pDAz linker (Figure 24A). It was found that the clicked enzyme in the presence of pDAz showed higher catalytic current with lower onset potential indicating that the increase in efficiency is due to the site-specific wiring of FGM. The wired enzymes have been tested for electrochemical communication with the electrode in high scan rates (100-500 mV/sec) and showed catalytic currents even in those scan rates, indicating very fast ET rate (Figure 25). The specificity of S247PrKFGM towards glucose was tested as well by introducing other sugars and measuring the chronoamperometric response (Figure 24B). It has been shown that the wired enzyme didn’t lose its specificity towards glucose.
[0326] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMS What is claimed is:
1. A recombinant protein, comprising (a) alpha subunit of an FAD-GDH; and (b) a minimal c-type cytochrome peptide, comprising an amino acid sequence comprising a non-canonical amino acid (ncAA), wherein said amino acid sequence is set forth in SEQ ID NO: 20 or SEQ ID NO: 22, and wherein said ncAA is covalently bound to a mediator molecule being represented by Formula I:
Figure imgf000072_0001
wherein n is an integer in a range from 1 to 5.
2. The recombinant protein of claim 1, wherein said ncAA comprises Propargyl-lysine (PrK).
3. The recombinant protein of claim 1 or 2, wherein n equals 2.
4. The recombinant protein of any one of claims 1 to 3, wherein said mediator molecule is characterized by having a diethylene oxide group having a length of 6.6 A at most.
5. A polynucleotide comprising a nucleic acid sequence encoding the polypeptide of any one of claims 1 to 4.
6. The polynucleotide of claim 5, comprising a nucleic acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 17.
7. The polynucleotide of claim 5 or 6, being operably linked to a regulatory element.
8. The polynucleotide of claim 7, wherein said regulatory element is a T7 promoter.
9. An expression vector or a plasmid comprising the polynucleotide of any one of claims 5 or
8.
10. The expression vector or plasmid of claim 9, further comprising a nucleic acid sequence encoding a gamma subunit of an FAD-GDH.
11. The expression vector or plasmid of claim 9 or 10, wherein said nucleic acid sequence encoding said gamma subunit of an FAD-GDH is operably linked to a regulatory element.
12. The expression vector or plasmid of claim 11, wherein said regulatory element is a T7 promoter.
13. A transgenic or a transfected cell comprising any one of: a. the recombinant protein of any one of claims 1 to 4; b. the polynucleotide of any one of claims 5 to 8; c. the expression vector or plasmid of any one of claims 9 to 12; or d. both (a) and (b).
14. The transgenic or transfected cell of claim 13, being a prokaryotic cell.
15. An extract obtained or derived from the transgenic or transfected cell of claim 13 or 14.
16. A composition comprising any one of: a. the recombinant protein of any one of claims 1 to 4; b. the polynucleotide of any one of claims 5 to 8; c. the expression vector or plasmid of any one of claims 9 to 12; d. the transgenic or transfected cell of claim 13 or 14; e. the extract of claim 15; or f. any one of (a) to (d), and an acceptable carrier.
17. An electrode coupled to the polypeptide of any one of claims 1 to 4, wherein said coupled is by non-covalent interactions.
18. A device comprising the electrode of claim 17.
19. A method for determining presence, concentration, or both, of an analyte in a liquid medium, the analyte being capable of undergoing a biocatalytic oxidation or reduction reaction in the presence of an oxidizer or a reducer, respectively, the method comprising: i. providing said device of claim 18; ii. contacting said device with said liquid medium; iii. measuring the electric signal generated between a cathode and an anode, the electric signal being indicative of any one of the presence of the analyte, the concentration of the analyte, and both; and iv. determining the presence, concentration, or both, of said analyte in said liquid medium based on the electric signal measured in step (iii).
20. The method of claim 19, wherein said analyte comprises glucose.
PCT/IL2022/050559 2021-05-27 2022-05-26 Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof WO2022249186A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
IL308859A IL308859A (en) 2021-05-27 2022-05-26 Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof
EP22810794.2A EP4352211A1 (en) 2021-05-27 2022-05-26 Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US17/332,718 2021-05-27
US17/332,718 US20210292804A1 (en) 2017-08-02 2021-05-27 Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof

Publications (1)

Publication Number Publication Date
WO2022249186A1 true WO2022249186A1 (en) 2022-12-01

Family

ID=84229600

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2022/050559 WO2022249186A1 (en) 2021-05-27 2022-05-26 Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof

Country Status (3)

Country Link
EP (1) EP4352211A1 (en)
IL (1) IL308859A (en)
WO (1) WO2022249186A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019026082A1 (en) * 2017-08-02 2019-02-07 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University A recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019026082A1 (en) * 2017-08-02 2019-02-07 B. G. Negev Technologies And Applications Ltd., At Ben-Gurion University A recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALGOV ITAY, FEIERTAG AVIV, ALFONTA LITAL: "Site-specifically wired and oriented glucose dehydrogenase fused to a minimal cytochrome with high glucose sensing sensitivity", BIOSENSORS AND BIOELECTRONICS, ELSEVIER SCIENCE LTD, UK, AMSTERDAM , NL, vol. 180, 1 May 2021 (2021-05-01), Amsterdam , NL , pages 113117, XP093006757, ISSN: 0956-5663, DOI: 10.1016/j.bios.2021.113117 *
ALGOV ITAY, GRUSHKA JENNIFER, ZARIVACH RAZ, ALFONTA LITAL: "Highly Efficient Flavin–Adenine Dinucleotide Glucose Dehydrogenase Fused to a Minimal Cytochrome C Domain", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 139, no. 48, 15 September 2017 (2017-09-15), pages 17217 - 17220, XP055782617, ISSN: 0002-7863, DOI: 10.1021/jacs.7b07011 *
AMIR LIRON, CARNALLY STEWART A., RAYO JOSEP, ROSENNE SHAKED, MELAMED YERUSHALMI SARIT, SCHLESINGER ORR, MEIJLER MICHAEL M., ALFONT: "Surface Display of a Redox Enzyme and its Site-Specific Wiring to Gold Electrodes", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, vol. 135, no. 1, 9 January 2013 (2013-01-09), pages 70 - 73, XP093006756, ISSN: 0002-7863, DOI: 10.1021/ja310556n *

Also Published As

Publication number Publication date
EP4352211A1 (en) 2024-04-17
IL308859A (en) 2024-01-01

Similar Documents

Publication Publication Date Title
US9353395B2 (en) Glucose dehydrogenase/cytochrome fusion protein
JP5398004B2 (en) Modified glucose dehydrogenase gene
JP5243878B2 (en) Fructosylvalylhistidine measuring enzyme and method of use thereof
JP2010057427A (en) Glucose dehydrogenase and method for electrochemical measurement of glucose
JP5740754B2 (en) Enzyme crystal-immobilized electrode, method for producing enzyme crystal-immobilized electrode, and bio battery and biosensor equipped with enzyme crystal-immobilized electrode
US20220162569A1 (en) Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof
CN108350439A (en) Mutant 3-hydroxybutyrate dehydrogenase and its correlation technique from Alcaligenes faecalis and purposes
JP5593689B2 (en) Lactate oxidase composition
JP2010051312A (en) Electrochemically measuring method for glucose using glucose dehydrogenase
EP4067483A1 (en) Mutant glucose dehydrogenase having improved thermal stability, and use thereof
US11505596B2 (en) Mutant cytochrome protein lacking certain heme domains and use thereof
US20210292804A1 (en) Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof
US20240141303A1 (en) Recombinant lactate dehydrogenase and uses thereof
WO2022249186A1 (en) Recombinant flavin-adenine dinucleotide glucose dehydrogenase and uses thereof
JPWO2017183717A1 (en) HbA1c dehydrogenase
EP3572503B1 (en) Mutant glucose oxidase and use thereof
JP2011103770A (en) Glucose dehydrogenase and method for electrochemically measuring glucose
US20220306996A1 (en) Mutant Glucose Dehydrogenase Having Improved Thermal Stability, and Use Thereof
JP4478865B2 (en) Modified metalloenzyme
JP2025030829A (en) Lactic acid-responsive enzyme electrode
ES2803273T3 (en) Alcaligenes faecalis mutant 3-hydroxybutyrate dehydrogenase, as well as procedures and uses involving the same
JP2009095348A (en) Modified sarcosine oxidase

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22810794

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 308859

Country of ref document: IL

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2022810794

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2022810794

Country of ref document: EP

Effective date: 20240102