RU2746432C1 - Using cagfbfp as fluorescent label - Google Patents

Abstract

FIELD: biotech.

SUBSTANCE: present invention relates to the field of biotechnology, specifically to a fluorescent protein. The invention makes it possible to obtain a new fluorescent protein that contains the LOV domain and in which at least one cysteine is replaced by an amino acid that does not bind it covalently to flavin under illumination.

EFFECT: invention can be used as a fluorescent label to study the interaction between proteins, folding and localization of proteins.

2 cl, 3 dwg, 3 tbl, 1 ex

Description

Description of the invention:

The term "chromophore" refers to a group of atoms that determines the color of a chemical compound.

The term "fluorescence" means a phenomenon in which light is emitted by a substance that has absorbed light or other electromagnetic radiation.

The term "fluorophore" refers to a molecule that is capable of fluorescence.

The term "emission spectrum" is the dependence of the emission intensity on the wavelength for a fixed length of the exciting light.

The term "maximum emission" is the wavelength at which the emission intensity is maximum for a particular exciting wavelength.

The term "excitation spectrum" is the dependence of the radiation intensity (at a certain wavelength) depending on the excitation wavelength.

The term "maximum excitation" is the excitation wavelength at which the radiation intensity (at a particular wavelength) is maximum.

The term "quantum yield" is the ratio of the number of photons emitted and absorbed as a result of fluorescence.

The term "molar extinction coefficient" is a coefficient determined by the Bouguer-Lambert-Beer law, measured in mol ^-1 * cm ^-1 .

The term "visible light" is electromagnetic waves, the wavelength of which is in the range of 380 nm. - 780 nm.

The term "protein fluorescence brightness" is the product of the molar extinction coefficient and the quantum yield divided by 1000.

The term "amino acid sequence" is the order in which the amino acid residues occur in a protein.

The term "nucleic acid" is understood to mean a single-stranded or double-stranded molecule that consists of nucleotides. The most common nucleic acid is deoxyribonucleic acid (DNA) and its complementary deoxyribonucleic acid (cDNA), which also includes ribonucleic acid (RNA). DNA consists of nitrogenous bases adenine (A), guanine (G), cytosine (C), thymine (T). The RNA molecule consists of the same nitrogenous bases, with the exception of thymine, which is replaced by uracil (U).

By "complementary nucleic acid" is meant a nucleic acid, the nucleotide sequence of which consists of nitrogenous bases, each of which is capable of forming hydrogen bonds with each corresponding nitrogenous base of the original nucleotide acid. In the case of a DNA-DNA complex, adenine (T-A) is associated with thymine, and guanine-cytosine (G-C) and vice versa A-T and C-G. In the case of an RNA-DNA complex in the formation of hydrogen bonds in ribonucleic acid, the place of thymine is taken by uracil (U-A; A-U; G-C; C-G). As an example for a DNA fragment of AGTCATG, the complementary nucleotide sequence of the cDNA fragment would look like this: TCAGTAC:

5 'AGTCATG 3'

3 'TCAGTAC 5'

The term "silent mutation" means a mutation in a nucleic acid that does not lead to a change in the amino acid sequence encoded by this nucleic acid.

The term "fragment" is understood as a part of an amino acid or nucleotide sequence that lacks some portions of the sequence, while retaining the ability to fluoresce in the case of an amino acid sequence and encodes a polypeptide that retains the ability to fluorescence in the case of a nucleotide sequence.

The term "homologue" refers to a nucleic acid or amino acid sequence in the sequence of which at least one nucleotide or amino acid is added, removed, substituted, or otherwise modified from the original nucleic acid or amino acid sequence. It is taken into account that the homologue has the same properties as the original nucleic acid.

The term "optimized nucleic acid for a specific expression system" means a nucleic acid sequence that encodes the same amino acid sequence as the original nucleotide sequence, but consists of different nucleotide codons (a sequence of three nitrogenous bases that encode the amino acid), in accordance with what codons are used by the expression system to synthesize the polypeptide.

By "sequence identity X%" is meant the identity of the X% sequences according to the BLAST algorithm (peptide and nucleotide sequence comparison algorithm), which is available on the NCBI homepage (https://www.ncbi.nlm.nih.gov/).

GFP-like proteins.

GFP (Green Fluorescent Protein) is a protein that was obtained from the jellyfish Aequorea victoria and is widely used in the field of fluorescent labeling of both individual cells and various tissues [1].

There are a huge number of derivatives and homologues of GFP (GFP-like proteins), some of which are obtained from other organisms [1]. Among GFP-like proteins, there are those that demonstrate different fluorescent properties (maximum excitation, maximum emission, quantum yield, molar extinction coefficient) [2].

GFP-like proteins have a number of advantages: there are proteins with high fluorescence brightness, autocatalytic assembly of the internal chromophore occurs, and the presence of a large number of proteins with different optical characteristics (emission spectrum, fluorescence brightness, etc.):

1) Among GFP-like proteins, there are proteins with excitation maxima varying from 400 nm. up to 610 nm.

2) Among the GFP-like proteins, there are proteins whose emission maxima are in the range of 450 nm. up to 670 nm.

Thus, there are many GFP-like proteins that can fluoresce in different colors from red to violet.

Also, GFP-like proteins have a number of properties that limit the possibilities of their use:

1) In order for the fluorescence of a GFP-like protein to be observed, the presence of molecular oxygen in the solution is necessary. This property cannot be eliminated by genetic modifications [3]. Thus, there are restrictions on the use of GFP-like proteins in conditions of low oxygen content (tumor hypoxia, cerebral ischemia, pathogenesis and development of biofilms) [4].

2) GFP-like proteins have a weight of more than 25 kDa, which can cause various steric restrictions.

LOV proteins.

LOV domains are part of the large family of Per-Arnt-Sim (PAS) domains and bind various flavins (FMN - flavin mononucleotide; FAD-flavin adenine dinucleotide; riboflavin, which are shown in Fig. 1) as chromophores that, when exposed to ultraviolet and blue light form a covalent bond with the nearest cysteine of the LOV domain [5].

Most proteins containing LOV domains are oligomeric multidomain sensory systems consisting of a photosensitive LOV domain and attached effector domains. The typical size of LOV domains is 12-19 kDa. The core region (core) of LOV domains has a classic PAS folding, which consists of five antiparallel beta chains (Aβ; Bβ; Gβ; Hβ; Iβ), forming a beta framework, and alpha-helical connecting elements (Cα; Dα; Eα ; Fα), jointly non-covalently binding chromophores (Fig. 2).

Previously, some LOV-domain containing proteins have already been used for the development of fluorescent labels (reporter proteins) [6], [7]. These proteins are called FbFPs (FMN-binding fluorescent proteins or FMN-binding fluorescent proteins). These proteins include EcFbFP and BsFbFP, which were made on the basis of the LOV-domain-containing YtvA protein from the gram-positive soil bacterium Bacillus subtilis. PpFbFP was also created based on the PbSB2-LOV protein, but from the gram-negative soil bacterium Pseudomonas putida. All three fluorescent proteins are characterized by the substitution of cysteine for alanine, which does not bind covalently to flavin and thus allows one to observe the fluorescent emission of polypeptides all the time [8].

A LOV-domain-containing fluorescent protein iLOV was also obtained, but this time from the Arabidopsis thaliana plant [9], which was later structurally improved to the so-called phiLOV, whose photostability (the ability of a molecule to maintain its stability under prolonged exposure to visible light from 380 nm to 780 nm) was better than that of its predecessor [10].

In addition, LOV-domain-containing fluorescent peptides were created based on proteins found in thermostable organisms. One of the first such proteins was CreiLOV from the alga Chlamydomonas reinhardtii and VafLOV from another alga, Vaucheria frigida [11].

CagFbFP

The bacterial genome of Chloroflexus aggregans contains the Cagg_3753 protein (SEQ ID 4), which contains the LOV domain [12]. To create a fluorescent label, a gene (SEQ ID 1) was created that encodes a fragment of the Cagg_3753 protein (SEQ ID 4) from amino acid 47 to amino acid 153, resulting in SEQ ID 2 gene encodes a polypeptide fragment of SEQ ID 1.

In order to use the polypeptide sequence of SEQ ID 3 as a fluorescent label, replacement of cysteine (amino acid 83 in SEQ ID 4) with any other amino acid that will not covalently bind to flavin is required.

One of the possible options is the replacement of cysteine with alanine (SEQ ID 1).

In addition, the sequence SEQ ID 1 is allowed to contain up to 5 additional amino acid point mutations (in addition to mutations of cysteine to another amino acid).

The invention is also a LOV domain:

1) Which is encoded by the nucleic acid SEQ ID 2, as well as a fragment or homologue of this sequence.

2) Coded by a nucleic acid that is 70% identical to the sequences from point 1.

3) Coded by a nucleic acid that allows silent mutations in the sequences from points 1-2.

4) Encoded by the nucleic acid from items 1-3, which have been optimized for a specific expression system.

Fluorescent protein SEQ ID 1 has the following properties:

1) Its weight is no more than 12 kDa.

2) Has a maximum excitation in the range of 430 nm. - 470 nm.

3) Has a maximum radiation in the range 480-520 nm, when irradiated with a wavelength of 450 nm.

Compared to its analogs, CagFbFP was obtained from the thermophilic Chloroflexus aggregans and is the smallest monomer protein (Table 3), and its amino acid sequence CagFbFP is less than 55% identical with any other known LOV-domain-containing proteins that are used as a fluorescent label (Table 3).

The present invention will be further described using the following examples. It should be understood that these examples are given only to illustrate the present invention, and the present invention is not limited to these examples. Any modification can be made by a person skilled in the art in light of the present disclosure within the scope of the contemplated claims.

Example:

Nucleic acid (SEQ ID 2), which encodes a fragment of the protein SEQ ID 4 (complete sequence can be found at https://www.uniprot.org/ with unique identifier B8GAY9) from amino acid 47 to 153, in which cysteine is additionally replaced at position 85 on alanine, was assembled by genetic engineering using a plasmid.

For protein expression, the bacterial strain C41 (DE3) was used. The cells produced protein for 7 hours at 37 ° C. Then the cells with the protein were disrupted in a buffer containing 50 mM Tris-HCl pH 8.0; 300 mM NaCl. The lysate was purified from membranes by ultracentrifugation at 100,000 g for 1 hour. The resulting solution was additionally purified by metal affinity chromatography using Ni-NTA resin (Qiagen, Germany). Then the eluate with the protein was purified by gel filtration (a Superdex® 200 Increase 10/300 GL GE Healthcare Life Sciences, USA gel filtration column was used) in a buffer containing 10 mM NaCl and 10 mM sodium phosphate at pH 8.0.

The resulting purified protein CagFbFP (SEQ ID 1) can fluoresce in the green region. Such a protein has a maximum excitation in the 430-470 nm wavelength range and an emission maximum in the 480-520 range when CagFbFP is irradiated with 450 nm light (Fig. 3).

LINKS:

[1] M. V. Matz, K. A. Lukyanov, and S. A. Lukyanov, 'Family of the green fluorescent protein: journey to the end of the rainbow', Bioessays, vol. 24, no. 10, pp. 953-959, 2002.

[2] G. J. Kremers, S. G. Gilbert, P. J. Cranfill, M. W. Davidson, and D. W. Piston, 'Fluorescent proteins at a glance. ', J Cell Sci, vol. 124, no. 2, pp. 157-160, 2011.

[3] R. M. Wachter, 'Chromogenic cross-link formation in green fluorescent protein', Acc. Chem. Res., Vol. 40, no. 2, pp. 120-127, 2007.

[4] C. Coralli, M. Cemazar, C. Kanthou, G. M. Tozer, and G. U. Dachs, 'Limitations of the reporter green fluorescent protein under simulated tumor conditions', Cancer Res., Vol. 61, no. 12, pp. 4784-4790, 2001.

[5] M. Salomon, J. M. Christie, E. Knieb, U. Lempert, and W. R. Briggs, 'Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin', Biochemistry, vol. 39, no. 31, pp. 9401-9410, 2000.

[6] A. Mukherjee and C.M. Schroeder, 'Flavin-based fluorescent proteins: emerging paradigms in biological imaging', Curr. Opin. Biotechnol, vol. 31, pp. 16-23, 2015.

[7] A. M. Buckley, J. Petersen, A. J. Roe, G. R. Douce, and J. M. Christie, 'LOV-based reporters for fluorescence imaging', Curr. Opin. Chem. Biol, vol. 27, pp. 39-45,2015.

[8] T. Drepper et al., 'Reporter proteins for in vivo fluorescence without oxygen', Nat. Biotechnol, vol. 25, no. 4, pp. 443-445, 2007.

[9] S. Chapman et al., 'The photoreversible fluorescent protein iLOV outperforms GFP as a reporter of plant virus infection', Proc. Natl. Acad. Sci, vol. 105, no. 50, pp. 20038-20043, 2008.

[10] J. M. Christie et al, 'Structural tuning of the fluorescent protein iLOV for improved photostability', J. Biol. Chem., Vol. 287, no. 26, pp. 22295-22304, 2012.

[11] A. Mukherjee, K. B. Weyant, U. Agrawal, J. Walker, I. K. Cann, and C. M. Schroeder, 'Engineering and characterization of new LOV-based fluorescent proteins from Chlamydomonas reinhardtii and Vaucheria frigida', ACS Synth. Biol., Vol. 4, no. 4, pp. 371-377, 2014.

[12] S. T. Glantz et al., 'Functional and topological diversity of LOV domain photoreceptors', Proc. Natl. Acad. Sci., Vol. 113, no. 11, pp. E1442-E1451, 2016.

Using CagFbFP as a fluorescent label.

Table 1

SEQ ID 3 is the amino acid sequence of Cagg_3753 from amino acids 47 to amino acids 153.

SEQ ID 1 - Amino acid sequence of CagFbFP with cysteine (39 amino acids) replaced by alanine

SEQ ID 4 is the complete amino acid sequence of the Cagg_3753 protein, a portion of which (amino acids 47 to 153) contains CagFbFP.

table 2

Nucleic acid encoding SEQ ID 1, which is optimized for expression in E. coli.

Table 3.

The table shows the lengths and weights of the monomers of the main LOV-domain-containing proteins and their amino acid sequence identity with respect to CagFbFP (SEQ ID 1).

Claims (2)

1. Protein, the amino acid sequence of which is shown in SEQ ID NO: 1, intended for use as a fluorescent label, having an excitation maximum in the interval between 430-470 nm, having an emission maximum in the interval between 480-520 nm when the sample is irradiated with a wavelength of 450 nm. 2. A nucleic acid encoding a fluorescent protein according to claim 1.

Images