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EP2747901A1 - Procédés de revêtement de substrats par au moins une monocouche de protéines capables d'autoassemblage - Google Patents

Procédés de revêtement de substrats par au moins une monocouche de protéines capables d'autoassemblage

Info

Publication number
EP2747901A1
EP2747901A1 EP12753109.3A EP12753109A EP2747901A1 EP 2747901 A1 EP2747901 A1 EP 2747901A1 EP 12753109 A EP12753109 A EP 12753109A EP 2747901 A1 EP2747901 A1 EP 2747901A1
Authority
EP
European Patent Office
Prior art keywords
protein
self
assembling
solution
substrate
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP12753109.3A
Other languages
German (de)
English (en)
Inventor
Kai Ostermann
Leopold GRUNER
Gerhard RÖDEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Technische Universitaet Dresden
Original Assignee
Technische Universitaet Dresden
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 DE102011081524.4A external-priority patent/DE102011081524B4/de
Priority claimed from DE201110089241 external-priority patent/DE102011089241B3/de
Application filed by Technische Universitaet Dresden filed Critical Technische Universitaet Dresden
Publication of EP2747901A1 publication Critical patent/EP2747901A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D7/00—Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials
    • B05D7/24—Processes, other than flocking, specially adapted for applying liquids or other fluent materials to particular surfaces or for applying particular liquids or other fluent materials for applying particular liquids or other fluent materials
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
    • B05D3/007—After-treatment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/31504—Composite [nonstructural laminate]
    • Y10T428/31725—Of polyamide

Definitions

  • the invention relates to methods for coating a substrate with at least one monolayer of self-assembling proteins and substrates obtainable thereby.
  • the invention finds application in the chemical industry, biotechnology, sensor technology as well as in medical research.
  • Self-assembling proteins such as hydrophobins or surface layer proteins (S-layer proteins) tend in aqueous solutions to a rapid and uncontrollable aggregate formation (the uncontrolled assembly of protein monomers to multimers of self-assembling proteins), which is the production of homogeneous layers of self-assembling proteins on a Substrate difficult.
  • self-assembling proteins are also simply referred to simply as "proteins.”
  • Self-assembling proteins form a directed folding and association with one another through interactions of the protein molecules (molecular self-assembly) and are the most diverse of applications because of the formation of this highly ordered structure usable in nanotechnologies.
  • the Langmuir-Blodgett method, the Langmuir-Schafer technique, or the "layer-by-layer” method are commonly used to construct homogeneous layers of self-assembling proteins
  • the substrate is immersed in a solution containing protein monomers so that a layer of the protein monomers (monolayer) is deposited on the substrate.
  • the layer-by-layer techniques are based exclusively on exploiting the physisorption of the protein monomers on the substrate surface Since self-assembling proteins have a strong propensity for aggregate formation, long-term storage and use of previously prepared protein solutions is not possible, and the coating of hard-to-reach surfaces of the substrate (eg, pores of a protein) is lim It is difficult, because difficult to reach places on the substrate, such as pores, are caused by uncontamination Roll aggregated protein monomers can be closed, so that other monomers can not penetrate into pores.
  • DE 10 2005 051515 A1 discloses methods for coating surfaces with defined fusion hydrophobins.
  • An aqueous solution having a pH of> 4 is contacted with a surface and the solvent is subsequently removed.
  • the hydrophobin solution can be further additives, such as nonionic Surfactants and / or metal ions are added n.
  • the method does not permit controlled deposition of protein layers.
  • TFA trifluoroacetic acid
  • WO 01/57066 A2 discloses a process in which the disulfide bonds within a hydrophobin are cleaved by addition of a strongly denaturing agent, such as guanidine hydrochloride, so that the folding of the hydrophobin is dissolved.
  • a strongly denaturing agent such as guanidine hydrochloride
  • suitable sulfhydryl protecting agent-forming agents avoids re-formation of disulfide bonds in the hydrophobin solution so that the resulting hydrophobin monomer-containing solutions are stable.
  • the removal of the protective group is required, which is effected by addition of an oxidizing agent.
  • WO 96/41882 A1 discloses hydrophobin assembly on oil-water interphases. Furthermore, EP 1 252 516 A1 and WO 2005/068087 A1 disclose the immobilization of hydrophobins by means of thermal sample preparation or by shifting the pH to an acidic medium. However, both methods give no information about the number and homogeneity of generated hydrophobin deposits, which is why they can not be used for applications in sensor technology or biomedicine.
  • the object of the invention is a method which allows the coating of substrates with at least one monolayer of self-assembling proteins and to provide necessary solutions.
  • the object is achieved by a method for coating a substrate with at least one monolayer of self-assembling proteins with the following steps:
  • Self-assembling proteins in aqueous solutions tend to aggregate and form aggregates with more than 10 protein units. With these aggregates a controlled deposition of defined protein layers is not possible. It is therefore important that these aggregates are separated and preferably remain in the separated solution monomers and oligomers of self-assembling proteins.
  • the separation is advantageously carried out by centrifugation with a centrifugal acceleration of at least 5000 ⁇ g, preferably 10000 to 50,000 ⁇ g over a period of at least 5 minutes, preferably 15-60 minutes, at a temperature of -10 ° C. to 40 ° C., preferably 0. 10 ° C.
  • Monomers are the smallest indivisible protein units of a self-assembling protein. Oligomers according to the invention are assemblies of self-assembling proteins with a maximum size of 10 protein monomers.
  • a protein monolayer is a planar structure of surface-assembled protein units, preferably protein monomers, with the layer thickness of a protein monomer.
  • a solution of ionic surfactants and / or a salt-containing and / or alkaline and / or an acidic solution are added to the solution after separation of the protein aggregates.
  • ionic surfactants are added so that only the surface-active part of each active predominant protein oligomer is enveloped by surfactant particles.
  • the concentration of the self-assembling proteins in the aqueous solution is at least 5 ng / ⁇ , preferably at least 50, particularly preferably at least 90 ng / ⁇ .
  • the aqueous solution of the self-assembling proteins preferably has a pH of 7 to 9.5. This is preferably a buffered solution.
  • self-assembling proteins are hydrophobins and / or surface layer proteins (S-layer proteins) and / or recombinant fusion proteins having at least two domains, wherein at least one domain is a self-assembling protein domain and a domain is a functional domain.
  • S-layer proteins surface layer proteins
  • recombinant fusion proteins having at least two domains, wherein at least one domain is a self-assembling protein domain and a domain is a functional domain.
  • Preferred hydrophobins are class I hydrophobins, preferably Ccg2 from Neurospora crassa, or class II hydrophobins, preferably HFBI or HFBII from Trichoderma reesei.
  • Preferred S-layer proteins are SBSC from Bacillus stearothermophilus, S 13240 from Bacillus stearothermophilus or SslA from Sporosarcina ureae.
  • the invention is particularly advantageously suitable for functionalized self-assembling proteins, since the accessibility of the functionalization can be ensured by the coating according to the invention of the functionalized self-assembling protein. It is advantageously possible to carry out the coating of the substrate so that the functionalization is present on the side of the protein-containing coating remote from the substrate.
  • the self-assembling protein is preferably a chemically modified or a recombinant fusion protein, ie a protein which is produced by genetically modified organisms.
  • a recombinant fusion protein ie a protein which is produced by genetically modified organisms.
  • a recombinant fusion protein used in the invention comprises at least two domains, wherein at least one domain is a self-assembling protein domain (preferably selected from the above-mentioned self-assembling proteins) and a domain is a functional domain.
  • Preferred functional domains are fluorescent protein domains, catalytic domains or protein domains with enzyme activity.
  • the ionic surfactant used is a solution with anionic or cationic surfactants, preferably selected from the group of hydrocarbon-coupled sulfates, sulfonates or carboxylates or from the group of quaternary ammonium compounds. Particular preference is given to alkyl sulfates or tetraalkylammonium salts, very particular preference to using sodium dodecylsulfate (SDS) and cetyltrimethylammonium bromide (CTAB).
  • anionic or cationic surfactants preferably selected from the group of hydrocarbon-coupled sulfates, sulfonates or carboxylates or from the group of quaternary ammonium compounds. Particular preference is given to alkyl sulfates or tetraalkylammonium salts, very particular preference to using sodium dodecylsulfate (SDS) and cetyltrimethylammonium bromide (CTAB).
  • SDS sodium dodecyl
  • surfactants have a single hydrophilic moiety (also referred to herein as a "surfactant moiety”) and a single hydrophobic moiety
  • the surfactant is used in a concentration of at most 30 mmol / l, more preferably at most 15 mmol / l.
  • the surfactant-containing solution is added portionwise to the protein-containing solution. At least two, more preferably at least four, individual portions of the surfactant-containing solution are preferably added.
  • the addition in portions has the advantage that the critical micelle formation concentration of the free surfactants in the solution formed is not exceeded, so that the micelle formation of the surfactants is largely prevented and these are available for the coating of protein monomers and oligomers.
  • the invention offers the advantage of providing monomer or oligomeric compounds of self-assembling proteins in which the uncontrolled aggregation of the proteins is prevented.
  • these coating solutions can be used for position-independent coating of substrate surfaces without their pretreatment.
  • the proteinaceous coating on the substrate may contain one or more different proteins capable of self-assembly within the protein layer.
  • a multiplicity of molecules of a defined self-assembling protein preferably an S-layer protein or hydrophobin or fusion proteins generated therefrom, are used.
  • a mixture of various self-assembling proteins is used, for example, a mixture of a fusion protein containing an S-layer protein with the corresponding S-layer protein.
  • the surfactants In the incubation of the unstabilized protein-containing solution with the surfactant-containing solution added in several portions (in at least two, preferably at least three individual portions), the surfactants enter into a coordinative bond with the proteins contained in the aqueous solution, so that a protein-surfactant complex is formed.
  • the formation of the protein-surfactant complex prevents the uncontrolled formation of protein aggregates and stabilizes the protein-containing solution.
  • stabilization is understood as meaning the prevention of uncontrolled assembly of self-assembled proteins in aqueous solutions.
  • metal ions or anions preferably in the form of an aqueous solution.
  • the formation of protein-ion complexes masks protein charges and promotes the formation of predominant protein oligomers.
  • PSE Periodic Table of the Elements
  • water-soluble salts preferably in the form of chlorides, Nitrates, carbonates, acetates, citrates, gluconates, hydroxides, lactates, sulfates, succinates or tartrates.
  • inorganic anions in particular halides, hydroxides, nitrates, carbonates, sulfates, phosphates, but also organic anions, in particular acetates, citrates, succinates or tartrates, which have alkali metal ions as counterions.
  • the concentration of the metal ions or anions in the aqueous solution is preferably 0.01 mmol / l to 10 mmol / l, preferably 0.1 mmol / l to 5 mmol / l and particularly preferably 0, 5 mmol / l to 2 mmol / l.
  • the pH can be shifted in the direction of the isoelectric point of the proteins used.
  • the invention also provides an aqueous, protein-containing coating solution which contains monomers or oligomers of self-assembling proteins, wherein the surface-active part of the monomers or oligomers of self-assembling proteins is coated with ionic surfactants.
  • the invention also includes a substrate with a coating of at least one monolayer of self-assembling protein layer,
  • a layer containing at least one ionic surfactant is present on the surface of the at least one monolayer of self-assembling proteins, the surfactant being in coordinative association with the self-assembling protein.
  • At least one monolayer is assembled directly on the substrate surface.
  • a applied by pretreatment mediator layer, such. B a polylysine or secondary cell wall polymer layer (glycoprotein layer) is not required.
  • the self-assembling proteins are recombinant fusion proteins comprising at least two domains, wherein at least one domain is a self-assembling protein domain and a domain is a functional domain or self-assembling protein domain, wherein the functional domain is located on the side of the protein-containing coating remote from the substrate ,
  • the substrate is preferably a metallic, silicon-containing, ceramic or polymeric solid.
  • the invention also encompasses the use of a protein-containing coating solution according to the invention for coating substrate surfaces with at least one monolayer of a self-assembling protein.
  • the coating process comprises the steps of:
  • an aqueous solution containing at least one ionic surfactant is added in portions to the protein-containing solution from a), wherein the addition is the final surfactant concentration c Te nsid in mol / 1 in the following range: Cprotei '5.237
  • Ao.protein the surface of a molecule of the self-assembling protein corresponds A T KG corresponding to the cross-sectional area of the head group of the surfactant and c Pro tein the concentration of self-assembling protein in mol / 1 corresponds
  • the coating solution as a stabilized aqueous solution after step a is prepared from an aqueous solution of self-assembling proteins by separating aggregated protein units such that monomers or oligomers of self-assembling proteins preferably remain in the solution.
  • an aqueous solution comprising at least one ionic surfactant is added in portions to the protein-containing solution after separation of the protein aggregates, after which the final surfactant concentration c Te nsid in mol / 1 lies in the following range:
  • a 0 corresponds to the surface protein of a molecule of the self-assembling protein A T KG the cross sectional area of the head group of the surfactant and c corresponds to the concentration of the pro tein self-assembling protein in mol / 1 corresponds.
  • the surfactant portion of each active protein monomer is enveloped by surfactant particles.
  • an ordered monolayer of one or more self-assembling proteins on the surface of a substrate in one process step.
  • a Protein layer with homogeneous layer thickness understood relative standard deviation of the layer thickness preferably not more than 30%.
  • the mean thickness of the layer corresponds to the extent of a molecule of the self-assembling protein.
  • the coating on the substrate may contain one or more different proteins capable of self-assembly within the protein monolayer.
  • a multiplicity of molecules of a defined self-assembling protein preferably an S-layer protein or hydrophobin or fusion proteins generated therefrom, are inserted.
  • a mixture of various self-assembling proteins is used, for example a mixture of a fusion protein containing an S-layer protein with the corresponding S-layer protein.
  • a stabilized aqueous solution containing the at least one self-assembling protein as protein monomer is provided, the stabilization taking place by addition of at least one surfactant.
  • the disulfide bridges of the protein structure are not dissolved.
  • the surfactant serves to coat protein monomers with a surfactant layer. This is done in the absence (at most 0.01% by weight, based on the self-assembling protein, preferably at most 0.0001% by weight) of substances which are suitable for cleaving disulfide bridges (in particular reducing thiols, such as ⁇ -mercaptoethanol) , Dithiothreitol or dithioerythritol).
  • the entire coating process according to the invention is carried out in the absence of these substances.
  • the surfactants In the incubation of the unstabilized protein-containing solution with the surfactant-containing solution added in several portions (in at least two, preferably at least three individual portions), the surfactants enter into a coordinative bond with the proteins contained in the aqueous solution, so that a protein-surfactant complex is formed.
  • the formation of the protein-surfactant complex prevents the uncontrolled formation of multimers of proteins (aggregates) and stabilizes the protein-containing solution.
  • stabilization is to be understood as meaning the invention of an uncontrolled assembly of self-assembled proteins in aqueous solutions. It is possible with the method according to the invention to produce stabilized solutions with protein monomers.
  • the stabilized aqueous solutions obtainable by a process according to the invention are storage-stable and multiply to Coating of substrates usable.
  • a stabilized aqueous protein solution obtainable by a process according to the invention is likewise provided by the invention.
  • the concentration of the at least one self-assembling protein in the unstabilized aqueous solution after separation of the protein aggregates is preferably at least 5 ng / ⁇ , more preferably at least 50 ng / ⁇ , most preferably at least 90 ng / ⁇ .
  • the unstabilized aqueous solution containing the at least one self-assembling protein preferably has a pH of from 7 to 9.5. This is preferably a buffered solution.
  • the respectively added aqueous solution is filtered prior to carrying out the method according to the invention, preferably by a sterile filter, particularly preferably with a pore size of 0.01 ⁇ m - 0.5 ⁇ m.
  • ionic surfactants cationic or anionic surfactants
  • surfactants have a single hydrophilic moiety (also referred to herein as a "surfactant moiety") and at least one hydrophobic moiety
  • the surfactant is used in a concentration of at most 30 mmol / L, more preferably at most 15 mmol / L (Process b).
  • a surfactant solution (in process step b) is used, which is preferably at least 1 mmol / l, more preferably at most 15 mmol / l, more preferably at most 10 mmol / l, of the surfactant.
  • the surfactant-containing solution is added in step b) in portions to the protein-containing solution. At least two, more preferably at least four, individual portions of the surfactant-containing solution are preferably added.
  • the addition in portions has the advantage that the critical micelle formation concentration of the free surfactants in the solution formed is not exceeded, so that the micelle formation of the surfactants is largely prevented and these are available for the coating of the protein monomers.
  • Equation (1) takes into account that the amount of surfactant used to coat the protein monomers depends on the protein surface available and the hydrophilic or hydrophobic properties of the protein.
  • the radii ratio p K of the ideal sphere protein molecule with the radius of gyration R G to the hydrodynamic radius of the protein molecule R H is 0.774.
  • a threshold range takes into account that at least as much surfactant must be added as is necessary from the transition of an ideal sphere protein to completely unfolded protein, if only hydrophobic interactions of the surfactant with the protein predominate (left limit of formula 1).
  • the radii ratio p up from the radius of gyration of the unfolded protein molecule R G to the hydrodynamic radius of the protein molecule R H is 1.5.
  • the other limit area takes into account that maximally as much surfactant is added as is necessary to break up "random coil” protein aggregates until the monomeric form of the protein is present as an ideal sphere, if only hydrophilic interactions of the surfactant with the protein prevail (right In this case, the radii ratio p rc from the radius of gyration of the random coil present aggregated protein molecule R G to the hydrodynamic radius of the protein molecule R H is 2.05.
  • the surfactant concentration in the stabilized material set in the coating process according to the invention for coating with a monolayer of self-assembling proteins protein solution as a coating solution (after addition of the surfactant-containing solution to the protein-containing solution) in mol / l is in the range given in equation (3).
  • the protein- and surfactant-containing solution formed according to an advantageous embodiment of the method for coating with a monolayer of self-assembling proteins are preferably added positively charged metal ions or anions, preferably in the form of an aqueous solution.
  • metal ions or anions preferably in the form of an aqueous solution.
  • divalent metal ions especially alkaline earth metal ions, which are used in the form of water-soluble salts, preferably in the form of chlorides, nitrates, carbonates, acetates, citrates, gluconates, hydroxides, lactates, sulfates, succinates or tartrates.
  • inorganic anions in particular halides, hydroxides, nitrates, carbonates, sulfates, phosphates, but also organic anions, in particular acetates, citrates, succinates or tartrates, which have alkali metals as counterions.
  • the concentration of the metal ions or anions in the aqueous solution is preferably 0.01 mmol / l to 10 mmol / l, preferably 0, 1 mmol / l to 5 mmol / l and particularly preferably 0.5 mmol / l to 2 mmol / l ,
  • the contacting of the stabilized protein-containing solution with the surface of the substrate in the coating method according to the invention is preferably carried out for at least 15 minutes, preferably for a maximum of 48 hours.
  • This process step is preferably carried out at a temperature of 15 to 70 ° C, more preferably above the Krafft point (Krafft temperature) of the surfactant used.
  • the self-assembling protein molecules deposit in the form of a monolayer on the surface of the substrate.
  • the supernatant proteinaceous solution is removed from the substrate by preferably either stripping the solution or drying the coated substrate (removal of the solvent).
  • the coated substrate surface is freed from excess and non-localized molecules by repeated rinsing with deionized water.
  • the drying of the coated substrate is preferably carried out by treatment with compressed air.
  • substrates are metallic substrates, silicon-containing solids (in particular glass or silicon wafers), ceramic or polymeric solids (in particular polytetrafluoroethylene).
  • the coating method according to the invention produces a substrate which has a layer above the monolayer protein-containing coating which contains the surfactant (s).
  • the surfactant-containing layer present on the surface of the protein-containing coating protects the surface of the protein-containing coating and the self-assembling protein from thermal and chemical stress (and therefore acts as a protective layer).
  • the surfactant-containing layer is preferably bound by coordinate binding to the protein layer.
  • the surfactant-containing protective layer arranged on the surface of the proteinaceous coating on the substrate is preferably removed, with preference being given subsequently to method step iii):
  • the coated substrate is washed several times under mechanical action and / or
  • the surfactant layer is removed by buffer treatment in a precipitation reaction.
  • the surfactant or surfactants used can either be recovered from the aqueous solution withdrawn from the coated substrate in step iii) by known methods (for example by precipitation) and can thus be reused after a purification step.
  • the invention also encompasses a substrate having a protein-containing coating with a monolayer of a self-assembling protein.
  • the substrate according to the invention has
  • a substrate according to the invention is obtainable by a coating method according to the invention.
  • the monolayer of the at least one self-assembling protein is thus initially arranged on the substrate surface, and there is a further, surfactant-containing layer thereon.
  • the method is carried out with the following steps: i. Providing a protein-containing coating solution
  • a solution containing at least one ionic surfactant is added in portions to the protein-containing solution, after addition the surfactant concentration c Te nsid in mol / l in the following range:
  • a 0, Prot where AO.FI corresponds to the surface area of the liquid sintering phase, A 0 , p ra tein the cross-sectional area of the protein (circular area), V F i corresponds to the total volume of liquid used, and N A is the Avogadro constant.
  • a 0, p ra teinoiigomere corresponds to corresponds to the cross sectional area of the Tensidkopf administrat (circular surface) of the surface lessnessdominanter AD.TKG protein oligomers, and Cp ro tein of the protein concentration employed.
  • the one limit range takes into account that maximally as much surfactant is added as is necessary for the stabilization of predominant protein oligomers.
  • a calculable surfactant concentration (right limit of formula 5)
  • protein assemblies (spherical or undefined structure) are decomposed by direct interaction with surfactant particles with cleavage of spherical protein particles.
  • the relationship given in equation (5) takes into account that the amount of surfactant used to coat the hydrophobin multimers depends on the multimeric surface area.
  • the radii ratio p K of the ideal sphere of the protein particle with the radius of gyration R G to the hydrodynamic radius of the protein molecule R H is 0.774.
  • the radii ratio p rc from the radius of gyration of the aggregated hydrophobin multimer R G present in undefined form to the hydrodynamic radius of the protein molecule R H is 2.05.
  • AOJKG is determined by scattering experiments with electromagnetic waves or is taken from the literature.
  • the formed protein bilayer on the substrate is covered by a layer of surfactant and a further attachment of self-assembling proteins to the interphase is prevented.
  • each protein particle has a defined space on the substrate.
  • contact sites for other protein particles on the protein image coated substrate must be blocked by surfactant particles.
  • a minimum concentration of surfactant left border region of formula 5
  • a partial surfactant layer is formed on the fully formed protein image and a direct interaction with other protein assemblies prevented.
  • the area Ao, p ro tein corresponds to the space required by a Proteinteilchens on the substrate, which must be blocked by at least one surfactant particles to prevent further uncontrolled growth of the Proteinbilage.
  • Ao, p r otein can be calculated according to Equation 6.
  • R is H, p ra tein ⁇ hydrodynamic radius of the protein, which can be determined by scattering experiments with electromagnetic waves or can be found in the literature.
  • Equation (5) takes into account that the additive surfactant used to promote significant layer growth is taken from the total interphase available depends on the use of coming liquid volume.
  • a correction term x is inserted in equation (5), which can preferably assume the values 1 and 2.
  • the contacting of the stabilized protein-containing coating solution with the surface of the substrate in the coating method according to the invention is preferably carried out for at least 15 minutes, preferably for a maximum of 48 hours.
  • This process step is preferably carried out at a temperature of 15 to 70 ° C, more preferably above the Krafft point (Krafft temperature) of the surfactant used.
  • the protein oligomers are deposited in the form of a protein bilayer on the surface of the substrate.
  • the supernatant proteinaceous solution is removed from the substrate by preferably stripping off the solution.
  • the coated substrate surface is freed from excess, non-bound protein molecules by repeated rinsing with deionized water.
  • the drying of the coated substrate can take place; preferably by treatment with compressed air.
  • the process is advantageously carried out with the following steps: i.
  • Providing a stabilized aqueous solution containing hydrophobins of at least one hydrophobin type by a) providing the hydrophobins in an aqueous solution, and b) depending on the charge of the hydrophobins, a saline and / or alkaline or acid solution, for masking the hydrophobin charges or Adjusting a net neutral charge of the hydrophobins in portions and over a period of 1 to 60 minutes to form predominant hydrophobin multimers, ii.
  • a 0, Prot a where AO.FI corresponds to the total area of the liquid interphase, A 0 , p ra tein the cross-sectional area of the protein (circular area), V F i corresponds to the total volume of liquid used and N A is the Avogadro constant.
  • Ao, p ra teinmuitimer corresponds to the surface venezdominanter Hydrophobinmultimere, AD.TKG corresponds to the cross sectional area of the Tensidkopf distr (circular surface) and c per ton of Hydrophobinkonzentration used.
  • iii Removing the supernatant solution from the coated substrate and / or drying the coated substrate.
  • Predominant multimers refer to oligomers with a maximum of 10 protein units.
  • an hydrophobic layer having a homogeneous hydrophobin layer is thereby referred to as an ordered layer or (ordered) bilage Layer thickness understood (relative standard deviation of the layer thickness preferably not more than 30%).
  • the average thickness of the layer corresponds to the extent of two molecules of the hydrophobin.
  • hydrophobin monomers In order to be able to ensure the construction of a homogeneous hydrophobic insert, two opposing protein properties must be balanced out.
  • the assembly of hydrophobin monomers is based on the interaction of two different physical quantities.
  • the assembly of the same hydrophobin monomers strongly depends on the accessibility of two separated hydrophobin areas, which differ in their polarizability (hydrophilicity or hydrophobicity).
  • the interaction of two identically polarizable regions of hydrophobin particles leads to an attraction between the hydrophobin particles, which leads to repulsion of oppositely polarizable regions.
  • the net electrical charge of the hydrophobins must be taken into account.
  • the hydrophobins used To prevent repulsion of like-loaded hydrophobin particles, the hydrophobins used must have a net neutral charge.
  • the method according to the invention makes it possible to specifically intervene in these interactions and to enable a substrate coating with a defined hydrophobin bilayer.
  • a stabilized aqueous solution containing at least one hydrophobinic species is initially provided, the stabilization being effected by addition of at least one salt, one alkali or acid.
  • the addition of salts, lyes or acids promotes the formation of predominant hydrophobin multimers with the compensation of protein charges.
  • hydrophobin within hydrophobin bilayers.
  • a multitude of molecules of a defined hydrophobin or fusion proteins generated therefrom are used.
  • a mixture of different self-assembling hydrophobins is used, for example a mixture of a fusion protein containing at least one hydrophobin.
  • These molecules of the hydrophobins are first stabilized in aqueous solution, then an ordered formation of predominant assembly structures is induced and finally used to coat the substrate.
  • the counterions go with those contained in the aqueous solution Hydrophobins coordinate so as to form a hydrophobin counterion complex.
  • the formation of the hydrophobin-counterion complex initiates the controlled formation of multimers of the hydrophobins (assemblies) and stabilizes the protein-containing solution.
  • stabilization is thus understood as the prevention of an uncontrolled aggregation of hydrophobins in aqueous solutions. It is possible with the method according to the invention to produce stabilized solutions with hydrophobin multimers (preferably dimers or tetramers).
  • the stabilized aqueous solutions obtainable by a process of the invention are useful for coating substrates.
  • the concentration of the at least one hydrophobin type in the unstabilized aqueous solution is preferably at least 50 ng / ⁇ , particularly preferably at least 90 ng / ⁇ .
  • the unstabilized aqueous solution containing the at least one self-assembling hydrophobin type preferably has a pH of from 7 to 9.5. This is preferably a buffered solution.
  • hydrophobins In order to establish a neutral net charge of the hydrophobins, preferably positively charged metal ions or anions, preferably in the form of an aqueous solution, are added in the coating process according to the invention to provide the stabilized protein solution.
  • the formation of hydrophobin-ion complexes mask hydrophobin charges and promote the formation of predominant hydrophobin multimers.
  • Particularly preferred are solutions with divalent metal ions having a high ionic strength, in particular alkaline earth metal ions which are used in the form of water-soluble salts, preferably in the form of chlorides, nitrates, carbonates, acetates, citrates, gluconates, hydroxides, lactates, sulfates, succinates or tartrates.
  • inorganic anions in particular halides, hydroxides, nitrates, carbonates, sulfates, phosphates, but also organic anions, in particular acetates, citrates, succinates or tartrates, which have alkali metals as counterions.
  • the concentration of the metal ions or anions in the aqueous solution is preferably 0.01 mmol / l to 10 mmol / l, preferably 0.1 mmol / l to 5 mmol / l and particularly preferably 0.5 mmol / l to 2 mmol / l ,
  • the pH can be shifted in the direction of the isoelectric point of the hydrophobins used.
  • solutions of ionic surfactants preferably cationic and / or anionic surfactants, are added to the protein- and saline-containing solution.
  • surfactants have a hydrophilic part of the molecule (also referred to herein as a "surfactant head group") and a hydrophobic part of the moiety - containing solution containing a surfactant concentration, which preferably at least 1 ⁇ / ⁇ and at most 500 ⁇ / ⁇ , more preferably at most 100 ⁇ / ⁇ corresponds.
  • the coating method according to the invention only a maximum of surfactant is added, as necessary, to envelop the interphase of the solution containing protein molecules with a bilayer.
  • enough surfactant is added to the solution from b) that the final concentration of the surfactant in mol / l after addition to the protein and saline solution is in the following range:
  • the one boundary area takes account of the fact that maximally as much surfactant is added as is necessary for the departure of predomi- nant hydrophobin mu ltimers.
  • hydrophobinassemblates (spherical or undefined structure) are decomposed by direct interaction with surfactant particles with elimination of spherical hydrophobin particles.
  • the relationship given in equation (5) takes account of the fact that the amount of surfactant used to charge the hydrophobin polymers depends on the available multimer surface.
  • the radii ratio p K of the hydrophobin particle having the ideal radius and the radius of gyration R G to the hydrodynamic radius of the protein molecule R H is 0.774.
  • the radii ratio p rc from the radius of gyration of the aggregated hydrophobin multimer R G present in undefined form relative to the hydrodynamic radius of the protein molecule R H is 2.05.
  • AOJKG is determined by scattering experiments with electromagnetic waves or is taken from the literature.
  • the formed protein bilayer on the substrate is covered by a layer of surfactant and prevents any further attachment of hydrophobins to the interphase.
  • each hydrophobin particle has a defined space on the substrate.
  • contact sites for further hydrophobin particles on the hydrophobin bilayer coated substrate must be blocked by surfactant particles.
  • a minimum concentration of surfactant left boundary region of formula 5
  • a partial surfactant layer is formed on the fully formed hydrophobin bilayers and a direct interaction with other hydrophobinassemblates is prevented.
  • the area Ao, p ro tein corresponds to the space required by a Hydrophobinteilchens on the substrate, which must be blocked by at least one surfactant particles to prevent further uncontrolled growth of the Hydrophobinbilage.
  • Ao, p ro ton can be calculated using equation. 6
  • R is H, p ra tein ⁇ hydrodynamic radius of the hydrophobin, which can be determined by scattering experiments with electromagnetic waves or can be found in the literature.
  • Equation (5) takes account of the fact that the surfactant concentration to be added, which is used to prevent further layer growth, depends on the total, available interphase of the liquid volume used.
  • a correction term x is inserted in equation (5), which can preferably assume the values 1 and 2.
  • the contacting of the stabilized hydrophobin-containing solution with the surface of the substrate in the coating method according to the invention is preferably carried out for at least 15 minutes, preferably for a maximum of 48 hours.
  • This process step is preferred at a temperature of 15 to 70 ° C, more preferably above the Krafft point (Krafft-Te mpe rat ur) d introduced n surfactant carried out.
  • the hydrophobinassemblates are deposited in the form of a protein bilayer on the surface of the substrate.
  • the supernatant proteinaceous solution is removed from the substrate by preferably stripping off the solution.
  • the coated substrate surface is freed from excess, non-bound protein molecules by repeated rinsing with deionized water.
  • the drying of the coated substrate can take place; preferably by treatment with compressed air.
  • Substrates of various structures are suitable for the coating method according to the invention.
  • the coating method of the invention is also suitable for porous substrates and allows a homogeneous ordered layer structure even within the difficult to access pore system.
  • Preferred substrates are metallic substrates, silicon-containing solids (in particular glass or silicon wafers), ceramic or polymeric solids (in particular polytetrafluoroethylene).
  • the coating process according to the invention produces a substrate which, above the bilayered protein-containing coating, has a layer which contains the surfactant (s).
  • the surfactant-containing layer present on the surface of the protein-containing coating protects the surface of the protein-containing coating and the hydrophobin from thermal and chemical stress (and therefore acts as a protective layer).
  • the surfactant-containing layer is preferably bound by coordinate binding to the protein layer.
  • the surfactant-containing protective layer disposed on the surface of the hydrophobin-containing coating on the substrate is preferably removed, the preferred substrate subsequently being after step ii): the coated substrate being washed several times under mechanical action and / or
  • the surfactant layer is removed by buffer treatment in a precipitation reaction.
  • the surfactant (s) used can either be obtained from the aqueous solution withdrawn from the coated substrate in step iii) (for example, by precipitation) and can be reused after a cleaning step.
  • hydrophobins used are class I hydrophobins, preferably Ccg2 from Neurospora crassa, or class II hydrophobins, preferably HFBI or HFBII from Trichoderma reesei.
  • the invention is particularly advantageously suitable for functionalized hydrophobins, since the accessibility of the functionalization can be ensured by the ordered layer structure of bilayers of the functionalized hydrophobin. It is advantageously possible to carry out the coating of the substrate so that the functionalization is present on the side of the protein-containing coating remote from the substrate.
  • the hydrophobin used is a recombinant fusion protein comprising at least two domains, one domain being a self-assembling protein domain and one domain being a functional domain or self-assembling protein domain.
  • the hydrophobin is therefore preferably a recombinant fusion protein, ie a protein which is produced by genetically modified organisms.
  • a recombinant fusion protein ie a protein which is produced by genetically modified organisms.
  • the recombinant fusion protein can be obtained by expression of the fused gene.
  • the artificially generated reading frame of the DNA construct is flanked at the 5 ' position by a host-specific promoter and at the 3 ' position by a terminator.
  • a recombinant fusion protein employed in the invention comprises at least two domains, one domain being a self-assembling protein domain (preferably selected from the above hydrophobins) and one domain being a functional domain.
  • Preferred functional domains are fluorescent protein domains, catalytic domains or protein domains with enzyme activity.
  • other hydrophobins may also act as a fusion domain.
  • the choice of suitable fusion partner parts is not limited to proteins of certain organisms.
  • the amino acid sequence in the N- and / or C-terminal region may also have an affinity domain comprising 1-20 amino acids, which may interact as a specific anchor group with complementary groups on solid supports.
  • the aqueous solution containing at least one hydrophobin type, to which the salt-containing, basic and / or acidic and surfactant-containing solutions are added in one variant contains mixtures of different hydrophobin types, preferably at least one functionalized hydrophobin type in combination with at least one non-functionalized hydrophobin type. This improves the stability of the proteinaceous coating. This embodiment is particularly advantageous for functionalized hydrophobins with large functional domains to allow interaction between assembling domains of different protein molecules.
  • the functional domain of the recombinant fusion protein is preferably arranged on the side of the protein-containing coating remote from the substrate.
  • the hydrophobin domain of the recombinant fusion protein is arranged on the substrate-facing side of the proteinaceous coating.
  • the coating containing at least one surfactant is thus arranged in this case above the functional domain of the recombinant fusion protein.
  • the surfactant used is an anionic or cationic surfactant, preferably selected from sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB).
  • SDS sodium dodecyl sulfate
  • CTAB cetyltrimethylammonium bromide
  • the invention also encompasses a method for stabilizing monomers of at least one self-assembling protein in an aqueous solution (also referred to herein as "stabilization method")
  • a) at least one self-assembling protein is admixed with an aqueous solution, preferably an aqueous buffer solution , and subsequently
  • a 0 protein corresponds to the surface of a molecule of the self-assembling protein
  • a T KG corresponds to the cross-sectional area of the head group of the surfactant
  • Cp ro tein corresponds to the concentration of the self-assembling protein in mol / l.
  • a stabilized aqueous proteinaceous solution which is preferably obtainable by a process according to the invention and which contains at least one self-assembling protein and at least one surfactant, is likewise provided by the invention (the terms “stabilized aqueous solution” and “stabilized monomers in aqueous solution” are used synonymously herein ). Preference is given to stabilized protein monomer solutions.
  • the stabilized aqueous solution according to the invention contains at least one self-assembling protein, at least one ionic surfactant and contains no substances which are suitable for cleaving disulfide bridges (maximum 0.01% by weight based on the self-assembling protein, preferably not more than 0.0001% by weight). ).
  • monomers of the self-assembling protein are in coordinative binding with the ionic surfactant (protein-surfactant complex).
  • the surfactant concentration c Te nsid in mol / l in the aqueous solution is in the following range:
  • A corresponds to 0, p ra tein the surface of a molecule of the self-assembling protein A T KG the cross sectional area of the head group of the surfactant and Cp ro tein the concentration of self-assembling protein in mol / l.
  • the invention also includes a substrate having a coating of a hydrophobin bilayer, on the substrate surface is a bilayer of at least one Hydrophobinart, wherein on the surface of the hydrophobin bilayer is a layer containing at least one ionic surfactant, wherein the surfactant with the hydrophobin of the surface of Bilage is present in coordinate binding.
  • the hydrophobins of the at least one hydrophobin species are preferably recombinant fusion proteins which comprise at least two domains, one domain being a self-assembling protein domain and one domain being a functional domain or self-assembling protein domain, the functional domain being on the hydrophobin-containing side remote from the substrate Coating is arranged.
  • the substrate is preferably a metallic, silicon-containing, ceramic or polymeric solid.
  • the invention also encompasses the use of a stabilized protein-containing solution according to the invention for coating substrate surfaces with a monolayer of a self-assembling protein. Subsequent embodiments are preferred for the invention:
  • the self-assembling protein is preferably selected from hydrophobins and surface layer proteins (S-layer proteins).
  • Preferred hydrophobins are class I hydrophobins, preferably Ccg2 from Neurospora crassa, or class II hydrophobins, preferably HFBI or HFBII from Trichoderma reesei.
  • Preferred S-layer proteins are SBSC from Bacillus stearothermophilus, S13240 from Bacillus stearothermophilus or SslA from Sporosarcina ureae.
  • the invention is suitable for functionalized self-assembling proteins, since the accessibility of the functionalization can be ensured by the ordered layer structure of monolayers of the functionalized self-assembling protein. It is advantageously possible to carry out the coating of the substrate so that the functionalization is present on the side of the protein-containing coating remote from the substrate.
  • the self-assembling protein is therefore preferably a recombinant fusion protein, ie a protein which is produced by genetically modified organisms.
  • a recombinant fusion protein ie a protein which is produced by genetically modified organisms.
  • the recombinant fusion protein can be obtained by expression of the fused gene.
  • a recombinant fusion protein employed in the invention comprises at least two domains, one domain being a self-assembling protein domain (preferably selected from the above-mentioned self-assembling proteins) and one domain being a functional domain.
  • Preferred functional domains are fluorescent protein domains, catalytic domains or protein domains with enzyme activity.
  • the at least one self-assembling protein-containing aqueous solution, to which the surfactant-containing solution is added in one embodiment contains mixtures of blast-free bleaching proteins, preferably at least one structurally self-assembling protein in combination with at least one non-functionalized self-assembling protein. This improves the stability of the proteinaceous coating.
  • This embodiment variant is particularly advantageous for functionalized self-assembling proteins with large functional domains in order to enable the interaction between assembling domains of different protein molecules.
  • the functional domain of the recombinant fusion protein preferably arranged on the side facing away from the substrate side of the proteinaceous coating.
  • the self-assembling protein domain of the recombinant fusion protein was placed on the substrate in order to obtain the protein-containing coating.
  • the coating containing at least one surfactant is thus in this case located above the functional domain of the recombinant fusion protein.
  • the surfactant is an anionic or cationic surfactant, preferably selected from sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB).
  • SDS sodium dodecyl sulfate
  • CTAB cetyltrimethylammonium bromide
  • the invention offers the advantage of providing stabilized monomer solutions of self-assembled proteins in which the uncontrolled aggregation of the proteins is prevented. Through targeted monomerization and stabilization of the solution, these can be used for position-independent coating of substrate surfaces.
  • FIG. 1 SEM image of a silicon wafer coated with a monolayer of the S-layer protein SbsC- (R5P) 2 from Bacillus stearothermophilus with a layered SDS layer.
  • FIG. The homogeneous areas on the left and right show the protein monolayer from which the SDS layer (seen in the center) was removed.
  • FIG. 2 AFM image of a silicon wafer coated with a monolayer of Class 1 hydrophobin Ccg-2 from Aspergillus nidulans after removal of the SDS layer with exposed protein monolayer.
  • Fig. 3 height profile of a coated with a monolayer of Class 1 hydrophobin Ccg-2 from Aspergillus nidulans silicon wafer, determined by AFM.
  • Embodiment 1 Expression of Recombinant Compounding Fusion Proteins in E. coli and Purification
  • HFBI Trichoderma reesei
  • the DNA sequences coding for the respective domain were amplified by means of polymerase chain reaction (PCR) and a fusion gene was assembled by overlap PCR.
  • the flanking primers used contained recognition sequences for the restriction endonucleases Nde ⁇ and Xho ⁇ . After vector and fragment restriction via the corresponding endonucleases, ligation and transformation into Escherichia coli (E. coli) as the host organism, the presence of the fusion gene in the generated plasmids was checked.
  • the self-assembling protein fusion protein containing the plasmids containing the generated fusion correctly, were transformed into £ coli strain shuffle ® T7 Express.
  • the fusion protein is accumulated intracellularly in the bacterial cells.
  • the transformed E. coli cells were cultured at 30 ° C.
  • the expression of the fusion gene was induced by the addition of 0.4 mM (mmol / l) I PTG.
  • the cells were pelleted by centrifugation (at 4000 xg, 4 ° C, 10 min).
  • 50 mM Tris buffer (pH 7.5) After washing the cell pellet twice with 50 mM Tris buffer (pH 7.5), the cells were again pelleted by centrifugation.
  • the £. coli cells were subjected to a triple ultrasound treatment (9-eye, 70%, 4 ° C, 2 min) followed by a three-time French press treatment (20 ⁇ 00 psi).
  • the digested bacterial cells were washed twice with 50 mM Tris buffer (pH 7.5).
  • the purification of the fusion protein was carried out by means of nickel affinity chromatography.
  • the elution of the fusion protein was carried out in an isotype phosphate buffer (pH 4.5) with 250 mM imidazole.
  • the resulting eluate was dialyzed against 4000 times the volume of a dialysis buffer at 4 ° C for a maximum of 48 hours.
  • a Tris buffer 50 mM, pH 8.5 with 1 mM glutathione reduced and 0.2 mM glutathione oxidized
  • 10 mM Tris buffer pH 9.0 was used as the dialysis buffer.
  • fusion protein HFBI- (XSR5P) 3 was used, which contains as self-assembling domain a class I hydrophobin (HFBI from Trichoderma reesei) and contains as a functional domain a human influenza hemaglutinin tag.
  • centrifugation was carried out for sedimentation of larger protein aggregates (15 min. At 18,000 x g, 4 ° C.) and the protein concentration was determined (according to Lowry). The sediment was taken up in 50 mM Tris buffer pH 8.5. A protein concentration of 100 ng / ⁇ was set.
  • a surfactant concentration to be set for a 100 ng / ⁇ protein solution is between 0.5 mmol / l and 5.6 mmol / l.
  • the self-assembling fusion protein HFBI- (XSR5P) 3 has a molecular weight of about 14000 g / mol.
  • the concentration at a protein concentration of 100 ng / ⁇ in the solution is in mol / l, corresponding to 7.14 * 10 "6 mol / l.
  • the Tensidkopfen of the employed SDS molecule has an effective area of A T KG of 0.62 nm 2.
  • the stabilized solution thus obtained was clear and stable for at least five days when stored at 20 ° C.
  • the stabilized solution containing monomers of the self-assembling protein was used to coat a silicon wafer with the edge lengths 1 x 1 cm.
  • 100 ⁇ of the stabilized protein-containing solution were applied to the silicon wafer and incubated for at least 30 min.
  • the supernatant was then stripped off and the HFBI (XSR5P) 3 coated silicon wafer was washed in filtered bidistilled water.
  • the storage of the coated silicon wafer took place in the dialysis buffer mentioned in Example 1.
  • the stripped stabilized protein solution could then be used for further coating processes.
  • Embodiment 3 Characterization of the Coating Using HFBI- (XSR5P) 3 Produced in Embodiment 2 on a Silicon Wafer by Ellipsometry
  • the coated silicon wafer obtained in Example 2 was examined ellipsometrically (Multiskop from OPTREL (Berlin)).
  • the change in the polarization plane of a monochromatic laser beam (wavelength 632.8 nm) was determined by reflection at an angle of 136 °.
  • the intensity ratio (p) was determined.
  • the ellipsometric angles ⁇ and ⁇ are derived via FRENSEL's formulas.
  • ⁇ ⁇ and 5 S indicate the phase shift to the zero point of parallel and perpendicularly polarized light.
  • the optical constants known from the literature for silicon were used as substrate and silicon dioxide.
  • the refractive index of the protein film was 1, 375.
  • the determined layer thickness of HFBI- (XSR5P) 3 on the silicon wafer was 13.2 ⁇ 0.2 A. This corresponds to the literature values for a monolayer of the self-assembling protein.
  • Table 2 shows the ellipsometric data of an analysis of four different samples (1 to 4, the different values in the individual rows were recorded at different measuring points on the respective sample): Table 2:
  • EMBODIMENT 4 Scanning Electron Microscopy (SEM) of a Sbsc (R5P) 2 Coated Silicon Wafer
  • a silicon wafer analogously to Example 2 was coated with the self-assembling protein SbsC- (R5P) 2 (Table 1).
  • the obtained coated silicon wafer was examined by means of SEM (Zeiss DSM 982 Gemini).
  • SEM Zeiss DSM 982 Gemini
  • the coated silicon wafer was dried and then vapor-deposited with an atomic gold layer.
  • the results of the SEM analysis are shown in FIG. Bright areas show therein the protein layer with a layered SDS layer.
  • the dark areas show the protein as a monolayer from which the SDS layer has been removed.
  • a silicon wafer coated with Ccg2-HA analogously to Example 2 was investigated in an aqueous medium by means of AFM.
  • the results of the AFM analysis are shown in FIG. It can be seen a uniform protein monolayer.
  • the Ccg2 tube structures have a length of about 100 nm ( Figure 2, bright areas). From the height profile ( Figure 3), it can be seen that the self-assembled structures of the Ccg2 HA have a width of about 25 nm and a height of about 2.5 nm.
  • Example 2 With the self-assembling fusion protein HFBI- (R5P) 2 produced in Example 1, analogously to Example 2 , a silicon wafer with a monolayer of Proteins coated. The layer thickness, which was determined ellipsometrically analogously to Example 3, was 1 3.8 ⁇ 0.4 ⁇ . In the fusion protein is the functional domain, the R5P subunit of the silaffin fused with the hydrophilic part of the hydrophobin.
  • the contact angle in air was determined by the sessile drop method (Drop Shape Analysis System DSA10, Kruss GmbH, Germany). The contact angle in degrees of a drop of 2 ⁇ deionized water was determined.
  • Example 2 An ordered protein image of a hydrophobin, as obtained in Example 1, was generated on a silicon wafer.
  • the fusion protein HFBI- (R5P) 2 was used, which contains a class I hydrophobin (HFBI from Trichoderma reesei) as a self-assembling domain and contains a mineralization tag as the functional domain.
  • Centrifugation was first carried out for sedimentation of larger protein aggregates (15 min at 18,000 ⁇ g, 4 ° C.) and the protein concentration was determined (according to Lowry). The sediment was discarded. A protein concentration of 100 ng / ⁇ was set.
  • the stabilized solution containing multimers of hydrophobin was used to coat a silicon wafer with edge lengths of 1 x 1 cm.
  • 200 .mu. ⁇ of the stabilized protein-containing solution were applied to the silicon wafer and for at least Incubated for 30 min.
  • the supernatant was then stripped off and the HFBI (R5P) 2 coated silicon wafer was washed in filtered bidistilled water.
  • the storage of the coated silicon wafer took place in the dialysis buffer mentioned in Example 1.
  • the stripped stabilized protein solution could then be used for further coating processes.
  • Exemplary Embodiment 8 Characterization of the Coating Using HFBI- (R5P) 2 and HFBI- (XSR5P) 3 Produced in Embodiment 6 on a Silicon Wafer by Ellipsometry
  • the coated silicon wafer obtained in Example 2 was examined ellipsometrically (Multiskop from OPTREL (Berlin)).
  • the change in the polarization plane of a monochromatic laser beam (wavelength 632.8 nm) was determined by reflection at an angle of 136 °.
  • the intensity ratio (p) was determined.
  • the ellipsometric angles ⁇ and ⁇ are derived via FRENSEL's formulas.
  • the layer thickness of the uppermost layer was calculated.
  • ⁇ ⁇ and 5 S indicate the phase shift to the zero point of parallel and perpendicularly polarized light.
  • the optical constants known from the literature for silicon were used as substrate and silicon dioxide.
  • the refractive index of the protein film was 1, 375.
  • the determined layer thickness of HFBI- (R5P) 2 on the silicon wafer was 25.7 ⁇ 1.8 ⁇ , or 25.8 ⁇ 0.8 ⁇ for the construct HFBI- (XSR5P) 3 . These layer thicknesses correspond to the literature values for a bilayer of hydrophobin.
  • Table 3 shows the ellipsometric data of an analysis of seven different samples (1 to Z), the different values in the individual rows were recorded at different measuring points on the respective sample):
  • Table 3 Ellipsometric data for substrates coated with HFBI (R5P) 2
  • Example 2 With the self-assembling fusion protein HFBI- (R5P) 2 prepared in Example 1, a silicon wafer was coated with a bilage of the protein analogously to Example 7. The layer thickness, which was determined ellipsometrically analogously to Example 8, was 25.7 ⁇ 1.8 ⁇ . In the fusion protein, the funcional domain, the R5P subunit of the silaffin, is fused to the hydrophilic part of the hydrophobin.
  • the contact angle in air was determined by the sessile drop method (Drop Shape Analysis System DSA10, Kruss GmbH, Germany). The contact angle in degrees of a drop of 2 ⁇ deionized water was determined.
  • Exemplary Embodiment 10 Checking the Accessibility of the Fused HA Tag of a Ccg2-HA Coated Silicon Wafer.
  • Fluorescence microscopic images were taken with a Zeiss Axio Observer.ZI (CARL ZEISS MICROIMAGING GMBH, Jena, Germany) in combination with the associated filter set 38 GFP.
  • a green fluorescent substrate surface confirms the accessibility of the fused HA tag.
  • a substrate according to Example 2 was treated by analogy with BSA. After incubation with the antibody anti-HA ® AlexaFluor 488 conjugate no green fluorescent signals on the substrate surface could be detected ..
  • Exemplary embodiment 11 Checking the accessibility of a fused luciferase tag of a HFBI-GLuc coated plastic surface.
  • the bifunctional hydrophin chimera (HFB1-Gluc) and the subunit displayed a luminescent signal as expected.
  • the catalytically inactive HFB1 subunit shows no luminescence signal.
  • the proteins were deposited on a polystyrene surface as in Embodiment 2.
  • the substrates were overlaid with reaction buffer and the reaction was initiated by the addition of luciferol. Only the immobilized bifunctional hydrophobin chimera showed a significant luminescence signal.
  • Embodiment 12 Examination of the long-term stability of a protein-containing coating solution containing Ccg2-tRFP or HFBI-tRFP
  • the fluorescent fusion proteins consist of a hydrophobin (Ccg2 or HFBI), which represents the self-assembling domain and a red fluorescent peptide domain (tRFP).
  • Ccg2 or HFBI hydrophobin
  • tRFP red fluorescent peptide domain
  • the red-fluorescent peptide domain allows the influence of ionic surfactants on the structure of the fusion protein to be determined by fluorescence measurements. A change in the fluorescence intensity allows direct conclusions about conformational changes of the protein scaffold, which are caused by interactions with the ionic surfactant.
  • a proteinaceous solution containing a fusion protein (Ccg2-tRFP or H FB I-tRFP) or an unfused tRFP peptide sequence of concentration 200 ng / ml was optionally added in four portions with a solution of ionic surfactants including CTAB a final concentration of ionic surfactant of 1 mM added. All solutions were then filtered through a 0.22 ⁇ ". Filter and the effect of filtration and the influence of added ionic surfactants was subsequently examined over a period of one week.
  • the filtration of the protein-containing solutions containing only the fusion protein (Ccg2-tRFP or HFBI-tRFP) without the addition of ionic surfactant resulted in a 37% and 46% reduction in fluorescence intensity, respectively.
  • the proteinaceous solution containing only the unfused tRFP without the addition of ionic surfactant did not show a decrease in fluorescence intensity.
  • the fluorescence intensity was subsequently monitored every 24 hours for a period of one week. All samples showed a continuous decrease in fluorescence intensity. After one week, a fluorescence which was reduced by about 95% to the baseline was detectable in all samples.
  • the protein-containing solutions containing a fusion protein (Ccg2-tRFP or HFBI-tRFP) and an ionic surfactant showed no drop in fluorescence intensity over the entire experimental period.

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Abstract

L'invention concerne des procédés de revêtement d'un substrat par au moins une monocouche de protéines capables d'autoassemblage au moyen de solutions aqueuses stabilisées comprenant des protéines capables d'autoassemblage, ainsi que des substrats pouvant être obtenus par ces procédés. L'invention concerne également des procédés de stabilisation de solutions comprenant des protéines capables d'autoassemblage, ainsi que les solutions stabilisées pouvant être obtenues par ces procédés. Un procédé de revêtement selon l'invention consiste à former au moins une monocouche d'une protéine capable d'autoassemblage sur un substrat au moyen d'une solution aqueuse stabilisée contenant au moins une protéine capable d'autoassemblage. Pour préparer la solution de revêtement, des ensembles de protéines agrégées sont séparés d'une solution aqueuse de protéines capables d'autoassemblage et des monomères ou des oligomères des protéines capables d'autoassemblage sont produits et stabilisés par ajout d'une solution de tensioactifs ioniques et/ou d'une solution saline et/ou alcaline et/ou acide à la solution de revêtement protéique, le tensioactif ionique étant ajouté dans une quantité suffisante pour enrober uniquement la partie à surface active de chaque monomère de protéine actif ou oligomère de protéine prédominant par des particules de tensioactif.Une surface du substrat est ensuite mise au contact de la solution protéique stabilisée de manière à former un revêtement protéique sur le substrat.L'excédent de solution est éliminé du substrat revêtu et/ou le substrat revêtu est séché.
EP12753109.3A 2011-08-24 2012-08-24 Procédés de revêtement de substrats par au moins une monocouche de protéines capables d'autoassemblage Withdrawn EP2747901A1 (fr)

Applications Claiming Priority (3)

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DE102011081524.4A DE102011081524B4 (de) 2011-08-24 2011-08-24 Beschichtung von substraten mit einer monolage selbstassemblierender proteine
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