EP2747901A1

EP2747901A1 - Method for coating substrates with at least one monolayer of self-assembling proteins

Info

Publication number: EP2747901A1
Application number: EP12753109.3A
Authority: EP
Inventors: Kai Ostermann; Leopold GRUNER; Gerhard RÖDEL
Original assignee: Technische Universitaet Dresden
Current assignee: Technische Universitaet Dresden
Priority date: 2011-08-24
Filing date: 2012-08-24
Publication date: 2014-07-02
Also published as: US10000536B2; US20140255712A1; WO2013026919A1

Abstract

The invention relates to methods for coating a substrate with at least one monolayer of self-assembling proteins, using stabilized aqueous solutions with self-assembling proteins, and also to substrates obtainable as a result. Methods for stabilizing solutions with self-assembling proteins, and the stabilized solutions obtainable therefrom, are likewise provided by the invention. According to the coating method, at least one monolayer of a self-assembling protein is produced on a substrate by first providing a stabilized aqueous solution which comprises at least one self-assembling protein. To provide the coating solution, protein units aggregated from an aqueous solution of self-assembling proteins are separated off, and addition of a solution of ionic surfactants and/or of a salt-containing and/or alkaline and/or acidic solution to the protein-containing coating solution generates monomers or oligomers of the self-assembling proteins, and stabilizes them, the amount of ionic surfactant added being such that only the surface-active part of each active protein monomer or predominant protein oligomer is enveloped by surfactant particles. A substrate surface is then brought into contact with the stabilized, protein-containing solution, thus producing a protein-containing coating on the substrate. The supernatant solution is removed from the coated substrate and/or the coated substrate is dried.

Description

Process for coating substrates with at least one monolayer of self-assembling proteins

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. In the context of the invention, 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. However, the method does not permit controlled deposition of protein layers.

Methods are also known for stabilizing solutions of monomers of self-assembling proteins. Thus, a method is known to dissolve protein aggregates by adding trifluoroacetic acid (TFA) without damaging the proteins. With regard to environmental regulations and the highly corrosive properties of TFA, this method is only of limited suitability for industrial applications.

Furthermore, 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. The addition of 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. For the coating of a substrate surface with self-assembling protein monomers then 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.

According to the invention the object is achieved by a method for coating a substrate with at least one monolayer of self-assembling proteins with the following steps:

i. Providing a proteinaceous coating solution,

ii. Contacting the surface of the substrate with the proteinaceous solution, iii. Removing the supernatant solution from the coated substrate and / or drying the coated substrate. To provide the protein-containing coating solution, aggregated protein units are separated from an aqueous solution of self-assembling proteins in such a way that monomers or oligomers of self-assembling proteins preferably remain in the solution.

By subsequently adding a solution of ionic surfactants and / or a salt-containing and / or alkaline and / or acidic solution, monomers or oligomers of the self-assembling proteins are produced and stabilized in the protein-containing coating solution. In this process, enough ionic surfactant is added that only the surface-active part of each active protein monomer or protein oligomer is enveloped by surfactant particles.

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.

To form and stabilize monomers of the assembling proteins, ionic surfactants are added to the solution after separation of the protein aggregates in such a way that only the surface-active part of each active protein monomer is enveloped by surfactant particles. Such a coating solution makes it possible to deposit protein monolayers on substrates without pretreatment. A protein monolayer is a planar structure of surface-assembled protein units, preferably protein monomers, with the layer thickness of a protein monomer.

For the formation and stabilization of oligomers of the assembling proteins, 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. There will be so much ionic surfactants are added so that only the surface-active part of each active predominant protein oligomer is enveloped by surfactant particles.

The addition of salts, bases or acids promotes the formation of predominant protein oligomers with the compensation of protein charges. Upon incubation of the proteinaceous solution with the added solution, the counterions coordinate with the proteins contained in the aqueous solution to form a protein-counterion complex. The formation of the protein-counterion complex triggers and stabilizes the controlled formation of oligomers of the proteins (assemblies).

After separation of the protein aggregates, the concentration of the self-assembling proteins in the aqueous solution is at least 5 ng / μΙ, preferably at least 50, particularly preferably at least 90 ng / μΙ. The aqueous solution of the self-assembling proteins preferably has a pH of 7 to 9.5. This is preferably a buffered solution.

As 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.

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. By fusion of the genes of two or more protein domains that are not naturally present in this combination, which are usually linked together via flexible linker structures (linker peptides), expression of the fused gene, the recombinant fusion protein available. 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). These surfactants have a single hydrophilic moiety (also referred to herein as a "surfactant moiety") and a single hydrophobic moiety Preferably, 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. By targeted generation of monomers or oligomers and their stabilization, 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. For this purpose, a multiplicity of molecules of a defined self-assembling protein, preferably an S-layer protein or hydrophobin or fusion proteins generated therefrom, are used. Alternatively, 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. These molecules of self-assembling proteins are initially in aqueous Stabilized solution in monomeric or oligomeric form and then used to coat the substrate.

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. For the purposes of the invention, stabilization is understood as meaning the prevention of uncontrolled assembly of self-assembled proteins in aqueous solutions.

In order to establish a neutral net charge of the protein oligomers, it is preferable to add positively charged 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. Particular preference is given to solutions having divalent metal ions, ie having a high ionic strength, in particular alkaline earth metal ions or metal ions of the I. or II subgroup of the PSE (Periodic Table of the Elements) 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. Particular preference is given to 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. Depending on the concentration of the self-assembling proteins, 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. Alternatively, by adding alkalis or acids, the pH can be shifted in the direction of the isoelectric point of the proteins used. By means of both treatment steps, when using fusion proteins which contain at least one self-assembling and at least one functional domain, an orientation of the functional domain to the side remote from the substrate can be advantageously supported in the subsequent coating step.

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,

in which there is at least one monolayer of self-assembling proteins on the substrate surface,

and wherein 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.

Preferably, 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.

For example, in the present invention, in one of the methods used in the self-assembling protein, the coating process comprises the steps of:

i. Providing a stabilized aqueous solution containing at least one self-assembling protein by:

a) providing at least one self-assembling protein in an aqueous solution, and then

b) in the absence of substances which are suitable for the cleavage of disulfide bridges, 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

wherein 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,

Contacting the surface of the substrate with the stabilized proteinaceous solution, thereby producing a proteinaceous coating on the surface of the substrate,

iii. Removing the supernatant solution from the coated substrate and / or drying the coated substrate.

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.

For the formation and stabilization of monomers of the self-assembling proteins, 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:

wherein 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. With such an addition, preferably the surfactant portion of each active protein monomer is enveloped by surfactant particles.

With such a coating solution and the coating method, it is possible to selectively produce an ordered monolayer of one or more self-assembling proteins on the surface of a substrate in one process step. Under an ordered layer or (ordered) monolayer is in accordance with the invention 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. For this purpose, a multiplicity of molecules of a defined self-assembling protein, preferably an S-layer protein or hydrophobin or fusion proteins generated therefrom, are inserted. Alternatively, 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. These molecules of the self-assembling proteins are first stabilized in aqueous solution in monomeric form and then used to coat the substrate.

According to the method for coating with a monolayer of self-assembling proteins, first 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 β-mercaptoethanol) , Dithiothreitol or dithioerythritol). The entire coating process according to the invention is carried out in the absence of these substances.

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. Thus, 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.

Before the addition of the surfactant, 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 / μΙ, more preferably at least 50 ng / μΙ, most preferably at least 90 ng / μΙ. 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.

Optionally, 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 μm - 0.5 μm.

In the coating process according to the invention, ionic surfactants (cationic or anionic surfactants) are used to provide the stabilized protein solution. These surfactants have a single hydrophilic moiety (also referred to herein as a "surfactant moiety") and at least one hydrophobic moiety Preferably, the surfactant is used in a concentration of at most 30 mmol / L, more preferably at most 15 mmol / L (Process b). For the production of a superficial protein monolayer on the substrate, 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.

With the coating method according to the invention only a maximum amount of surfactant is added, as necessary, to envelop the protein molecules contained in the solution with a bilayer of the surfactant. For this, as much surfactant is added to the solution from a) that the final concentration of the surfactant in mol / l after addition to the protein-containing solution is in the following range: D abeisind: Ao, p _ra tein ■■■ O berfläche of sel bstassem bl i ng the P rotei n Molecule ls (spherical surface), A _T KG ■■■ cross-sectional area of the Tensidkopfgruppe (circular surface), N _Prot a number of ■■■ Monomers of self-assembled protein per liter, N _A ... Avogad ro constant (N _A = 6.022x10 ²³ particles / mol), p _rc = 2.05, p _K = 0.774, p _up = 1.5.

The relationship given in 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). In this case, 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.

Under knowledge of the protein concentration in mol / l in the protein-containing solution from a)

and with the fixed parameters p _rc , p _K and p _up, this results solely in dependence on the self-blinding protein and its surface, the transverse surface of the surfactant used and the concentration of the self-assembling protein in the protein-containing solution of formula (1) the following relationship: i C protein "Vi XO S. _cognac ig S i ^ protein"

^A TKG ^A -TEG ()

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 surface of the self-assembling protein molecule results here under consideration as a ball from the following equation (4), wherein R is _H, p _ra tein the hydrodynamic radius of the self-assembling protein molecule is:

Following the process step b), 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. Particularly preferred are 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. Particular preference is given to 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 , As a result, in the case of the use of fusion proteins which contain at least one self-assembling and at least one functional domain, an orientation of the functional domain to the side remote from the substrate can be advantageously supported in the subsequent coating step.

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. During this process step, the self-assembling protein molecules deposit in the form of a monolayer on the surface of the substrate. Subsequently, the supernatant proteinaceous solution is removed from the substrate by preferably either stripping the solution or drying the coated substrate (removal of the solvent). If the protein-containing solution is removed from the substrate, 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 of various structures are suitable for coating processes according to the invention. Particularly advantageously, 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 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.

For some applications, it is necessary to remove the surfactant-containing protective layer to ensure the accessibility of the proteins. For this purpose, in a coating method according to the invention, the surfactant-containing 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

on the substrate surface, a monolayer of at least one self-assembling protein and

On the surface of the monolayer of the at least one self-assembling protein is another layer containing at least one ionic surfactant. In this case, the surfactant is in coordinative binding with the self-assembling protein. Preferably, 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.

For coating a substrate with a bilayer of self-assembling proteins, the method is carried out with the following steps: i. Providing a protein-containing coating solution

ii. Contacting the surface of the substrate with the proteinaceous solution, iii. Removing the supernatant solution from the coated substrate and / or drying the coated substrate.

wherein to provide the coating solution

a) protein units aggregated from an aqueous solution of self-assembling proteins are separated so that monomers or oligomers of self-assembling proteins preferably remain in the solution b) by adding a solution of ionic surfactants and / or a saline and / or alkaline and / or acidic solution

in the protein-containing coating solution, oligomers of the self-assembling proteins are produced and stabilized

wherein so much ionic surfactant is added that only the surfactant portion of each active predominant protein oligomer is enveloped by surfactant particles.

During contacting or before, 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 Tensidkopfgruppe (circular surface) of the surface prädominanter AD.TKG protein oligomers, and Cp _ro tein of the protein concentration employed. With 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. For this purpose, as much surfactant is added to the solution from a) that the final concentration of the surfactant in mol / l after addition to r protein and saline solution is in the following range:

(Z'ACJCF + X'AQl ^ ^J OK Protsi-Rmiiitims-r "V -rotsin PS

A ₀ .Pr _{O tsi} n ^' ^. ^ - ™ ^ifi - AO.TKC' * Λ 'Prc'^>

The following are: ■■■ contact area of the liquid with substrate, AO, LI ■■■ remaining interphase of the liquid, V _F L ■■■ volume of the liquid, Ao, p _ro teinmuitimer the surface of predominant protein oligomers, AOJKG ■■■ cross-sectional area of the surfactant head group (Circular area), N _Pro t _e in ■■■ Number of protein particles in solution, N _A ... Avogadro constant (N _A = 6.022x10 ²³ particles / mol), x ... multiplication factor. p _K = 0.774, p _rc = 2.05.

The one limit range takes into account that maximally as much surfactant is added as is necessary for the stabilization of predominant protein oligomers. At 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.

In the second limiting case, 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. To build an ordered protein map, each protein particle has a defined space on the substrate. To counteract disordered growth of the protein layer, contact sites for other protein particles on the protein image coated substrate must be blocked by surfactant particles. By adding 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. Here, 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.

A 0, p ro TKG ^{= π ■} Ά Pro fine (6)

The relationship given in 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. The contact surface of the liquid with the substrate and the remaining adjacent interphases (AO, LI), calculated from their geometric parameters.

Taking into account that the preferred orientation of the surfactant particles at interphase depends on their wettability (hydrophilic or hydrophobic), a correction term x is inserted in equation (5), which can preferably assume the values 1 and 2. At hydrophilic-hydrophilic interfaces, surfactant particles preferably accumulate in a bilayer on the substrate (x = 2). On the other hand, the formation of a surfactant monolayer (x = 1) predominates at hydrophilic-hydrophobic interfaces.

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. During this process step, the protein oligomers are deposited in the form of a protein bilayer on the surface of the substrate. Subsequently, the supernatant proteinaceous solution is removed from the substrate by preferably stripping off the solution. Subsequently, the coated substrate surface is freed from excess, non-bound protein molecules by repeated rinsing with deionized water. As an alternative to storage in aqueous solutions, the drying of the coated substrate (removal of the solvent) can take place; preferably by treatment with compressed air. To coat a substrate with a hydrophobin bilayer of at least one hydrophobin type, 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. Contacting the surface of the substrate with the stabilized hydrophobin-containing solution to produce a proteinaceous coating on the substrate, wherein either during contacting or before, a solution containing at least one ionic surfactant is added in portions to the proteinaceous solution, after addition the surfactant concentration c _Te nsid in mol / l lies in the following range:

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 prädominanter Hydrophobinmultimere, AD.TKG corresponds to the cross sectional area of the Tensidkopfgruppe (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.

With this coating method, it is possible to selectively produce an ordered bilayer of one or more "fusion hydrophobi" on the surface of a substrate in one process step. In the context of the invention, 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.

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. On the one hand, 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. On the other hand, the net electrical charge of the hydrophobins must be taken into account. 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.

According to the process of the invention, 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.

There are several different types of hydrophobin within hydrophobin bilayers. For this purpose, a multitude of molecules of a defined hydrophobin or fusion proteins generated therefrom are used. Alternatively, 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.

In the incubation of the hydrophobin-containing solution with the salt-containing solution added in several portions (in at least two, preferably at least three individual portions), 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. For the purposes of the invention, 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.

Before adding the salt, the alkali or acid, the concentration of the at least one hydrophobin type in the unstabilized aqueous solution is preferably at least 50 ng / μΙ, particularly preferably at least 90 ng / μΙ. 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.

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. Particular preference is given to 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 , Alternatively, by adding alkalis or acids, the pH can be shifted in the direction of the isoelectric point of the hydrophobins used. By means of both treatment steps, when using fusion proteins which contain at least one self-assembling and at least one functional domain, an orientation of the functional domain to the side remote from the substrate can be advantageously supported in the subsequent coating step. The addition is carried out in portions, preferably in equal portions and preferably in at least two parts, more preferably in 3 portions and over a period of 1 to 60 minutes in order to prevent uncontrolled hydrophobin aggregation.

During the contacting of the substrate or before, solutions of ionic surfactants, preferably cationic and / or anionic surfactants, are added to the protein- and saline-containing solution. These 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 μηηοΙ / Ι and at most 500 μηηοΙ / Ι, more preferably at most 100 μηηοΙ / Ι corresponds.

With 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. For this purpose, 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:

Here are: ■■■ contact surface of the liquid with substrate, AO, LI ■■■ remaining interphase of the liquid, V _F L ■■■ volume of the liquid, Ao, p _ro teinmuitimer the surface of predominant hydrophobin multimers, Ao, TKG ■■■ cross-sectional area of the surfactant head group (circular area), N _Pro t _e in ■■■ Number of protein particles in solution, N _A ... Avogadro constant (N _A = 6.022x10 ²³ particles / mol), x ... Multiplication factor. p _K = 0.774, p _rc = 2.05,

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. Above a calculable surfactant concentration (right-hand limit of formula 5), 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.

In the second limiting case, the formed protein bilayer on the substrate is covered by a layer of surfactant and prevents any further attachment of hydrophobins to the interphase. To build an ordered hydrophobin bilayer, each hydrophobin particle has a defined space on the substrate. To counteract the disordered growth of the hydrophobin layer, contact sites for further hydrophobin particles on the hydrophobin bilayer coated substrate must be blocked by surfactant particles. By adding 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 Here, 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.

A 0, p ro TKG ^{= π ■} Ά Pro fine (6)

The relationship given in 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. The contact surface of the liquid with the substrate and the remaining adjacent interphases (AO, LI), calculated from their geometric parameters.

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. During this process step, the hydrophobinassemblates are deposited in the form of a protein bilayer on the surface of the substrate. Subsequently, the supernatant proteinaceous solution is removed from the substrate by preferably stripping off the solution. Subsequently, the coated substrate surface is freed from excess, non-bound protein molecules by repeated rinsing with deionized water. As an alternative to storage in aqueous solutions, the drying of the coated substrate (removal of the solvent) can take place; preferably by treatment with compressed air.

Substrates of various structures are suitable for the coating method according to the invention. Particularly advantageously, 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.

For some applications, it is necessary to remove the surfactant-containing protective layer to ensure the accessibility of the proteins. In a coating method according to the invention, the surfactant-containing 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 (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.

The 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.

Preferably, 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. By fusing the genes of two or more protein domains that are not naturally present in this combination, which are usually connected to one another via flexible linker structures (linker peptides), the recombinant fusion protein can be obtained by expression of the fused gene. For heterologous gene expression, 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. To generate a double hydrophobin, other hydrophobins may also act as a fusion domain. The choice of suitable fusion partner parts is not limited to proteins of certain organisms. For selective isolation of the fusion proteins from the cell lysate, 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.

In a coated substrate according to the invention which contains recombinant fusion protein as hydrophobin, 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).

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") In the stabilization method a) at least one self-assembling protein is admixed with an aqueous solution, preferably an aqueous buffer solution , and subsequently

b) in the absence of substances which are suitable for the cleavage of disulfide bridges, an aqueous solution containing at least one ionic surfactant added in portions to the protein-containing solution from a), wherein after the addition, the final surfactant concentration c _Te nsid in mol / l in the following range is: 5,297,

where 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 and Cp _ro tein corresponds to the concentration of the self-assembling protein in mol / l.

Further embodiments of the stabilization method according to the invention are analogous to the embodiments of the coating method according to the invention. 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). ). In the aqueous solution according to the invention, 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:

^■ 0.516 <c _surfactant < ^■ 5.297

Here, 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 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.

Particularly advantageously, 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. By fusion of the genes of two or more protein domains which are not naturally present in this combination and which usually have the linker polypeptides (linker peptides), 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.

In a coated substrate according to the invention which contains recombinant fusion protein as a self-assembling protein, 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).

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.

Reference to the following figures and embodiments, the invention will be explained in more detail, without limiting the invention to these.

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

The recombinant fusion proteins mentioned in Table 1 were generated, which each contain the self-assembling protein domain N-terminal and contain a functional domain designated C-terminally therein. Table 1 :

Fusion protein Self-assembling domain Functional domain

HFBI- (R5P) ₂ class II hydrophobin subunit of silaffin

HFBI (Trichoderma reesei) (Cylindrotheca fusiformis)

HFBI Class II Hydrophobin Truncated subunit of (XSR5P) ₃ HFBI (Trichoderma reesei) Silaffins (Cylindrotheca fusiformis)

Ccg2-HA Class I Hydrophobin Human influenza hemagglutinin Ccg2 (Neurospora crassa) day

S13240-HA S-Layer Protein Human influenza hemagglutinin

$>?> 2W (Geobacillus day

stearothermophilus)

SbsC-HA S-Layer Protein Human influenza hemagglutinin-SbsC {Geobacillus tag

stearothermophilus)

SbsC- (R5P) ₂ S-layer protein subunit of silaffin

SbsC {Geobacillus (Cylindrotheca fusiformis) stearothermophilus)

SbsC- S-layer protein subunit of Shortened (XSR5P) ₃ SbsC {Geobacillus Silaffins (Cylindrotheca stearothermophilus) fusiformis)

SslA S-layer protein no functional domain

SslA (Sporosacina ureae)

For this purpose, 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.

For the production of 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. After 4-6 h, the cells were pelleted by centrifugation (at 4000 xg, 4 ° C, 10 min). After washing the cell pellet twice with 50 mM Tris buffer (pH 7.5), the cells were again pelleted by centrifugation. For cell disruption, 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 (20Ό00 psi). The digested bacterial cells were washed twice with 50 mM Tris buffer (pH 7.5). For protein extraction, the taken up in 1 ml lysis buffer and incubated at 23 ° C for 30 min. 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.

To convert the protein to an active state, the resulting eluate was dialyzed against 4000 times the volume of a dialysis buffer at 4 ° C for a maximum of 48 hours. For dialysis of hydrophobins, a Tris buffer (50 mM, pH 8.5 with 1 mM glutathione reduced and 0.2 mM glutathione oxidized) was used. For dialysis of surface-layer proteins, 10 mM Tris buffer (pH 9.0) was used as the dialysis buffer.

Exemplary Embodiment 2 Generation of a Protein Monolayer on a Silicon Wafer

An ordered protein monolayer of self-assembling protein as obtained in Embodiment 1 was generated on a silicon wafer. For this purpose, the 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.

At first, 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 / μΙ was set.

To this protein containing solution was added a filtered 8 mM sodium dodecylsulfate (SDS) in four portions over a period of 10 minutes. For HFBI (XSR5P) ₃ , the amount of surfactant added per liter of the solution adjusted to a protein concentration of 100 ng / μΙ was 2 mmol, so that a final surfactant concentration of 2 mmol / l in the solution was established.

From equation (3), a surfactant concentration to be set for a 100 ng / μΙ 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 / μΙ in the solution is in mol / l, corresponding to 7.14 ^* 10 ^"6 mol / l. The Tensidkopfgruppe of the employed SDS molecule has an effective area of A _T KG of 0.62 nm ^2. The protein molecule has a hydrodynamic radius R _H of 2.7 nm. It follows from equation (4), a protein surface Ao, p _ra ton = 91, 6 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. For this purpose, 100 μΙ 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.

In order to access the functional domain of the self-assembling fusion protein on the substrate, remaining surfactant was removed from the coated substrate by precipitation reaction.

Exemplary 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 °. By intensity measurement of the normal (R _s ) and parallel (R _p ) reflected planes of incidence of the polarized light beam, the intensity ratio (p) was determined. The ellipsometric angles Δ and Ψ are derived via FRENSEL's formulas. By calculating the refractive indices of the individual layers, the layer thickness of the uppermost layer was calculated.

IL

R, IR _s \

In this case, δ _ρ 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.

The following 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:

1 2 3 4

□ SiO 2 [°] 175.87 175.772 175.761 175.879

d _S i02 [E] 16.5 17.0 17.0 16.5

173,216 173,172 173,138 173,168

173.26 173.297 173.138 173.509

173.324 173.09 173.362 173.166

173.334 173.21 1 173.45

173.37 173.096 173.436

173.06

Protein [°] 173,301 173,154 173,213 173,346

0.062 0.090 0.129 0.166 mean standard deviation d protein [A] 13.2 13.4 13.1 13 13.2 0.2

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). For the analysis, 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.

Exemplary Embodiment 5 Atomic Force Microscopy (AFM) of a Ccg2-HA Coated Silicon Wafer

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.

Exemplary Embodiment 6: Exposure of Functional Domains to Self-Assembling Fusion Proteins

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 Å. In the fusion protein is the functional domain, the R5P subunit of the silaffin fused with the hydrophilic part of the hydrophobin.

It was checked on the basis of the coated silicon wafer by means of contact angle measurement, whether the hydrophilic domain of the self-assembling protein and thus also the functional domain R5P is oriented in the medium, ie on the side facing away from the silicon wafer. For this purpose, 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 μΙ deionized water was determined.

An uncoated silicon wafer cleaned with ethanol had a contact angle of Θ = 35.2 ± 2 °. The silicon wafer coated with HFBI (R5P) ₂ has a contact angle of Θ = 56.4 ± 0.7 °. This is a clear indication that the hydrophilic domain is exposed in the medium.

Exemplary Embodiment 7 Generation of a Hydrophobin Sheet on a Silicon Wafer

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 / μΙ was set.

To this protein-containing solution was added a filtered 10 mM sodium sulfate solution (Na ₂ SO ₄ ) in four portions over a period of 10 minutes. Subsequently, an 8 mM sodium dodecyl sulfate solution (SDS) is added. For HFBI- (R5P) ₂ , the added salt concentration was 0.05 mM and the amount of surfactant per liter of the solution adjusted to a protein concentration of 100 ng / μΙ 20 μηηοΙ, so that a final surfactant concentration of 20 μηηοΙ / Ι was set in the solution.

The stabilized solution thus obtained was clear.

The stabilized solution containing multimers of hydrophobin was used to coat a silicon wafer with edge lengths of 1 x 1 cm. For this purpose, 200 .mu.Ι 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.

To access the functional domain of the self-assembling fusion protein on the substrate

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 °. By intensity measurement of the normal (R _s ) and parallel (R _p ) reflected planes of incidence of the polarized light beam, the intensity ratio (p) was determined. The ellipsometric angles Δ and Ψ are derived via FRENSEL's formulas. By calculating the refractive indices of the individual layers, the layer thickness of the uppermost layer was calculated. p = - ^£ = i ^£, ** p - **> = tan-OF) e ^iJ

n j R ^ J

The determined layer thickness of HFBI- (R5P) ₂ on the silicon wafer was 25.7 ± 1.8 Å, or 25.8 ± 0.8 Å for the construct HFBI- (XSR5P) ₃ . These layer thicknesses correspond to the literature values for a bilayer of hydrophobin.

The following 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

EMBODIMENT 9 Determination of the Functional Domain Exposure of Self-Assembling Fusion Proteins

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 Å. In the fusion protein, the funcional domain, the R5P subunit of the silaffin, is fused to the hydrophilic part of the hydrophobin.

An uncoated silicon wafer cleaned with ethanol had a contact angle of Θ = 35.2 ± 2 °. The HFBI (R5P) ₂ coated silicon wafer has a contact angle of 0 = 54, 1 ± 0.9 °. This is a clear indication that the hydrophilic domain is exposed in the medium. to make remaining surfactant from the coated substrate

Removed precipitation reaction. Exemplary Embodiment 10: Checking the Accessibility of the Fused HA Tag of a Ccg2-HA Coated Silicon Wafer.

To determine the orientation of deposited protein monomers on a silicon wafer coated with Ccg2-HA analogously to Example 2, the coupling of a fluorescently labeled antibody to the fused protein domain of a hydrophobin fusion protein was carried out. (Darmstadt, Germany GMBH INVITROGEN) diluted according to the manufacturer and incubated for 30 min on the substrate for the fluorescence microscopic analysis of the accessibility of the HA tag antibody Anti-HA ^® AlexaFluor 488 conjugate. Subsequently, the substrate was washed three times with double distilled water to remove unbound antibody residues. 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.

As a reference, 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.

To determine the accessibility of protein domains of a fusion protein, the coating of a silicon wafer with HFBI-GLuc was carried out in analogy to embodiment 2. The fused luciferase domain (GLuc) catalyses the chemical conversion of luciferin with the emission of light with the wavelength 475 nm. In addition to a visual assessment quantitative fluorescence analysis was carried out by Infinite M200 plate reader (TECAN GROUP LTD., Männedorf, Germany). The unfused protein domains (HFBI or GLuc) served as references.

Upon addition of luciferol, the bifunctional hydrophin chimera (HFB1-Gluc) and the subunit displayed a luminescent signal as expected. The catalytically inactive HFB1 subunit shows no luminescence signal. In a second experiment, 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

In order to investigate the effects of self-assembling proteins, CTAB was added to fluorescent fusion proteins. The fluorescent fusion proteins consist of a hydrophobin (Ccg2 or HFBI), which represents the self-assembling domain and a red fluorescent peptide domain (tRFP). 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.

Provision of 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 0.22μη ". Filter and the effect of filtration and the influence of added ionic surfactants was subsequently examined over a period of one week.

Due to the properties of self-assembled proteins, 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. In contrast, 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.

A contrasting picture emerged in the case after adding an ionic surfactant. The fluorescence intensity of the proteinaceous solution containing the unfused tRFP peptide sequence and CTAB decreased by 25%. The filtration of this sample had no influence on the fluorescence intensity. In the case of proteinaceous solutions containing a fusion protein (Ccg2-tRFP or HFBI-tRFP), the addition of an ionic surfactant had no effect on the fluorescence intensity. Even after filtration of these solutions, the fluorescence intensity remained unaffected. The fluorescence intensity was subsequently monitored every 24 hours for a period of one week. 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. The fluorescence intensity of the proteinaceous solutions, including the unfused tRFP peptide sequence and an ionic surfactant, decreased continuously over the entire experimental period. After one week, a fluorescence which was reduced by 67% from baseline was detectable in this sample.

Claims

claims

1 . Process for coating a substrate with at least one monolayer of self-assembling proteins, comprising the steps:

i. Providing a proteinaceous coating solution,

ii. Contacting the surface of the substrate with the proteinaceous solution, iii. Removing the supernatant solution from the coated substrate and / or drying the coated substrate,

characterized in that aggregated protein units are separated from an aqueous solution of self-assembling proteins to provide the coating solution a) and, b) by adding a solution of ionic surfactants and / or a saline and / or alkaline and / or acidic solution in the protein-containing coating solution M On the other hand, oligomers of the self-assembling proteins are generated and stabilized with enough ionic surfactant added to encase only the surfactant portion of each active protein monomer or protein oligomer of surfactant particles.

2. The method according to claim 1, characterized in that as self-assembling proteins hydrophobins and / or surface 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.

3. The method according to claim 1 or 2, characterized in that the surfactants are selected from the group of the hydrocarbon-coupled sulfates, S u l fo n e d e r C a r b o xy l a te o d e r a of the group of quaternary ammonium compounds.

4. The method according to any one of claims 1 to 3, characterized in that the saline and / or alkaline and / or acidic solution of alkaline earth metal ions or metal ions of subgroups I and II in the form of chlorides, nitrates, carbonates, acetates, citrates, gluconates, hydroxides , Lactates, sulfates, succinates or Tartrates or 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 contains.

5. protein-containing coating solution containing monomers or oligomers of self-assembling proteins, wherein the surface-active part of the monomers or oligomers of ionic surfactants is coated

6. Use of a protein-containing coating solution according to claim 5 for coating substrate surfaces.

7. substrate having a protein-containing coating of at least one self-assembling protein, characterized by

that there is at least one monolayer of self-assembling proteins on the substrate surface,

in that on the surface of the at least one monolayer of self-assembling proteins there is a layer which contains at least one ionic surfactant, the surfactant being in coordinative binding with the self-assembling protein.

8. Substrate according to claim 7, characterized in that the self-assembling protein is a hydrophobin and / or a surface layer protein and / or recombinant fusion protein comprising at least two domains, wherein a domain is a self-assembling protein domain and a domain is a functional domain , wherein the functional domain is disposed on the side facing away from the substrate of the proteinaceous coating.