DE102005051515A1 - Coating surfaces with hydrophobins involves coating with hydrophobin in form of fusion hydrophobin and using formulation with pH value of greater than or equal to 4 - Google Patents

Abstract

The method involves providing water containing a formulation or an aqueous solution mixture as well as a hydrophobin, treating the surface with the formulation and removing the solvent. The hydrophobin is a fusion hydrophobin and the formulation has a pH value of greater than or equal to 4.

Description

The The present invention relates to a method for coating of surfaces with fusion hydrophobins at a pH ≥ 4 and surfaces with a coating comprising fusion hydrophobins.

hydrophobins are small proteins of about 100 to 150 amino acids that are characteristic for filamentous mushrooms, for example, Schizophyllum commune. They point in all Rule 8 cysteine units on. Hydrophobins can be made, for example, from natural Sources are isolated.

hydrophobins have a pronounced affinity to interfaces and are therefore suitable for coating surfaces. So let yourself For example, Teflon using hydrophobins to obtain a hydrophilic surface coat.

in the The state of the art is the use of hydrophobins for various purposes Applications have been proposed.

WHERE 96/41882 proposes the use of hydrophobins derived from edible fungi as emulsifiers, thickeners, surface-active substances, for Hydrophilizing hydrophobic surfaces to improve the resistance to water hydrophilic substrates, for the preparation of oil-in-water emulsions or of water-in-oil emulsions in front. Furthermore, pharmaceutical applications such as the production of Ointments or creams as well as cosmetic applications such as skin protection or the production of hair shampoos or hair rinses.

EP-B 1 252 516 discloses the coating of windows, contact lenses, Biosensors, medical devices, containers for carrying out Try or for storage, ship hulls, solid particles or Frame or body of passenger car with a hydrophobin containing solution at a temperature of 30 to 80 ° C. In addition, a preferred surfactants Substance used as a coating aid. In the examples is a hydrophobin obtained from fungi (Schizophyllum commune) used by the type SC3. To prepare the solution for coating is freeze-dried SC3 used, dissolved with trifluoroacetic acid, the mixture in a stream of nitrogen dried and then in Water or a buffer solution solved. This procedure is cumbersome.

WHERE 2005/068087 beats as an alternative to heating the coating in the acidic pH range before. The Scripture reveals a method of coating surfaces with hydrophobins a pH less than 7, preferably less than 4 and more preferably smaller 2. Furthermore, a method for optimizing the coating conditions under variation of the parameters pH value, incubation time, concentration and the presence of a buffer. In the examples For example, a SC3-type hydrophobin is used from natural sources.

The The present application relates to the coating of surfaces with a novel class of non-naturally occurring hydrophobins. These are fusion hydrophobins, in which naturally occurring Hydrophobins with at least 20 amino acid long peptide sequences are linked which naturally not linked to a hydrophobin. Also such fusion hydrophobins are suitable for coating surfaces.

Surprisingly was found that the quality the coatings obtained with fusion hydrophobins also at increased pH values does not decrease. This naturally occurring hydrophobin allows inverse behavior it, also in the alkaline range high quality coatings to obtain.

More specifically, the following is to be accomplished for the invention:
For carrying out the present invention, "fusion hydrophobins" are used which have the following general structural formula (I), X _n -C ¹ -X _1-50 -C ² -X _0-5 -C ³ -X _1-100 -C ⁴ -X _1-100 -C ⁵ -X _1-50 -C ⁶ -X _0-5 -C ⁷ -X _1-50 -C ⁸ -X _m (I) where X is for each of the 20 naturally occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly). X may be the same or different. Here, the indices standing at X each represent the number of amino acids, C stands for cysteine, alanine, serine, glycine, methionine or threonine, wherein at least four of the radicals named C are cysteine. The indices n and m independently of one another represent natural numbers of 0 and 500, preferably of 15 to 300, with the proviso that at least one of the peptide sequences designated X _n and X _{m represents a} peptide sequence of at least 5, preferably at least 20 amino acids naturally not associated with a hydrophobin.

The Polypetides according to formula (I) are further characterized by the property that they at room temperature after coating a glass surface Magnification of the Contact angle of a water droplet of at least 20 °, preferably at least 25 ° and particularly preferably 30 ° cause each compared with the contact angle of a water droplet of the same size with the uncoated glass surface.

The amino acids designated C ¹ to C ⁸ are preferably cysteines; but they can also be replaced by other amino acids of similar space filling, preferably by alanine, serine, threonine, methionine or glycine. However, at least four, preferably at least 5, more preferably at least 6 and in particular at least 7, of the positions C ¹ to C ^{8 should} consist of cysteines. Cysteines can either be reduced in the proteins used according to the invention or form disulfide bridges with one another. Particularly preferred is the intramolecular formation of CC bridges, in particular those with at least one, preferably 2, more preferably 3 and most preferably 4 intramolecular disulfide bridges. In the exchange of cysteines described above by amino acids of similar space filling, it is advantageous to exchange in pairs those C positions which are capable of forming intramolecular disulfide bridges with one another.

If in the positions indicated by X, also cysteines, serines, alanines, Glycine, methionine or threonine may be used Numbering of the individual C positions in the general formulas change accordingly.

Preference is given to fusion hydrophobins of the general formula (II) X _n -C ¹ -X _3-25 -C ² -X _0-2 -C ³ -X _5-50 -C ⁴ -X _2-35 -C ⁵ -X _2-15 -C ⁶ -X _0-2 -C ⁷ -X _3-35 -C ⁸ -X _m (II) used for carrying out the present invention, wherein X, C and the indices standing at X and C are as defined above, however, the indices n and m are numbers between 0 and 300, and the proteins are further characterized by the above-mentioned contact angle change, with the proviso that at least one of the peptide sequences designated X _n and X _m is an at least 15, preferably at least 35 amino acid long peptide sequence which is not naturally linked to a hydrophobin.

Particular preference is given to fusion hydrophobins of the general formula (III) X _n -C ¹ -X _5-9 -C ² -C ³ -X _11-39 -C ⁴ -X _2-23 -C ⁵ -X _5-9 -C ⁶ -C ⁷ -X _6-18 -C ⁸ -X _m (III) where X, C and the indices at X and C have the above meaning, the indices n and m are numbers between 0 and 200, and the proteins are further characterized by the above-mentioned contact angle change, provided that at least one of the peptide sequences designated X _n and X _m is a peptide sequence which is at least 20 amino acids long, preferably at least 50 amino acids, which is not naturally linked to a hydrophobin, and at least 6 of the radicals named C are cysteine. Most preferably, all C radicals are cysteine.

The naturally radicals not linked to a hydrophobin are described below also be referred to as fusion partners. This is to express that the proteins consist of at least one hydrophobin part and one May exist which do not occur together in nature in this form.

The fusion partner can be selected from a variety of proteins. Several fusion partners can also be linked to a hydrophobin part, for example at the amino terminus (X _n ) and at the carboxy terminus (X _m ) of the hydrophobin part. However, for example, two fusion partner parts can also be linked to a position (X _n or X _m ) of the hydrophobin.

Especially suitable fusion partner parts are proteins that are naturally in microorganisms, in particular in E. coli or Bacillus subtilis occurrence. examples for Such fusion partner parts are the sequences yaad (SEQ ID NO: 15 and 16), yaae (SEQ ID NOs: 17 and 18), and thioredoxin. Well suited are also fragments or derivatives of said sequences which only a part, for example 70 to 99%, preferably 5 to 50% and particularly preferably comprise 10 to 40% of said sequences, or where individual amino acids, or nucleotides changed the said sequence are, wherein the percentages in each case refers to the number of amino acids.

In a further preferred embodiment, in addition to the fusion partner, the fusion hydrophobin also has a so-called affinity domain (affinity tag / affinity tail) as a group X _n or X _m . These are in a manner known in principle to anchor groups, which can interact with certain complementary groups and can be used to facilitate processing and purification of the proteins. Examples of such affinity domains include (His) _k , (Arg) _k , (Asp) _k , (Phe) _k or (Cys) _k groups, where k is generally a natural number from 1 to 10. It may preferably be a (His) _k group, where k is 4 to 6.

The used according to the invention Fusion hydrophobins can also be modified in their polypeptide sequence, for example by glycosylation, acetylation or by chemical cross-linking for example with glutaric dialdehyde.

A essential property of the fusion proteins used according to the invention is the change of Surface properties when the surfaces coated with the fusion proteins. The change the surface properties let yourself experimentally determine that the contact angle of a water drop measured before and after coating the surface with the protein and the difference between the two measurements is determined.

The execution Contact angle measurements are known in principle to the person skilled in the art. The measurements refer to room temperature and water droplets of 5 μl and the use of glass slides as Substrate. The exact experimental conditions for an example suitable method for measuring the contact angle are in the experimental Part shown. Under the conditions mentioned there have the used according to the invention Fusion proteins the property, the contact angle by at least 20 °, preferably at least 25 °, more preferably at least 30 ° to enlarge, respectively compared with the contact angle of an equal drop of water with the uncoated glass surface.

preferred Fusion hydrophobins for execution of the present invention are those having a hydrophobin moiety of the type dewA, rodA, hypA, hypB, sc3, basf1, basf2, which are described below Sequence listing are structurally characterized. It may be even just parts or derivatives of it. There can also be several Hydrophobin parts, preferably 2 or 3, the same or different Structure linked together.

Especially suitable for implementation In the present invention, the fusion proteins having the sequences shown in SEQ ID NO: 20, 22, 24 polypeptide sequences shown and the coding therefor Nucleic acid sequences, in particular the sequences according to SEQ ID NO: 19, 21, 23. Also, proteins derived from the in SEQ ID NO. 20, 22 or 24 represented by polypeptide sequences Exchange, insertion or deletion of at least one, all the way through to 10 preferably 5, more preferably 5% of all amino acids, and the biological properties of the source proteins still at least 50% are particularly preferred embodiments. Under biological Property of the proteins here is the already described enlargement of the Contact angle by at least 20 ° understood.

The used according to the invention Fusion hydrophobins can be prepared by known methods of peptide synthesis, for example, by Merrifield solid phase synthesis, chemically produce.

Prefers the production of the fusion hydrophobins by genetic engineering takes place Procedures in which a for the fusion partner and one for the hydrophobin part coding nucleic acid sequence, in particular DNA sequence, be combined so that in a host organism by gene expression the combined nucleic acid sequence the wished Fusion hydrophobin is generated.

Suitable host organisms (production organisms) for said production process may be prokaryotes (including archaea) or eukaryotes, especially bacteria including halobacteria and methanococci, fungi, insect cells, plant cells and mammalian cells, especially before Escherichia coli, Bacillus subtilis, Bacillus. megaterium, Aspergillus oryzea, Aspergillus nidulans, Aspergillus niger, Pichia pastoris, Pseudomonas spec., Lactobacilli, Hansenula polymorpha, Trichoderma reesei, SF9 (or related cells) and others.

object The invention is also the use of expression constructs containing under genetic control of regulatory nucleic acid sequences, a polypeptide used in the invention coding nucleic acid sequence, and vectors comprising at least one of these expression constructs.

Preferably include inserted constructs 5'-upstream of the respective coding Sequence one promoter and 3'-downstream one Terminator sequence and optionally other common regulatory elements, each operatively linked with the coding sequence.

Under an "operational Linking "one understands the Sequential arrangement of promoter, coding sequence, terminator and optionally other regulatory elements such that each the regulatory elements have their function in the expression of the coding Sequence can meet as intended.

Examples for operational linkable Sequences are targeting sequences as well as enhancers, polyadenylation signals and the same. Other regulative elements include selectable ones Markers, amplification signals, origins of replication, and the like. suitable regulatory sequences are e.g. As described in Goeddel, Gene Expression Techno-logy: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).

In addition to These regulatory sequences may be the natural regulation of these sequences be present before the actual structural genes and optionally genetically modified have been so natural Regulation switched off and the expression of genes was increased.

One preferred nucleic acid construct contains advantageously also one or more of the already mentioned "enhancer" sequences, functionally linked to the promoter, which increased one Expression of the nucleic acid sequence enable. Also at the 3'-end the DNA sequences can additional advantageous sequences are inserted, such as further regulatory Elements or terminators.

The nucleic acids can be contained in one or more copies in the construct. In the construct can still additional markers, such as antibiotic resistance or complementing auxotrophs Genes, optionally be included for selection on the construct.

advantageous Regulatory sequences for the method are for example in promoters such as cos-, tac-, trp-, tet, trp-tet, lpp, lac, lpp-lac, laclq-T7, T5, T3, gal, trc, ara, rhaP (rhaPBAD) SP6, lambda PR or imlambda P promoter that are beneficial in gram-negative bacteria application Find. Further advantageous regulatory sequences are, for example in the gram-positive promoters amy and SP02, in the yeast or Fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH included.

It can also artificial Promoters for the regulation can be used.

The nucleic acid construct is advantageously for expression in a host organism into a vector, such as a plasmid or a phage inserted, which allows optimal expression of genes in the host. Among vectors are out of the box Plasmids and phages also all other vectors known in the art, So z. Viruses, such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, Cosmids, and linear or circular DNA, as well as the Agrobacterium system to understand.

These vectors can be autonomously replicated in the host organism or replicated chromosomally. These vectors represent a further embodiment of the invention. Suitable plasmids are described, for example, in E. coli pLG338, pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290, pIN-III''3-B1, tgt11 or pBdCl, in Streptomyces pIJ101, pIJ364, pIJ702 or pIJ361, in Bacillus pUB110, pC194 or pBD214, in Corynebacterium pSA77 or pAJ667, in fungi pALS1, pIL2 or pBB116, in yeasts 2alpha, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHlac +, pBIN19, pAK2004 or pDH51. The plasmids mentioned represent a small selection of the possible plasmids. Further plasmids are known to the person skilled in the art and can be found, for example, in the book Cloning Vectors (Eds. Pouwels PH et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). ent be taken.

Advantageous contains the nucleic acid construct For expression of the other genes contained additionally 3'- and / or 5'-terminal regulatory Sequences to increase expression, depending on the selected host organism and gene or genes for optimal expression selected become.

These Regulatory sequences are designed to target the expression of genes and enable protein expression. For example, depending on the host organism, this may mean that the gene is expressed or overexpressed after induction, or that it expresses immediately and / or overexpressed becomes.

The Regulatory sequences or factors can preferably the Gene expression of the introduced Positively influence genes and thereby increase them. So can a reinforcement of the regulatory elements advantageously at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. But there is also a reinforcement Translation possible, for example, by improving the stability of the mRNA.

In In a further embodiment of the vector, the nucleic acid construct or the nucleic acid containing vector also advantageously in the form of a linear DNA in the microorganisms are introduced and over heterologous or homologous recombination into the genome of the host organism to get integrated. This linear DNA can be linearized Vector such as a plasmid or only from the nucleic acid construct or the nucleic acid consist.

For an optimal Expression of heterologous genes in organisms, it is advantageous the nucleic acid sequences according to the specific "codon usage" used in the organism. The "codon usage" can be on the basis of computer evaluations of other, known genes of the concerned Easily detect organism.

The Production of an expression cassette takes place by fusion of a suitable promoter with a suitable coding nucleotide sequence as well as a terminator or Polyadenylation signal. For this purpose, conventional recombination and cloning techniques are used, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

The recombinant nucleic acid construct or gene construct is for expression in a suitable host organism, advantageously inserted into a host-specific vector which has a optimal expression of the genes in the host allows. Vectors are the specialist well known and can for example from "Cloning Vectors" (Pouwels P.H. et al., eds, Elsevier, Amsterdam-New York-Oxford, 1985) become.

With The vectors can be used to produce recombinant microorganisms. which transforms, for example, with at least one vector are used and for the production of the proteins used in the invention can be. Advantageously, the recombinant constructs described above become introduced and expressed in a suitable host system. there are preferably known in the art known cloning and transfection methods, such as co-precipitation, Protoplast fusion, electroporation, retroviral transfection and the like, ver used to the said nucleic acids in respective expression system for expression. suitable Systems are used, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Eds., Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning: A Laboratory Manual. Second Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Homologously recombined microorganisms can also be produced. For this purpose, a vector is prepared which contains at least a portion of a gene or a coding sequence to be used according to the invention, wherein optionally at least one amino acid deletion, addition or substitution has been introduced to alter the sequence, e.g. B. functionally disrupted ("knockout" vector). The introduced sequence can, for. Also be a homologue from a related microorganism or be derived from a mammalian, yeast or insect source. Alternatively, the vector used for homologous recombination may be designed such that the endogenous gene is mutated or otherwise altered upon homologous recombination but still encodes the functional protein (eg, the upstream regulatory region may be altered such that expression of the endogenous protein is changed). The altered portion of the gene used according to the invention is in the homologous recombination vector. The construction of suitable vectors for homologous recombination is e.g. As described in Thomas, KR and Capecchi, MR (1987) Cell 51: 503.

When recombinant host organisms for the invention used nucleic acid or the nucleic acid construct In principle, all prokaryotic or eukaryotic organisms come in question. Advantageously, microorganisms are used as host organisms like bacteria, fungi or yeasts used. Advantageous are gram-positive or gram-negative bacteria, preferably bacteria of the family Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferred bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus used.

The in the manufacturing process for Fusion hydrophobins used organisms will vary depending on the host organism attracted or bred in the manner known in the art. microorganisms are usually in a liquid Medium containing a carbon source mostly in the form of sugars, a Nitrogen source mostly in the form of organic nitrogen sources like yeast extract or salts like ammonium sulfate, trace elements like Iron, manganese and magnesium salts and optionally vitamins contains at temperatures between 0 and 100 ° C, preferably between 10 to 60 ° C below Attracted oxygen fumigation. In this case, the pH of the nutrient fluid be kept at a fixed value, that is regulated during the cultivation be or not. The cultivation can be done batchwise, semi-batchwise or continuously. nutrient can submitted at the beginning of the fermentation or semicontinuously or be continuously refilled. The enzymes can according to the method described in the examples from the organisms be isolated or used as crude extract for the reaction.

Used according to the invention Fusion proteins or functional, biologically active fragments thereof, can by means of a recombinant process in which a Cultured protein-producing microorganism, optionally induces the expression of the proteins and isolates them from the culture. The proteins can so also in large-scale scale produced if desired. The recombinant Microorganism can be cultured and fermented by known methods become. Bacteria can For example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9 are increased. In detail will be suitable cultivation conditions for example in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) described.

The Cells are then, if the proteins used in the invention not are secreted into the culture medium, open-minded and the product obtained from the lysate according to known protein isolation method. The cells can optionally by high-frequency ultrasound, by high pressure, such as z. B. in a French pressure cell, by osmolysis, by action detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods be open-minded.

A Purification of the invention used Fusion proteins can be detected by known chromatographic methods achieved, such as molecular sieve chromatography (gel filtration), such as Q-Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other common ones Methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are, for example in Cooper, F.G., Biochemical Working Methods, Water de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

It may be particularly advantageous to provide the fusion hydrophobins with special anchor groups to facilitate isolation and purification, which can bind to corresponding complementary groups on solid supports, in particular suitable polymers. Such solid carriers can be used, for example, as a filling for chromatography columns, and in this way the efficiency of the separation can generally be increased significantly. Such separation methods are also known as affinity chromatography. For the incorporation of the anchor groups can be used in the production of proteins vector systems or oligonucleotides that extend the cDNA to certain nucleotide sequences and thus encode altered proteins or fusion proteins. Proteins modified for ease of purification include so-called "tags" acting as anchors, such as the modification known as hexa-histidine anchors. For example, fusion hydrophobins modified with histidine anchors can be chromatographically purified using columnar nickel-Sepharose. The fusion hydrophobin can then by means of suitable means for eluting, such as an imidazole solution, are eluted again from the column.

up methods can Of course also be combined with each other. For example, you can first by means of Separate chromatography, and the resulting solution then by dialysis of substances used for elution.

In A simplified purification process can be applied to the chromatographic Cleaning can be dispensed with. For this purpose, the cells are first using separated from the Fermetationsbrühe a suitable method, for example by microfiltration or by centrifugation. Subsequently, the Cells by suitable methods, for example by means of already above methods, open-minded, and cell debris of the inclusion bodies (inclusion bodies) are separated. The latter can be advantageous by Centrifuging done. After all can the inclusion bodies be unlocked in a basically known manner to the fusion hydrophobins release. This can be done, for example, by acids, bases and / or detergents respectively. The inclusion bodies with the inventively used Fusion-hydrophobins can usually already using 0.1 M NaOH within 1 hour complete solved become. The purity of the obtained by this simplified method Fusion hydrophobins is usually at 60 to 80 wt.% With respect. the amount of all proteins. The described after the simplified Cleaning methods obtained solutions can used without further cleaning for coating surfaces become. As a rule, disturb the secondary components do not and influence the coating result at most insignificant.

The obtained hydrophobin solutions usually a concentration of 0.1 mg / ml to 50 mg / ml of fusion hydrophobins on.

The Fusion hydrophobins can from the solutions also be isolated as a solid. This can be done, for example, in in principle known manner by freeze or spray drying respectively.

In a preferred embodiment According to the invention, the isolation can be effected by spray-drying. The spray drying can be carried out with the chromatographically purified solution, preferably can but also after the simplified cleaning process by treatment the inclusion body (inclusion bodies) obtained solutions be used.

to execution spray-drying can the solutions be neutralized if necessary. A pH range of 7 to 9 has proved to be particularly advantageous.

Farther As a rule, it is advisable to concentrate the starting solutions a bit. Has proven yourself in the starting solution a solids concentration of up to 30% by weight. A solids content of> 5% generally results to a fine powdery product. Subsequently, the solution in spray-dried in a basically known manner. suitable Apparatus for spray drying are commercially available. The optimal spray drying conditions vary with device type and desired throughput. Inlet temperatures of 130 to 180 ° C and outlet temperatures from 50 to 80 ° C have become involved in hydrophobin solutions as cheap exposed. Optionally for spray drying Excipients such as sugar, mannitol, dextran or maltodextrin be used. proven has an amount of 0 to 30 wt.%, Preferably 5 to 20 wt. % of such excipients of the hydrophobin.

to execution the method according to the invention for coating a formulation (F) is used, which at least Water or watery Solvent mixture and a fusion hydrophobin.

suitable aqueous Solvent mixtures include water and one or more water-miscible solvents. The selection of such components is limited only insofar as the fusion hydrophobins and the other components in the mixture sufficiently soluble have to be. As a rule, such mixtures comprise at least 50% by weight, preferably at least 65% by weight, and more preferably at least 80% by weight. % Water. Most preferably, only water is used. Of the Professional meets with water-miscible solvents depending on the desired Properties of formulation F a suitable choice. Examples suitable water-miscible solvents include monoalcohols such as methanol, ethanol or propanol, higher alcohols such as ethylene glycol or polyether polyols and ether alcohols such as butyl glycol or methoxypropanol.

According to the invention the formulation used for the treatment has a pH ≥ 4, preferably ≥ 6 and especially preferably ≥ 7 on. For example, the pH may be 4, 5, 6, 7, 8, 9, 10, 11. In particular, the pH is in the range of 4 to 11, preferably 6 to 10, more preferably 7 to 9.5, and most preferably For example, the pH may be 7.5 to 8.5 or 8.5 to 9.

to Adjustment of the pH preferably comprises the formulation suitable buffer. The expert selects depending on the coating provided a suitable buffer pH range. To call are for example potassium dihydrogen phosphate buffer, tris (hydroxymethyl) aminomethane buffer (Tris buffer), Borax buffer, sodium bicarbonate buffer or sodium hydrogen phosphate buffer. Preferred is Tris buffer.

The Concentration of the buffer in the solution will vary depending on the professional the wished Characteristics of the formulation determined. The expert is in the Usually pay attention to a sufficient buffer capacity, if possible to achieve constant coating conditions. Has proven a concentration of 0.001 mol / l to 1 mol / l, preferably 0.005 mol / l to 0.1 mol / l and particularly preferably 0.01 mol / l to 0.05 mol / l.

Farther For example, the formulation comprises at least one fusion hydrophobin. Fusion hydrophobins as well Preferred fusion hydrophobins have already been mentioned at the beginning. Of course can also mixtures of different fusion Hydro-phobine be used. Especially suitable for execution In the present invention, the fusion hydrophobin is yaad-Xa-dewA-his (SEQ ID NO: 20), or derived proteins in which the Fusion partner yaad shortens is.

The Concentration of the fusion hydrophobins in the solution will depend on the skilled person the wished Properties of the coating chosen. With higher concentrations can be usually achieve a faster coating. Has proven As a rule, a concentration of 0.1 ug / ml to 1000 ug / ml, preferably 1 μg / ml up to 500 μg / ml, particularly preferably 10 μg / ml up to 250 μg / ml, most preferably 30 μg / ml to 200 μg / ml and for example 50 to 100 μg / ml.

The Formulation F can about it optionally optionally comprise further components or additives.

Examples of additional components include surfactants. Suitable surfactants are, for example, nonionic surfactants which comprise polyalkoxy groups, in particular polyethylene oxide groups. Examples include polyoxyethylene stearates, alkoxylated phenols and the like. Further examples of suitable surfactants include polyethyleneglycol (20) sorbitan monolaurate (Tween ^® 20), polyethyleneglycol (20) sorbitan monopalmitate (Tween ^® 40), polyethyleneglycol (20) sorbitan monostearate (Tween ^® 60), polyethyleneglycol (20) sorbitan monooleate (Tween ^® 80), cyclohexyl -methyl-β-D-maltoside, cyclohexyl-ethyl-β-D-maltoside, cyclohexyl-n-hexyl-β-D-maltoside, n-undecyl-β-D-malto-sid, n-octyl-β-D-maltopyranoside, n-octyl -β D-glucopyranoside, n-octyl-α D-glucopyranoside, n-dodecanoylsucrose. Further surfactants are disclosed, for example, in WO 2005/68087 page 9, line 10 to page 10, line 2. The concentration of surfactants is generally 0.001% by weight to 0.5% by weight, preferably 0.01% by weight to 0.25% by weight and particularly preferably 0.1% by weight to 0.2% by weight. %, in each case based on the amount of all components of the formulation.

Furthermore, it is also possible to add metal ions, in particular divalent metal ions, to the formulation. Metal ions can contribute to a more uniform coating. Examples of suitable divalent metal ions include, for example, alkaline earth metal ions such as Ca ²⁺ ions. Such metal ions may preferably be added as formulation-soluble salts, for example in the form of chlorides, nitrates or carbonate, acetate, citrate, gluconate, hydroxide, lactate, sulfate, succinate, tartrate. For example, CaCl ₂ or MgCl _{2 may be} added. The solubility can optionally also be increased by suitable auxiliaries, for example complexing agents. If present, the concentration of such metal ions is generally 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.

at additional Components may also be naturally occurring hydrophobins, which are used in admixture with the fusion hydrophobins.

For the preparation of the formulations F, in principle, those solutions can be used which are incurred in the preparation or processing of the hydrophobins. It may be both chromatographically purified fusion hydrophobins, or to the solutions which are obtained by completing the inclusion bodies. Such solutions may also white in addition to the fusion hydrophobin Tere components from the workup contain, for example, buffers, residues of the aids used for elution or auxiliaries from the spray drying. Unless such components interfere with the coating process, they need not be removed.

The solutions from the workup usually have a significantly higher hydrophobin concentration on, as required for coating. You can add water, other, water-miscible solvents or buffer solutions the desired Diluted concentration become.

In a preferred embodiment of the method are used to prepare the formulation F solid fusion hydrophobins used, preferably the above, by spray drying prepared fusion hydrophobins. Particularly advantageous can be the spray-dried Dissolve fusion hydrophobin in water or in the aqueous solvent mixture. This is a distinct advantage to solid, naturally occurring hydrophobins, which in the prior art using trifluoroacetic acid (TFA) or formic acid solved Need to become. TFA / formic acid but is for the coating of a number of substrates undesirable, so that TFA or formic acid after loosening the hydrophobin again consuming must be removed.

Further Components can solved in the formulation be, for example, by simply stirring. Of course it is it also possible additional To trigger components and then the solutions too unite. Various spray dried Materials can be mixed before dissolving. The spray-dried Fusion hydrophobin may also be added in a further step with additional Components are provided, e.g. by spraying other compounds and followed by Dry. Conversely, fusion hydro-phobin can also be applied to existing ones Particles of excipients are applied. A modification of the spray-dried Hydrophobins e.g. in the form of granulation is also possible.

According to the invention is the Coating treated the surface to be coated with the formulation.

The Selection of surfaces is not limited here. It may be, for example, the surfaces of moldings such as Act plates, foils or the like. The surfaces can, for example made of plastics such as Teflon, polyethylene, polypropylene, polystyrene or other polymeric materials, of metals such as Steel, aluminum, zinc, tin, copper or metal alloys like for example, brass, made of natural or changed natural Materials such as leather, textiles (e.g., cotton), Paper, as well as for the cosmetically relevant surfaces (e.g., skin, hair, teeth, Mucous membranes) or made of glass or ceramic materials. To be coated objects can also surfaces made of different materials, for example combinations made of glass, metal and plastics.

The Method for the treatment of the surface is depending on the expert the type of surface selected. For example, the article to be coated in the formulation be dipped, or the formulation can be sprayed on the surface be applied. This type of surface treatment is suitable as well as level as for irregular shaped Surfaces. extensive Shaped body like For example, plates or films can also be advantageous be treated by coating or rolling. Excess formulation can be removed again by means of suitable methods, for example by doctoring or crimping. Particularly preferably, the coating by spraying be made. Suitable spray apparatuses are those skilled in the art known.

In Typically, a certain exposure time is required to complete the fusion hydrophobin on the surface deposit. The specialist chooses depending on the desired Result a suitable exposure time. Examples of typical exposure times are from 0.1 to 12 h, without the invention being limited thereto should.

in the The rule is the exposure time of the temperature and the Concentration of the fusion hydrophobin in solution dependent. The higher the temperature and the higher the Concentration in the course of the coating process, the shorter it can be be the exposure time. The temperature in the course of the coating process may be at room temperature or it may be elevated temperatures act. For example, it can be temperatures of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 ° C act. Prefers it is temperatures of 15 to 120 ° C, more preferably 20 to 100 ° C, and for example 40 to 100 ° C or 70 to 90 ° C. The temperature may be, for example, by heating the bath in which the object to be coated is immersed. But you can also heat a submerged object later, for example using IR emitters.

To The coating is still in the coating existing solvent removed from the coating. This can be done, for example, by simple Evaporate in air. The removal of the solvent can also be through Increase the temperature and / or with suitable gas streams and / or applying a Vacuum be relieved. The evaporation can be relieved for example, by placing coated objects in a drying oven heated or with a heated one Gas stream blows. The methods can also be combined, for example by placing in a convection oven or a drying channel dries. Furthermore, the coating for removing the solvent also be heated by means of radiation, in particular IR radiation. You can do this All types of broadband IR emitters, for example. NIR, MIR or NIR steelers are used. But it can also, for example IR laser can be used. Such radiation sources are in various radiation geometries commercially available.

The Temperature and the drying time in the course of drying is from the Specialist. Has proven a drying temperature of 30 to 130 ° C, preferably 50 to 120 ° C, especially preferably 70 to 110 ° C, most preferably 75 to 105 ° C and for example 85 to 100 ° C. Meant Here is the temperature of the coating itself. The temperature in a dryer can of course be higher. The Drying time is naturally shorter, depending higher the Drying temperature is.

The Temperature treatment in the course of coating and drying can be advantageously combined with each other. So, for example a surface first be treated with the formulation F at room temperature and then at increased Temperatures are dried and tempered. In a preferred embodiment of the process is elevated in at least one of the two steps "treatment" or "drying" elevated temperature applied. Preferably, higher temperature is used in both steps.

By treating the surface with the method according to the invention is a fusion hydrophobin coated surface available, which is the material of the surface and includes a layer immediately adjacent thereto, the at least one fusion hydrophobin and possibly other constituents having the formulation. It can hereby use the entire surface be covered by the hydrophobin, or even only part of the surface. The quality can be judged by various methods, for example by means of the already mentioned Contact angle measurement. The contact angle changes as in the case of the coating with course occurring hydrophobins clearly. Further methods are the expert from known in the art cited above (e.g., "AFM" atomic force microscopy for direct detection of the protein layer on the surface).

The fusion hydrophobin layer may be further chemically modified before or after removal of the solvent. For example, it is possible to crosslink the layer using suitable crosslinkers. Examples of suitable crosslinkers include glutardialdehyde, formaldehyde, and other homo and heterobifunctional protein crosslinkers known in protein chemistry. As a result, the stability of the layer can be increased. In the case of protein-containing substrates such as, for example, leather, certain textiles, as well as surfaces relevant to cosmetics, the connection to the substrate can additionally be improved. The crosslinking can be carried out, for example, by treating the layer with the fusion hydrophobin after coating with a second solution with the crosslinker and then drying. Furthermore, it is also possible to pretreat protein-containing but also other substrates such that protein-reactive functional groups are formed on the surface of the substrate. This can be used, for example, the above-mentioned crosslinkers, but other chemicals such as ozone, peroxides or aldehydes. Another possibility is a coupling or amplification of the coupling via metal ions. Corresponding protein sequences with affinity for metal ions are known to the person skilled in the art (eg His ₆ to Ni, Co, Fe, etc.) and can be attached to the hydrophobins by means of standard molecular biological techniques or protein-chemical coupling. The metal ions may be coupled in advance to the surface to be coated or used simultaneously with the Hydrophobinkopplung.

The The following examples are intended to illustrate the invention in more detail:

Part A) Preparation of the used according to the invention Fusion hydrophobins

example 1

Preliminary work for the cloning of yaad-His ₆ / yaaE-His ₆

Using the oligonucleotides Hal570 and Hal571 (Hal 572 / Hal 573), a polymerase chain reaction was carried out. As template DNA genomic DNA of the bacterium Bacillus subtilis was used. The PCR fragment obtained contained the coding sequence of the gene yaaD / yaaE from Bacillus subtilis, and at the ends in each case an NcoI or BglII restriction cleavage site. The PCR fragment was purified and cut with the restriction endonucleases NcoI and BglII. This DNA fragment was used as an insert and cloned into the vector pQE60 from Qiagen, previously linearized with the restriction endonucleases NcoI and BgIII. The thus obtained vectors pQE60YAAD # 2 / pQE60YaaE # 5 can be used to express proteins consisting of, YAAD :: HIS ₆ and YAAE :: HIS ₆ , respectively.
Hal570: gcgcgcccatggctcaaacaggtactga
Hal571: gcagatctccagccgcgttcttgcatac
Hal572: ggccatgggattaacaataggtgtactagg
Hal573: gcagatcttacaagtgccttttgcttatattcc

Example 2

Cloning of yaad hydrophobin DewA-His ₆

With The oligonucleotides KaM 416 and KaM 417 became a polymerase Chain reaction performed. The template DNA was genomic DNA of the mold Aspergillus used nidulans. The resulting PCR fragment contained the coding Sequence of the hydrophobin gene dewA and an N-terminal factor Xa Proteinase interface. The PCR fragment was purified and washed with the restriction endonuclease BamHI cut. This DNA fragment was used as an insert, and previously with the restriction endonuclease BglII linearized vector pQE60YAAD # 2 cloned.

The thus constructed vector # 508 can be used to express a fusion protein consisting of, YAAD :: Xa :: dewA :: HIS ₆ .
KaM416: GCAGCCCATCAGGGATCCCTCAGCCTTGGTACCAGCGC
KaM417: CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCGC

Example 3

Cloning of yaad hydrophobin RodA-His ₆

The cloning of plasmid # 513 was carried out analogously to plasmid # 508 using the oligonucleotides KaM 434 and KaM 435.
KaM434: GCTAAGCGGATCCATTGAAGGCCGCATGAAGTTCTCCATTGCTGC
KaM435: CCAATGGGGATCCGAGGATGGAGCCAAGGG

Example 4

Cloning of yaad hydrophobin BASF1-His ₆

The Cloning of plasmid # 507 was carried out analogously to plasmid # 508 Use of the oligonucleotides KaM 417 and KaM 418.

The template DNA used was an artificially synthesized DNA sequence - hydrophobin BASF1 (see Appendix).
KaM417: CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCGC
KaM418: CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG

Example 5

Cloning of yaad hydrophobin BASF2-His ₆

The Cloning of plasmid # 506 was carried out analogously to plasmid # 508 Use of the oligonucleotides KaM 417 and KaM 418.

The template DNA used was an artificially synthesized DNA sequence - hydrophobin BASF2 (see Appendix).
KaM417: CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCGC
KaM418: CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG

Example 6

Cloning of yaad hydrophobin SC3-His ₆

The Cloning of plasmid # 526 was carried out analogously to plasmid # 508 Use of the oligonucleotides KaM464 and KaM465.

The template DNA used was Schyzophyllum commune cDNA (see Appendix).
KaM464: CGTTAAGGATCCGAGGATGTTGATGGGGGTGC
KaM465: GCTAACAGATCTATGTTCGCCCGTCTCCCCGTCGT

Example 7

Fermentation of the recombinant E. coli strain yaad hydrophobin DewA-His ₆

Inoculate 3 ml of LB liquid medium with a yaad-hydrophobin DewA-His ₆ expressing E. coli strain in 15 ml Greiner tubes. Incubate for 8 h at 37 ° C on a shaker at 200 rpm. 2 1 l Erlenmeyer flasks with baffles and 250 ml LB medium (+ 100 μg / ml ampicillin) are inoculated with 1 ml each of the preculture and incubated for 9 h at 37 ° C. on a shaker with 180 rpm.

13.5 l LB medium (+100 μg / ml ampicillin) in a 20 l fermenter with 0.5 l preculture (OD _600nm 1:10 measured against H ₂ O). At an OD _60nm of ~ 3.5 add 140ml 100mM IPTG. After 3 h fermenter, cool to 10 ° C and centrifuge the fermentation broth. Use cell pellet for further purification.

Example 8

Purification of the recombinant Hydrophobin Fusion Protein

(Purification of hydrophobin fusion proteins, having a C-terminal His6 tag)

100 g cell pellet (100-500 mg hydrophobin) are made up to 200 ml total volume with 50 mM sodium phosphate buffer, pH 7.5 and resuspended. The suspension is treated with an Ultraturrax type T25 (Janke and Kunkel, IKA-Labortechnik) for 10 minutes and then for 1 hour at room temperature with 500 units of benzonase (Merck, Darmstadt, Order No. 1.01697.0001) to break down the nucleic acids incubated. Before cell disruption, filter with a glass cartridge (P1). For homogenization of the cells and for shearing the remaining genomic DNA, two homogenizer runs are carried out at 1500 bar (Microfluidizer M-110EH, Microfluidics Corp.). The homogenate is centrifuged (Sorvall RC-5B, GSA rotor, 250 ml centrifuge beaker, 60 minutes, 4 ° C, 12,000 rpm, 23,000 g), the supernatant placed on ice and the pellet resuspended in 100 ml sodium phosphate buffer, pH 7.5 , Centrifugation and resuspension are repeated 3 times with the sodium phosphate buffer containing 1% SDS at the third repetition. After resuspension, stir for one hour and perform a final centrifugation (Sorvall RC-5B, GSA rotor, 250 ml centrifuge beaker, 60 minutes, 4 ° C, 12,000 rpm, 23,000 g). According to SDS-PAGE analysis, the hydrophobin is contained in the supernatant after the final centrifugation ( 1 ). The experiments show that the hydrophobin is probably contained in the form of inclusion bodies in the corresponding E. coli cells. 50 ml of the hydrophobin-containing supernatant are applied to a 50 ml Nickel-Sepharose High Performance 17-5268-02 column (Amersham) equilibrated with 50 mM Tris-Cl pH 8.0 buffer. The column is washed with 50 mM Tris-Cl pH 8.0 buffer and the hydrophobin subsequently eluted with 50 mM Tris-Cl pH 8.0 buffer containing 200 mM imidazole. To remove the imidazole is the Solution dialyzed against 50 mM Tris-Cl pH 8.0 buffer.

1 shows the purification of the prepared fusion hydrophobin:
Lane 1: Order nickel-Sepharose column (1:10 dilution)
Lane 2: run = eluate wash step
Lanes 3 - 5: OD 280 maxima of the elution fractions

The fusion hydrophobin of 1 has a molecular weight of about 53 kD. The smaller bands partially represent degradation products of hydrophobin.

Example 9

simplified cleaning process

Of the E. coli cell pellet in water obtained in Example 7 is 1000 bar through a nozzle pressed. The cells are completely disrupted. through Centrifugation is obtained in inclusion bodies (inclusion bodies) Hydrophobin be separated from the remaining cell debris. at A g-number of 5000 separate 2 phases after 30 minutes. The lower, fusion hydrophobin-containing phase is redone suspended with water and centrifuged as above. The inclusion bodies are subsequently in 0.1 M NaOH for Incubated for 60 minutes and so completely dissolved. The pH is raised with phosphoric acid 8 and the protein concentration adjusted to 20 mg / ml. The purity (in terms of total protein) of the fusion hydrophobin thus produced is 70%.

Example 10

Spray drying of hydrophobin

The obtained in Example 9 Hydrophobinlösung is in a commercial spray dryer further processed.

The Spray drying with an addition of 10% w / w mannitol at an inlet temperature of 160 ° C and Outlet temperatures of 70 ° C. A finely powdered product was obtained.

Example 11

Application testing; characterization of the fusion hydrophobin by changing the contact angle of a water drop on glass

substrate:

Glass (window glass, South German Glass, Mannheim):

• - Spray-dried hydrophobin according to Example 10 is received (8 + 0.1 mM CaCl ₂ (final concentration) + 0.1% polyoxyethylene (20) sorbitan monolaurate (Tween ^® 20) 50 mM Tris, pH) in an aqueous buffer solution and at a concentration of 100 μg / mL
• - Incubation of glass slides overnight (Temperature 80 ° C) then wash the coating in distilled water
• - after that Incubation 10min / 80 ° C / 1% Sodium dodecyl sulfate (SDS) solution in dest. water
• - To wash in dest. water

The Samples are dried in air and the contact angle (in degrees) a drop of 5 μl of water determined at room temperature.

The Contact angle measurement was done on a Dataphysics Contact Angle device System OCA 15+, Software SCA 20.2.0. (November 2002). The Measurement was carried out according to the manufacturer's information.

Untreated Glass gave a contact angle of 30 ± 5 °; the coated glass had one Contact angle of 75 ± 5 ° on.

Example 12

coating tests with the fusion hydrophobin by spraying:

1. Spray from Polyethylene plates:

For the experiments became one according to example 8 solution obtained from yaad-xa-dewA-his (SEQ ID NO: 20) used. The solution also contained sodium phosphate buffer at one concentration of 50 mM. The concentration of fusion hydrophobin in the solution was 11.23 mg / ml, the pH of the solution 7.5.

Farther became the according to the simplified Cleaning procedure according to example 9 solution obtained used.

For the spray experiments, the solutions were diluted about 100-fold to a concentration of 100 μg / ml fusion hydrophobin. For dilution, the following solutions or solvents were used in each case:

These solutions 10-1 to 10-5 were then sprayed on polyethylene (Simona ^® PE-HWU) with a laboratory sprayer (Desaga SG1) to form a thin, even film on the surface. This liquid film dried completely within 2 h at RT. After a rest period of a further 2 hours, the plates were rinsed thoroughly with plenty of water and dried in air overnight.

to Assessment of quality The contact angle of the coated surface was measured as described above. Furthermore, the film formation of water on the surface became optical assessed. The results are summarized in Table 1.

Tab. 1: Beschichtung von Polyethylen-Platten mit Fusions-Hydrophobinen

Tab. 1: Coating of polyethylene plates with fusion hydrophobins

With all solutions the fusion hydrophobins settled a hydrophilization of the surfaces to reach. This is the effect with a buffered solution, though without surfactant most clearly.

2. Spray from aluminum sheets

For the experiments were commercially available Aluminum sheets used (Elastogran).

In the same manner as described above, the aluminum sheets were sprayed with the solution 10-1 (water only) and 10-2 (fusion hydrophobin in Tris buffer), dried and rinsed with demineralized water. The consumption of solution was 150 mL EV 60646 (100 μg / ml) for 1.2m ² sheet, this corresponds to about 12.5 mg hydrophobin / m ² .

Tab. 2: Beschichtung von Aluminium-Platten mit Fusions-Hydrophobinen

Tab. 2: Coating of aluminum plates with fusion hydrophobins

It is a slight hydrophobing of the aluminum surface by means of To recognize contact angle measurement. Regarding the film formation of Water on the aluminum surface a clear modification is evident.

The two differently processed solutions of the fusion hydrophobin do not differ in their effectiveness.

Example 13

Cross-linking of surfaces and hydrophobin

Substrate: Leather (Wet Blue)

• - Spray dried Hydrophobin is absorbed in water and concentrated of 100 μg / mL adjusted
• - incubation of leather pieces overnight (at room temperature) in 50 mM Tris pH 8 + 0.1 mM CaCl ₂ (final concentration) + 0.1% polyoxyethylene (20) sorbitan monolaurate (Tween ^® 20)
• - after that Wash the coating in distilled water
• - after that Incubation 10min / 80 ° C / 1% Sodium dodecyl sulfate (SDS) solution in dest. water
• - To wash in dest. water
• - Incubation with 0.01% glutaraldehyde solution in water (2 hours at room temperature)
• - To wash in dest. water

It comes to a significant hydrophilization of the leather by the cross-linking additional mechanical stability wins. The hydrophilization can be carried out in a known manner by means of Water drop recording to be determined. While a drop of water on Untreated leather took about 4 minutes to move in, moved in same size Drops of water into the hydrophobin treated within less than 1 minute Leather.

Example 15

desiccation by means of IR radiation

substrate:

Glass (window glass, South German Glass, Mannheim):

• - Spray dried Hydrophobin according to example 9 is taken up in 10mM Tris pH 8 and adjusted to a concentration of 50 μg / mL set.
• - Glass slides are with the hydrophobin solution wetted and by means of IR radiation (IR125R from the Philips brand) dried within 10 min. Temperature of the surface about 100 to 120 ° C

Of the Contact angle (in degrees) of a drop of 5 μl of water is at room temperature certainly.

The Contact angle measurement was done on a Dataphysics Contact Angle device System OCA 15+, Software SCA 20.2.0. (November 2002). The Measurement was carried out according to the manufacturer's information.

Untreated Glass gave a contact angle of 30 ± 5 °; the treated glass gave a contact angle of 75 ± 15 °.

Assignment of sequence names to DNA and polypeptide sequences in the sequence listing

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (13)

Process for coating surfaces with hydrophobins comprising at least the following process steps: (1) providing a formulation (F) comprising water or an aqueous solvent mixture and a hydrophobin, (2) treating the surface with the formulation, and (3) removing the solvent, characterized in that the hydrophobin is a fusion hydrophobin and the formulation has a pH ≥ 4. Method according to claim 1, characterized in that the formulation has a pH ≥ 7. Method according to claim 1 or 2, characterized in that the formulation continues includes a buffer. Method according to one the claims 1 to 3, characterized in that the formulation by Release from solid fusion hydrophobin. Method according to claim 4, characterized in that it is spray-dried fusion hydrophobin is. Method according to one the claims 1 to 3, characterized in that for the preparation of the formulation a solution which by separating the cells from the fermentation broth, digestion the cells as well as dissolve the inclusion body (inclusion bodies) is produced. Method according to one the claims 1 to 6, characterized in that the removal of the solvent at 15 to 120 ° C performs. Method according to one the claims 1 to 6, characterized in that the coating at 20 up to 100 ° C performs. Method according to one the claims 1 to 8, characterized in that the coating in one additional Networked process step. Method according to one the claims 1 to 9, characterized in that it is the fusion hydrophobin yaad-Xa-dewA-his6 (SEQ ID NO: 20), or a protein with a shortened yaad fusion partner is. Surface, comprising a coating comprising at least one fusion hydrophobin. surface according to claim 11, characterized in that the coating is crosslinked. surface according to claim 11 or 12, characterized in that it is the fusion hydrophobin yaad-Xa-dewA-his (SEQ ID NO: 20), or a protein with a shortened yaad merger partner.