# Patent

## Title
Use of polypeptides in the form of adhesive agents

## Abstract

A multilayered composite or coated substrate, comprising compounds of which at least 40% by weight are composed of alpha-amino acids linked via peptide bonds as adhesion promoters between at least two adjacent layers of the composite or between the coating and the substrate.

## Background
RELATED APPLICATIONS
This application is a national stage application under 35 U.S.C. §371 of PCT/EP2006/061085, filed Mar. 28, 2006, which claims priority to German application 10 2005 015 043.8, filed Mar. 31, 2005, and German application 10 2005 045 770.3, filed Sep. 23, 2005.
The invention relates to a multilayered composite or a coated substrate, comprising compounds of which at least 40% by weight are composed of alpha-amino acids linked via peptide bonds (referred to as polypeptide for short hereinbelow) as adhesion promoters between at least two adjacent layers of the composite or between the coating and the substrate. More specifically, the invention relates to the use of hydrophobins as adhesion promoters.
Hydrophobins are small proteins of from about 100 to 150 amino acids, which are characteristic for filamentous fungi, for example Schizophyllum commune. They most usually have 8 cysteine units.
Hydrophobins have a marked affinity for interfaces and are therefore suitable for coating surfaces. Thus it is possible to coat, for example, Teflon by means of hydrophobins to obtain a hydrophilic surface.
Hydrophobins may be isolated from natural substances. Our previous application, DE 10 2005 007 480.4, discloses a process for preparing hydrophobins.
The use of hydrophobins for various applications has been proposed in the prior art.
WO 96/41882 proposes the use of hydrophobins as emulsifiers, thickeners, surfactants, for hydrophilizing hydrophobic surfaces, for improving the water resistance of hydrophilic substrates, for preparing oil-in-water emulsions or water-in-oil emulsions. Pharmaceutical applications such as the preparation of ointments or creams and also cosmetic applications such as skin protection or the preparation of hair shampoos or hair rinses are also proposed.
EP 1 252 516 discloses the coating of windows, contact lenses, biosensors, medical apparatus, containers for carrying out experiments or for storage, ship hulls, solid particles or the chassis or bodywork of passenger vehicles with a hydrophobin-containing solution at a temperature from 30 to 80° C.
WO 03/53383 discloses the use of hydrophobin for treating keratin materials in cosmetic applications.
WO 03/10331 discloses a hydrophobin-coated sensor, for example a measuring electrode, to which further noncovalent substances, for example electroactive substances, antibodies or enzymes, have been attached.
Previously, very different adhesion promoters have been used for improving the adhesion of, for example, coatings to a large variety of substrates. Suitable are, according to Römpp Chemie Lexikon (1990 edition), for example, titanates, silanes, chromium complexes of unsaturated carboxylic acids. Specially mentioned adhesion promoters for adhesives are ethylene/acylamide copolymers, polymeric isocyanates or reactive organosilicon compounds.
Polyurethanes and polyethyleneimines are also known adhesion promoters.
The object of the present invention was to provide alternative adhesion promoters which have very good application properties and which effect, in particular, good adhesion of the individual layers of a multilayered composite or of a coating on a substrate.
Accordingly, the multilayered composite or the coated substrate, as defined at the outset, were found.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows the results of the performance tests using steel sheets as metallic substrates.
FIG. 2 shows the results of the performance tests using Galvanized steel sheets as metallic substrates.
FIG. 3 shows the results of the performance tests using aluminum sheets as metallic substrates.



THE ADHESION PROMOTER
The multilayered composite or the coated substrate comprises the polypeptide defined at the outset as an adhesion promoter.
The polypeptide consists of at least 40% by weight, preferably at least 70% by weight, particularly preferably at least 90% by weight, and very particularly preferably at least 95 or 99% by weight, of alpha-amino acids linked via peptide bonds.
In a particular embodiment, the polypeptide consists exclusively of alpha-amino acids linked via peptide bonds.
Particularly suitable alpha-amino acids are glycine, alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, proline, hydroxyproline, serine, threonine, cysteine, cystine, methionine, tryptophan, aspartic acid, glutamic acid, arginine, lysine and histidine.
The polypeptide preferably comprises the alpha-amino acid cysteine in a mixture with other alpha-amino acids.
The polypeptide particularly preferably consists of at least 0.1% by weight, particularly preferably at least 0.5, very particularly preferably at least 1% by weight, of cysteine. The cysteine content in the polypeptide generally does not exceed 15% by weight, in particular 10% by weight, and very particularly preferably does not exceed 7% by weight.
In a particular embodiment, the polypeptides are hydrophobins.
The term “hydrophobins” in accordance with the present invention means hereinbelow proteins of the general structural formula (I)

Xn—C1—X1-50—C2—X0-5—C3—X1-100—C4—X1-100—C5—X1-50—C6—X0-5—C7—X1-50—C8—Xm  (I),

where X may be any 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 also in each case be identical or different. The indices next to X indicate in each case the number of amino acids, C is cysteine, alanine, serine, glycine, methionine or threonine, with at least four of the residues denoted C being cysteine, and the indices n and m are independently of one another natural numbers of 0 and 500, preferably from 15 to 300.

The polypeptides according to formula (I) are furthermore characterized by the property of their increasing, at room temperature, after coating of a glass surface, the contact angle of a water drop by at least 20°, preferably at least 25° and particularly preferably 30°, in each case compared to the contact angle of a water drop of similar size with the uncoated glass surface.
The amino acids denoted C1 to C8 are preferably cysteines; they may, however, also be replaced with other amino acids of similar spatial dimensions, preferably alanine, serine, threonine, methionine or glycine. However, at least four, preferably at least 5, particularly preferably at least 6 and in particular at least 7, of the positions C1 to C8 should consist of cysteines. Cysteines may either be in a reduced state or may form disulfide bridges between each other. Particular preference is given to the intramolecular formation of C—C bridges, in particular that with at least one, preferably 2, particularly preferably 3 and very particularly preferably 4, intramolecular disulfide bridges. The above-described substitution of cysteines by amino acids of similar spatial dimensions, involves advantageously substituting in pairs those C positions which can form intramolecular disulfide bridges between each other.
If cysteines, serines, alanines, glycines, methionines or threonines are also used in the positions indicated by X, the numbering of the individual C positions in the general formulae may change accordingly.
Preference is given to employing hydrophobins of the general formula (II)

Xn—C1—X3-25—C2—X0-2—C3—X5-50—C4—X2-35—C5—X2-15—C6—X0-2—C7—X3-35—C8—Xm  (II),

to carry out the present invention, wherein X, C and the indices next to X and C are as defined above, but the indices n and m are numbers between 0 and 300 and the proteins are furthermore distinguished by the abovementioned contact angle change.

Particular preference is given to employing hydrophobins of the formula (III)

Xn—C1—X5-9—C2—C3—X11-39—C4—X2-23—C5—X5-9—C6—C7—X6-18—C8—Xm  (III),

wherein X, C and the indices next to X and C are as defined above, the indices n and m are numbers between 0 and 200 and the proteins are furthermore distinguished by the abovementioned contact angle change, and at least 6 of the residues denoted C are cysteines. Particular preference is given to all residues C being cysteines.

The residues Xn and Xm may be peptide sequences which are naturally linked to a hydrophobin. However, either or both residues may also be peptide sequences which are not naturally linked to a hydrophobin. This also includes those residues Xn and/or Xm in which a peptide sequence naturally occurring in a hydrophobin has been extended by a peptide sequence which does not naturally occur in a hydrophobin.
If Xn and/or Xm are peptide sequences which are not naturally linked to hydrophobins, such sequences are usually at least 20, preferably at least 35, particularly preferably at least 50 and very particularly preferably at least 100, amino acids in length. A residue of this kind which is not naturally linked to a hydrophobin will also be referred to as fusion partner hereinbelow. This is intended to express the fact that the proteins can consist of at least one hydrophobin part and one fusion partner which in nature do not occur together in this form.
The fusion partner may be selected from a multiplicity of proteins. It is also possible for a plurality of fusion partners to be linked to one hydrophobin part, for example to the amino terminus (Xn) and to the carboxy terminus (Xm) of said hydrophobin part. However, it is also possible to link, for example, two fusion partner parts to one position (Xn or Xm) of the protein of the invention.
Particularly suitable fusion partner parts are proteins which occur naturally in microorganisms, in particular in E. coli or Bacillus subtilis. Examples of such fusion partner parts are the sequences yaad (SEQ ID NO:15 and 16), yaae (SEQ ID NO: 17 and 18), and thioredoxin. Fragments or derivatives of said sequences, which comprise only a part, preferably 70-99%, particularly preferably 80-98%, of said sequences or in which individual amino acids or nucleotides have been altered compared to the sequence mentioned, are also well suited, with the percentages given referring in each case to the number of amino acids.
It is furthermore also possible that the polypeptide sequence of the proteins used according to the invention has been modified, for example by glycosylation, acetylation or else by chemical crosslinking, for example with glutaraldehyde.
One characteristic of the proteins used according to the invention is the change in surface properties when the surfaces are coated with said proteins. The change in surface properties can be determined experimentally by measuring the contact angle of a water drop before and after coating of the surface with the protein and determining the difference of the two measurements.
The measurement of contact angles is known in principle to the skilled worker. The measurements are based on room temperature and water drops of 5 l. The precise experimental conditions for a method, suitable by way of example, of measuring the contact angle are illustrated in the experimental section. Under the conditions mentioned there, the proteins used according to the invention have the property of increasing the contact angle by at least 20°, preferably at least 25°, particularly preferably at least 30°, in each case compared to the contact angle of a water drop of similar size with the uncoated glass surface.
The positions of the polar and nonpolar amino acids in the hydrophobin part of the hydrophobins known to date are preserved, resulting in a characteristic hydrophobicity plot. Differences in biophysical properties and hydrophobicity resulted in the classification of the hydrophobins known to date into two classes, I and II (Wessels et al. 1994, Ann. Rev. Phytopathol., 32, 413-437).
The assembled membranes of class I hydrophobins are to a large extent insoluble (even to 1% sodium dodecyl sulfate (SDS) at an elevated temperature) and can only be dissociated again by means of concentrated trifluoroacetic acid (TFA) or formic acid. In contrast, the assembled forms of class II hydrophobins are less stable. They may be dissolved again even by 60% strength ethanol or 1% SDS (at room temperature).
Comparison of the amino acid sequences reveals that the length of the region between cysteine C3 and C4 is distinctly shorter in class II hydrophobins than in class I hydrophobins. Class II hydrophobins furthermore have more charged amino acids than class I.
Hydrophobins which are particularly preferred for carrying out the present invention are those of types dewA, rodA, hypA, hypB, sc3, basf1, basf2 which are structurally characterized in the sequence listing below. They may also be only parts or derivatives of said types. It is also possible to link a plurality of hydrophobin parts, preferably 2 or 3, of the same or a different structure to one another and to a corresponding suitable polypeptide sequence which is not naturally connected to a hydrophobin.
Particularly suitable for carrying out the present invention are furthermore the fusion proteins having the polypeptide sequences indicated in SEQ ID NO: 20, 22, 24 and also the nucleic acid sequences coding therefor, in particular the sequences according to SEQ ID NO: 19, 21, 23. Particularly preferred embodiments are also proteins which, starting from the polypeptide sequences indicated in SEQ ID NO. 20, 22 or 24, result from the substitution, insertion or deletion of at least one, up to 10, preferably 5, particularly preferably 5% of all, amino acids and which still have at least 50% of the biological property of the starting proteins. Biological property of the proteins here means the above-described increase in the contact angle by at least 20°.
The proteins used according to the invention can be prepared chemically by known processes of peptide synthesis, for example by solid phase synthesis according to Merrifield.
Naturally occurring hydrophobins can be isolated from natural sources by means of suitable methods. By way of example, reference is made to Wösten et. al., Eur. J Cell Bio. 63, 122-129 (1994) or WO 96/41882.
Fusion proteins may preferably be prepared by genetic engineering processes in which one nucleic acid sequence, in particular DNA sequence, coding for the fusion partner and one coding for the hydrophobin part are combined in such a way that the desired protein is generated by gene expression of the combined nucleic acid sequence in a host organism. A preparation process of this kind is disclosed in our previous application DE 102005007480.4.
Host organisms (producer organisms) which may be suitable here for the preparation process mentioned are prokaryotes (including Archaea) or eukaryotes, particularly bacteria including halobacteria and methanococci, fungi, insect cells, plant cells and mammalian cells, particularly preferably 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.
The invention moreover relates to the use of expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a polypeptide used according to the invention and also to vectors comprising at least one of these expression constructs.
Constructs used preferably comprise a promoter 5′ upstream of the particular coding sequence and a terminator sequence 3′ downstream and, if appropriate, further customary regulatory elements, in each case operatively linked to the coding sequence.
An “operative linkage” means the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements is able to fulfill its function as required in expressing the coding sequence.
Examples of operatively linkable sequences are targeting sequences and also enhancers, polyadenylation signals and the like. Other regulatory elements comprise selectable markers, amplification signals, origins of replication and the like. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).
In addition to these regulatory sequences, the natural regulation of these sequences may still be present upstream of the actual structural genes and, if appropriate, may have been genetically altered in such a way that the natural regulation has been switched off and expression of the genes has been increased.
A preferred nucleic acid construct also advantageously comprises one or more of the previously mentioned enhancer sequences which are functionally linked to the promoter and which enable expression of the nucleic acid sequence to be increased. Additional advantageous sequences such as further regulatory elements or terminators may also be inserted at the 3′ end of the DNA sequences.
The nucleic acids may be present in the construct in one or more copies. The construct may also comprise additional markers such as antibiotic resistances or auxotrophy-complementing genes, if appropriate for the purpose of selecting said construct.
Regulatory sequences which are advantageous for the process are present, for example, in promoters such as the cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq-T7, T5, T3, gal, trc, ara, rhaP (rhaPBAD)SP6, lambda-PR or in the lambda-P promoter, which promoters are advantageously used in Gram-negative bacteria. Further advantageous regulatory sequences are present, for example, in the Gram-positive promoters amy and SPO2, in the yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.
It is also possible to use artificial promoters for regulation.
For the purpose of expression in a host organism, the nucleic acid construct is advantageously inserted into a vector such as a plasmid or a phage, for example, which enables the genes to be expressed optimally in the host. Vectors mean, in addition to plasmids and phages, also any other vectors known to the skilled worker, i.e., for example, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA, and also the Agrobacterium system.
These vectors may be replicated autonomously in the host organism or replicated chromosomally. These vectors constitute a further embodiment of the invention. Examples of suitable plasmids are, 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. Said plasmids are a small selection of the possible plasmids. Other plasmids are well known to the skilled worker and can be found, for example, in the book Cloning Vectors (Eds. Pouwels P. H. et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
For the purpose of expressing the other genes which are present, the nucleic acid construct advantageously also comprises 3′-terminal and/or 5′-terminal regulatory sequences for increasing expression, which are selected for optimal expression in dependence on the host organism and the gene or genes selected.
These regulatory sequences are intended to enable the genes and protein expression to be specifically expressed. Depending on the host organism, this may mean, for example, that the gene is expressed or overexpressed only after induction or that it is expressed and/or overexpressed immediately.
In this connection, the regulatory sequences or factors may preferably influence positively and thereby increase expression of the genes which have been introduced. Thus, the regulatory elements may advantageously be enhanced at the level of transcription by using strong transcription signals such as promoters and/or enhancers. However, in addition to this, it is also possible to enhance translation by improving the stability of the mRNA, for example.
In a further embodiment of the vector, the vector which comprises the nucleic acid construct of the invention or the nucleic acid of the invention may also advantageously be introduced into the microorganisms in the form of a linear DNA and be integrated into the genome of the host organism by way of heterologous or homologous recombination. This linear DNA may consist of a linearized vector such as a plasmid or only of the nucleic acid construct or the nucleic acid.
In order to express heterologous genes optimally in organisms, it is advantageous to alter the nucleic acid sequences in accordance with the specific codon usage employed in the organism. The codon usage can readily be determined with the aid of computer analyses of other known genes of the organism in question.
An expression cassette is prepared by fusing a suitable promoter to a suitable coding nucleotide sequence and to a terminator signal or polyadenylation signal. Common recombination and cloning techniques, as are described, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) and also in T. J. Silhavy, M. L. Berman and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987), are used for this purpose.
In order to achieve expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables the genes to be expressed optimally in the host. Vectors are well known to the skilled worker and may be found, for example, in “Cloning Vectors” (Pouwels P. H. et al., Eds., Elsevier, Amsterdam-New York-Oxford, 1985).
It is possible to prepare, with the aid of the vectors, recombinant microorganisms which are, for example, transformed with at least one vector and which may be used for producing the proteins used according to the invention. Advantageously, the above-described recombinant constructs of the invention are introduced into a suitable host system and expressed. In this connection, familiar cloning and transfection methods known to the skilled worker, such as, for example, coprecipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used in order to cause said nucleic acids to be expressed in the particular expression system. Suitable systems are described, 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. 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
It is also possible to prepare homologously recombined microorganisms. For this purpose, a vector which comprises at least one section of a gene to be used according to the invention or of a coding sequence in which, if appropriate, at least one amino acid deletion, amino acid addition or amino acid substitution has been introduced in order to modify, for example functionally disrupt, the sequence (knockout vector), is prepared. The introduced sequence may, for example, also be a homolog from a related microorganism or be derived from a mammalian, yeast or insect source. Alternatively, the vector used for homologous recombination may be designed in such a way that the endogenous gene is, in the case of homologous recombination, mutated or otherwise altered but still encodes the functional protein (e.g. the upstream regulatory region may have been altered in such a way that expression of the endogenous protein is thereby altered). The altered section of the gene used according to the invention is in the homologous recombination vector. The construction of vectors which are suitable for homologous recombination is described, for example, in Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503.
Recombinant host organisms suitable for the nucleic acid used according to the invention or the nucleic acid construct are in principle any prokaryotic or eukaryotic organisms. Advantageously, microorganisms such as bacteria, fungi or yeasts are used as host organisms. Gram-positive or Gram-negative bacteria, preferably bacteria of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae, particularly preferably bacteria of the genera Escherichia, Pseudomonas, Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium or Rhodococcus, are advantageously used.
The organisms used in the process of preparing fusion proteins are, depending on the host organism, grown or cultured in a manner known to the skilled worker. Microorganisms are usually grown in a liquid medium which comprises a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as iron salts, manganese salts and magnesium salts and, if appropriate, vitamins, at temperatures of between 0° C. and 100° C., preferably between 10° C. and 60° C., while being supplied with oxygen. In this connection, the pH of the nutrient liquid may or may not be kept at a fixed value, i.e. may or may not be regulated during cultivation. The cultivation may be carried out batchwise, semibatchwise or continuously. Nutrients may be initially introduced at the beginning of the fermentation or be fed in subsequently in a semicontinuous or continuous manner. The enzymes may be isolated from the organisms by the process described in the examples or be used for the reaction as a crude extract.
Proteins used according to the invention or functional, biologically active fragments thereof may be prepared by means of a recombinant process, with a protein-producing microorganism being cultured, expression of the proteins being induced if appropriate and said proteins being isolated from the culture. The proteins may also be produced in this way on an industrial scale if this is desired. The recombinant microorganism may be cultured and fermented by known methods. Bacteria may, for example, be propagated in TB medium or LB medium and at a temperature of from 20 to 40° C. and a pH of from 6 to 9. Suitable culturing conditions are described in detail, for example, in T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989).
If the proteins used according to the invention are not secreted into the culture medium, the cells are then disrupted and the product is obtained from the lysate by known protein isolation processes. The cells may be disrupted, as desired, by means of high-frequency ultrasound, by means of high pressure, such as, for example, in a French pressure cell, by means of osmolysis, by the action of detergents, lytic enzymes or organic solvents, by means of homogenizers or by a combination of two or more of the processes listed.
The proteins used according to the invention may be purified using known chromatographic methods such as molecular sieve chromatography (gel filtration), for example Q Sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, and also using other customary methods such as ultrafiltration, crystallization, salting-out, dialysis and native gel electrophoresis. Suitable processes are described, for example, in Cooper, F. G., Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, N.Y. or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.
It may be advantageous to isolate the recombinant protein by using vector systems or oligonucleotides which extend the cDNA by particular nucleotide sequences and thereby code for altered proteins or fusion proteins which are used, for example, to simplify purification. Examples of suitable modifications of this kind are “tags” acting as anchors, such as the modification known as the hexa-histidine anchor, or epitopes which can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Other suitable tags are, for example, HA, calmodulin-BD, GST, MBD; chitin-BD, steptavidin-BD-avi-tag, Flag-tag, T7 etc. These anchors may be used for attaching the proteins to a solid support such as a polymer matrix, for example, which may, for example, be packed in a chromatography column, or may be used on a microtiter plate or on another support. The corresponding purification protocols can be obtained from the commercial affinity tag suppliers.
The proteins prepared as described may be used either directly as fusion proteins or, after cleaving off and removing the fusion partner, as “pure” hydrophobins.
If the fusion partner is intended to be removed, it is recommended to incorporate a potential cleavage site (specific recognition site for proteases) into the fusion protein between the hydrophobin part and the fusion partner part. Suitable cleavage sites are in particular those peptide sequences which otherwise occur neither in the hydrophobin part nor in the fusion partner part, which can be readily determined by means of bioinformatics tools. Particularly suitable are, for example, BrCN cleavage on methionine or protease-mediated cleavage with factor Xa, enterokinase cleavage, thrombin, TEV cleavage (tobacco etch virus protease).
Assignment of sequence names to DNA and polypeptide sequences in the sequence listing













dewA DNA and polypeptide sequences
SEQ ID NO: 1


dewA polypeptide sequence
SEQ ID NO: 2


rodA DNA and polypeptide sequences
SEQ ID NO: 3


rodA polypeptide sequence
SEQ ID NO: 4


hypA DNA and polypeptide sequences
SEQ ID NO: 5


hypA polypeptide sequence
SEQ ID NO: 6


hypB DNA and polypeptide sequences
SEQ ID NO: 7


hypB polypeptide sequence
SEQ ID NO: 8


sc3 DNA and polypeptide sequences
SEQ ID NO: 9


sc3 polypeptide sequence
SEQ ID NO: 10


basf1 DNA and polypeptide sequences
SEQ ID NO: 11


basf1 polypeptide sequence
SEQ ID NO: 12


basf2 DNA and polypeptide sequences
SEQ ID NO: 13


basf2 polypeptide sequence
SEQ ID NO: 14


yaad DNA and polypeptide sequences
SEQ ID NO: 15


yaad polypeptide sequence
SEQ ID NO: 16


yaae DNA and polypeptide sequences
SEQ ID NO: 17


yaae polypeptide sequence
SEQ ID NO: 18


yaad-Xa-dewA-his DNA and polypeptide sequences
SEQ ID NO: 19


yaad-Xa-dewA-his polypeptide sequence
SEQ ID NO: 20


yaad-Xa-rodA-his DNA and polypeptide sequences
SEQ ID NO: 21


yaad-Xa-rodA-his polypeptide sequence
SEQ ID NO: 22


yaad-Xa-basf1-his DNA and polypeptide sequences
SEQ ID NO: 23


yaad-Xa-basf1-his polypeptide sequence
SEQ ID NO: 24









The Multilayered Composite

Multilayered composites are employed for very different purposes, for example as packaging means (in particular composite films) or self-adhesive articles (multilayered composite of at least support and an adhesive layer).
The multilayered composites comprise at least two, preferably two to five, layers, it being possible for the individual layers to have a thickness of from 0.01 to 5 mm, for example. The individual layers may consist of natural or synthetic polymers or else of metal. The layers are in particular polymer films, paper, metal foils, metallized polymer films etc.
The above polypeptides are used as adhesion promoters between at least two adjacent layers of the multilayered composite. Preferably, at least one of the adjacent layers is a layer of a natural or synthetic polymer. Suitable polymers are in particular polycondensates such as polyesters, polyadducts such as polyurethanes, polyamides, polycarbonates or polyphenylene ethers or polyphenylene sulfides or polymers obtainable by free-radical or ionic polymerization of ethylenically unsaturated compounds (referred to as free-radical polymers for short). Such free-radical polymers consist preferably of at least 60% by weight, particularly preferably at least 80% by weight, of “main monomers” selected from C1 to C20 alkyl (meth)acrylates, vinyl esters of carboxylic acids comprising up to 20 carbon atoms, vinyl aromatics with up to 20 carbon atoms, ethylenically unsaturated nitriles, vinyl halides, vinyl ethers of alcohols comprising from 1 to 10 carbon atoms, aliphatic hydrocarbons with from 2 to 8 carbon atoms and one or two double bonds, in particular ethylene and propylene.
The polypeptides of the invention are suitable in particular also as adhesion promoters for nonpolar polymers; therefore, preferably at least one of the adjacent layers consists of a nonpolar polymer.
A measure of the polarity of polymers is the surface tension in air (21° C.). The lower the surface tension, the more nonpolar the polymer.
Therefore, at least one of the adjacent layers preferably consists of a nonpolar polymer having a surface tension of less than 50 mN/m (millinewton/meter), in particular less than 40 mN/m. Examples of nonpolar polymers of this kind are polyamide 66 (42.5 mN/m), polystyrene (43.5 mN/m), PVC (38.4 mN/m), polyethylene (36.1 mN/m), polypropylene (29 mN/m) or polytetrafluoroethane (22.5 mN/m).
Particular preference is given to both adjacent layers consisting of such a nonpolar polymer.
The amount of polypeptide required for adhesion promotion usually from 0.01 to 1000 mg (milligram/m2 (square meter)), in particular 0.01 to 100 mg/m2 and particularly preferably 0.1 to 10 mg/m2.
The polypeptide may be applied to either of the two adjacent layers and, alternatively, may also be applied to both layers if both layers have already been preformed.
The polypeptide is preferably in the form of an aqueous solution; the polypeptide content of the solution is preferably from 0.01 to 5 parts by weight of polypeptide to 100 parts by weight of water. For the use according to the invention, the solution is preferably diluted further to a concentration of from 1 to 10 000 μg/ml of water, in particular 10 to 1000 μg/ml of water.
The application may therefore be followed first by a drying process in order to remove the water.
Subsequently, the two adjacent layers may be bonded by customary methods, for example by laminating.
The polypeptide may in particular also be applied to one of the adjacent layers (first layer), and the other adjacent layer (second layer) may then be prepared by applying a polymer dispersion, polymer solution or a solvent-free polymer to the first layer provided with the adhesion promoter and subsequently forming a film and/or thermal or photochemical curing. For this purpose, the polymer in particular is in the form of an aqueous dispersion or solution, particularly preferably an aqueous dispersion of an emulsion polymer, preferably of any of the free-radical polymers listed above. After the polymer has been applied, a drying process is then carried out, if appropriate.
The Coated Substrate
Substrates are coated for very different purposes. Mention should be made in particular of decorative coatings or protective coatings (generic term: coatings) or else adhesive coatings, it being possible for the adhesive to be applied as such to the substrate or to be bonded to it, for example, in the form of a self-adhesive article (label or adhesive tape).
The substrate, or the substrate surface, may consist of any material. Likewise, the coating or the substrate-facing surface of the coating may consist of any material.
Preferably, the substrate surface or the coating, or the substrate-facing surface of the coating, consists of natural or synthetic polymers.
Suitable polymers are in particular polycondensates such as polyesters, polyadducts such as polyurethanes, polyamides, polycarbonates or polyphenylene ethers or polyphenylene sulfides or polymers obtainable by free-radical or ionic polymerization of ethylenically unsaturated compounds (referred to as free-radical polymers for short).
With regard to the structure of the free-radical polymers and their main monomer content, the comments made above apply.
The polypeptides of the invention are suitable in particular also as adhesion promoters for nonpolar polymers; therefore at least the substrate surface or the substrate-facing surface of the coating preferably consists of a nonpolar polymer.
With respect to the contact angle as a measure for polarity, the comments made above likewise apply.
Particular preference is given to both the substrate surface and the substrate-facing surface of the coating consisting of such a nonpolar polymer.
The amount of polypeptide required for adhesion promotion corresponds to the amount indicated above.
The polypeptide may be applied to the substrate surface, to the substrate-facing surface of the coating or to both. Application to the substrate-facing surface of the coating is possible if said coating has been preformed, i.e. if a simple film or a multilayered composite (see above) is to be applied to the substrate.
The polypeptide is preferably in the form of an aqueous solution; the content of said solution is as indicated above.
It is therefore possible, after application, to carry out first a drying process in order to remove the water.
The coating may be applied to the substrate by customary methods; films or multilayered composites may, for example, be laminated on.
In particular, the coating may be prepared by applying a polymer dispersion, polymer solution or a solvent-free polymer to the substrate-facing surface provided with the adhesion promoter and by subsequently forming a film and/or thermal or photochemical curing. For this purpose, the polymer in particular is in the form of an aqueous dispersion or solution, particularly preferably an aqueous dispersion of an emulsion polymer, preferably of any of the free-radical polymers listed above. After the polymer has been applied, a drying process is then carried out, if appropriate.
The multilayered composites and coated substrates of the invention have a markedly increased strength. By using the polypeptides as adhesion promoters, adhesion of the coating to the substrate is stronger and adhesion of the individual layers of the multilayered composite to one another is superior.
In a further preferred embodiment of the invention, the substrate is a metal. In principle, this may be any metals. Examples include iron, steel, zinc, tin, aluminum, copper and alloys of said metals with themselves and with other metals. They may be, in particular, steel, steel coated with zinc, zinc alloys, aluminum or aluminum alloys. Particular preference is given to zinc or zinc alloys or substrates therewith, such as, for example, galvanized steel.
Zn or Al alloys are known to the skilled worker. The skilled worker selects the type and amount of alloy. components depending on the application purpose desired. Typical components of zinc alloys include in particular Al, Pb, Si, Mg, Sn, Cu or Cd. Typical components of aluminum alloys include in particular Mg, Mn, Si, Zn, Cr, Zr, Cu or Ti. The alloys may also be Al/Zn alloys which comprise approximately the same amount of Al and Zn. Steel coated with alloys of this kind is commercially available.
The metals may have any shape, with preference given, however, to metal foils, metal strips or metal sheets. The metal may also be a composite material with a metallic surface. It may be, for example, a composite of a polymer film and a metal.
The metallic surfaces will be coated with the polypeptides to be used according to the invention, preferably with hydrophobins, as adhesion promoters. This may be carried out using aqueous solutions of said polypeptides. Details of the coating process have already been mentioned above.
The coating may be in particular typical paints or paint systems for coating metallic surfaces. They may be paints cured thermally, photochemically or by other mechanisms.
Typical paints for coating metal surfaces comprise at least one binder and also crosslinkable components. The crosslinkable components may be crosslinkers which are employed in addition to a binder or they may be crosslinkable groups linked to the binder. The binder may of course also have crosslinkable groups and a crosslinker may be employed additionally. In this case, various possible combinations are conceivable. For example, binders and crosslinkers may be employed separately from one another. The binder in this case comprises reactive functional groups which can react with complementary, reactive functional groups in the crosslinkers. An alternative are self-crosslinking binders which comprise reactive functional groups capable of undergoing crosslinking reactions with groups of their kind (“with themselves”) or with complementary, reactive functional groups on the same polymer. It is also possible for the crosslinkers exclusively to react with themselves.
Examples of suitable binders comprise (meth)acrylate (co)polymers, partially hydrolyzed polyvinyl esters, polyesters, alkyd resins, polylactones, polycarbonates, polyethers, epoxide resin-amine adducts, polyureas, polyamides, polyimides or polyurethanes. It is of course also possible to use mixtures of various polymers, provided that said mixture does not produce any undesired effects.
The crosslinking components may have thermally crosslinking groups or photochemically crosslinking groups. Examples of suitable thermal crosslinkers are crosslinkers based on epoxides, on melamine or on blocked isocyanates. Suitable crosslinkers for photochemical crosslinking are in particular compounds having multiple ethylenically unsaturated groups, in particular di- or polyfunctional acrylates.
The use according to the invention of polypeptides, preferably of hydrophobins, improves in an advantageous manner adhesion of the paint on the substrate. An improved resistance of the paint layer to creep in anticorrosion tests is also achieved.
The following examples are intended to illustrate the invention in more detail:
Part A) Preparation of Hydrophobins
Example 1
Preliminary Work for the Cloning of yaad-His6/yaaE-His6 
A polymerase chain reaction was carried out with the aid of the oligonucleotides Hal570 and Hal571 (Hal 572/Hal 573). The template DNA used was genomic DNA of the bacterium Bacillus subtilis. The PCR fragment obtained comprised the coding sequence of the Bacillus subtilis yaaD/yaaE gene and, at their termini, in each case an NcoI and, respectively, BglII restriction cleavage site. The PCR fragment was purified and cut with the restriction endonucleases NcoI and BglII. This DNA fragment was used as insert and cloned into the vector pQE60 from Qiagen, which had previously been linearized with the restriction endonucleases NcoI and BglII. The vectors thus obtained, pQE60YAAD#2/pQE60YaaE#5, may be used for expressing proteins consisting of YAAD::HIS6 and YAAE::HIS6, respectively.









(SEQ ID NO: 25)










Hal570:
gcgcgcccatggctcaaacaggtactga



 









(SEQ ID NO: 26)










Hal571:
gcagatctccagccgcgttcttgcatac



 









(SEQ ID NO: 27)










Hal572:
ggccatgggattaacaataggtgtactagg



 









(SEQ ID NO: 28)










Hal573:
gcagatcttacaagtgccttttgcttatattcc






Example 2
Cloning of yaad Hydrophobin DewA-His6 
A polymerase chain reaction was carried out with the oligonucleotide KaM 416 and KaM 417. The template DNA used was genomic DNA of the mold Aspergillus nidulans. The PCR fragment obtained comprised the coding sequence of the hydrophobin gene dewA and an N-terminal factor Xa proteinase cleavage site. The PCR fragment was purified and cut with the restriction endonuclease BamHI. This DNA fragment was used as insert and cloned into the pQE60YAAD#2 vector previously linearized with the restriction endonuclease BglII.
The vector thus obtained, #508, may be used for expressing a fusion protein consisting of YAAD::Xa::dewA::HIS6.







(SEQ ID NO: 29)








KaM416:
GCAGCCCATCAGGGATCCCTCAGCCTTGGTACCAGCGC


 







(SEQ ID NO: 30)








KaM417:
CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTC



CGTCTCCGC






Example 3
Cloning of yaad Hydrophobin RodA-His6 
The plasmid #513 was cloned analogously to plasmid #508, using the oligonucleotides KaM 434 and KaM 435.







(SEQ ID NO: 31)








KaM434:
GCTAAGCGGATCCATTGAAGGCCGCATGAAGTTCTCCATTG



CTGC


 







(SEQ ID NO: 32)








KaM435:
CCAATGGGGATCCGAGGATGGAGCCAAGGG






Example 4
Cloning of yaad Hydrophobin BASF1-His6 
The plasmid #507 was cloned analogously to plasmid #508, using the oligonucleotides KaM 417 and KaM 418. The template DNA employed was an artificially synthesized DNA sequence—hydrophobin BASF1—(see appendix).







(SEQ ID NO: 30)








KaM417:
CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTC



CGTCTCCGC


 







(SEQ ID NO: 33)








KaM418:
CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG






Example 5
Cloning of the yaad Hydrophobin BASF2-His6 
The plasmid #506 was cloned analogously to plasmid #508, using the oligonucleotides KaM 417 and KaM 418. The template DNA employed was an artificially synthesized DNA sequence—hydrophobin BASF2 (see appendix).







(SEQ ID NO: 30)








KaM417:
CCCGTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTC



CGTCTCCGC


 







(SEQ ID NO: 33)








KaM418:
CTGCCATTCAGGGGATCCCATATGGAGGAGGGAGACAG






Example 6
Cloning of the yaad Hydrophobin SC3-His6 
The plasmid #526 was cloned analogously to plasmid #508, using the oligonucleotides KaM464 and KaM465. The template DNA employed was Schyzophyllum commune cDNA (see appendix).







(SEQ ID NO: 34)








KaM464:
CGTTAAGGATCCGAGGATGTTGATGGGGGTGC


 







(SEQ ID NO: 35)








KaM465:
GCTAACAGATCTATGTTCGCCCGTCTCCCCGTCGT






Example 7
Fermentation of the Recombinant E. coli Strain yaad Hydrophobin DewA-His6 
Inoculation of 3 ml of LB liquid medium with an E. coli strain expressing yaad hydrophobin DewA-His6 in 15 ml Greiner tubes. Incubation at 37° C. on a shaker at 200 rpm at 37° C. for 8 h. In each case 2 1 l Erlenmeyer flasks with baffles and 250 ml of LB medium (+100 μg/ml ampicillin) are inoculated with 1 ml of preculture and incubated on a shaker at 180 rpm at 37° C. for 9 h. Inoculate 13.5 l of LM medium (+100 μg/mi ampicillin) with 0.5 l of preculture (OD600 nm 1:10 measured against H2O) in a 20 l fermenter. Addition of 140 ml of 100 mM IPTG at an OD60 nm of  ̃3.5. After 3 h, cool fermenter to 10° C. and remove fermentation broth by centrifugation. Use cell pellet for further purification.
Example 8
Purification of the Recombinant Hydrophobin Fusion Protein (Purification of Hydrophobin Fusion Proteins Possessing a C-Terminal His6 Tag)
100 g of cell pellet (100-500 mg of hydrophobin) are made up with 50 mM sodium phosphate buffer, pH 7.5, to a total volume of 200 ml and resuspended. The suspension is treated with an Ultraturrax type T25 (Janke and Kunkel; IKA-Labortechnik) for 10 minutes and subsequently, for the purposes of degrading the nucleic acids, incubated with 500 units of benzonase (Merck, Darmstadt; order No. 1.01697.0001) at room temperature for 1 hour. Prior to cell disruption, a filtration is carried out using a glass cartridge (P1). For the purposes of disrupting the cells and of shearing of 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 is put on ice and the pellet is resuspended in 100 ml of sodium phosphate buffer, pH 7.5. Centrifugation and resuspension are repeated three times, the sodium phosphate buffer comprising 1% SDS at the third repeat. After resuspension, the solution is stirred for one hour, followed by 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 present in the supernatant after the final centrifugation. The experiments show that hydrophobin is present in the corresponding E. coli cells probably in the form of inclusion bodies. 50 ml of the hydrophobin-comprising supernatant are applied to a 50 ml nickel-Sepharose High Performance 17-5268-02 column (Amersham) equilibrated with 50 mM Tris-Cl buffer, pH 8.0. The column is washed with 50 mM Tris-Cl buffer, pH 8.0, and the hydrophobin is subsequently eluted with 50 mM Tris-Cl buffer, pH 8.0, comprising 200 mM imidazole. For the purpose of removing the imidazole, the solution is dialyzed against 50 mM Tris-Cl buffer, pH 8.0.
The hydrophobin has a molecular weight of approx. 53 kD. Some of the smaller bands represent degradation products of hydrophobin.
Example 9
Performance Testing; Characterization of the Hydrophobin by Changing the Contact Angle of a Water Droplet on Glass
Substrate:


    Glass (window glass, Süddeutsche Glas, Mannheim, Germany):
    Hydrophobin concentration: 100 μg/mL
    Incubation of glass slides overnight (temperature 80° C.) in 50 mM sodium acetate pH 4+0.1% Tween 20
    followed by coating, washing in distilled water
    followed by incubation: 10 min/80° C./1% SDS solution in dist. water
    washing in dist. water


The samples are dried in air and the contact angle (in degrees) of a droplet of 5 μl of water is determined.
The contact angle was measured on a Dataphysics Contact Angle System OCA 15+ instrument, software SCA 20.2.0. (November 2002). The measurement was carried out according to the manufacturer's instructions.
Untreated glass resulted in a contact angle of 30±5°; a coating with the functional hydrophobin according to Example 8 (yaad-dewA-his6) resulted in contact angles of 75±5°.
Part B) the Use of Polypeptides as Adhesion Promoters
Example 10
Polyethylene Substrate
Materials Used:
Solution Used:
A solution of the fusion protein prepared according to Example 8, yaad-Xa-dewA-his (SEQ ID NO: 19), in water was employed in the performance experiments. Hydrophobin concentration in solution: 100 μg/ml (0.01% by weight).
Substrate: Shaped Bodies (Small Plates) of Polyethylene
Coating:
A polyester film was coated with Acronal A 240, a commercial, aqueous polyacrylate dispersion from BASF for pressure-sensitive adhesives, and dried and cut into strips of 2.5 cm in width. The adhesive strips obtained were used for coating the PE platelets.
Procedure:
The polypeptide solution was applied to polyethylene plates and dried (pretreated polyethylene plates).
Subsequently, adhesive strips were bonded to pretreated and, for comparison, to untreated polyethylene plates and the force necessary to remove the adhesive strips was determined (peel strength in N)

    Peel strength with polypeptide as adhesion promoter: 4.7 N
    Peel strength without polypeptide as adhesion promoter: 2.6 N


Example 11
Metallic Substrates
A solution of the fusion protein prepared according to Example 8, yaad-Xa-dewA-his (SEQ ID NO: 19), in water was employed in the performance experiments. Hydrophobin concentration in solution: 100 μg/ml (0.01% by weight).
The following test sheets were used as metallic substrates:











No.
Substrate







Example 11-1
Steel (type ST 2 (materials No. 1.0330))


Example 11-2
Galvanized steel (sendzimir-galvanized steel



sheets GARDOBOND OE HDG/2 (Chemetall))


Example 11-3
Aluminum (AlMgSi AA 6016 GARDOBOND untreated



(Chemetall))









The paint used was a baked topcoat based on alkyd melamine.
Experimental Description
The aluminum sheets were pickled in an alkaline cleaning dipping bath (60 g/l NaOH, 60° C., 1 min) and rinsed with distilled water. The sheets were then descaled in an acidic descaling bath with HNO3/H2O (1:1) at room temperature for 15 seconds, rinsed with distilled water and blown dry with pressurized air.
The galvanized steel sheets and the steel sheets were rinsed with hot river water, subsequently rinsed with distilled water and blown dry with pressurized air.
The sheets were coated with the hydrophobins by dipping the former into the abovementioned solution, in each case at room temperature. The aluminum sheets were dipped for 16 h, the galvanized steel sheets and the steel sheets were dipped for 4 h. The sheets were subsequently rinsed with distilled water and blown dry with pressurized air.
After coating with the hydrophobin as adhesion promoter, the sheets were dipped into a baked topcoat based on alkyd melamine in a plastic bowl for 15 s and predried in air for approx. 1 h. This was followed by drying in a drying oven at 190° C. for 30 min and curing.
For comparison, in each case a further sample was prepared in the same manner with the paint but without the hydrophobin adhesion promoter.
Performance Test
The performance of the sheets was assessed by EN ISO 2409 (crosscut), EN ISO 4628-8 (creep) and DIN 53156 (Erichsen cupping).
The crosscut test assesses the appearance of the paint surface on the basis of predefined standards, after a crosscut has been cut into said surface. This involves evaluating the extent to which the paint flakes off the surface due to making the crosscut. The evaluation is done in the known manner with the aid of grades from 0 to 5, with 0 being the best and 5 the worst score.
The creep of the paint layer is determined by a standard corrosion test (exposure in the salt spray chamber (SS DIN 50021)) of the sheets. The creep of the aluminum sheets was evaluated after 298 h, that of the steel sheets was evaluated after 50 h and that of the galvanized steel sheets was evaluated after 190 h, in each case on the basis of predefined standards and using grades from 0 to 5, with 0 being the best and 5 being the worst score.
The Erichsen cupping comprises pressing a ball against the back of the sample and making a depression. The appearance of the paint at the mark is evaluated on the basis of predefined standards, with in this case 5 being the best and 0 being the worst score.
The results of the performance tests are summarized below.
Example 11-1
Steel Substrate
As shown in FIG. 1, using a hydrophobin as adhesion promoter does not change the creep (grade 5) compared with a sample without hydrophobin. The crosscut test (lower value) and the Erichsen cupping test (higher value) in each case give a superior result.
Example 11-2
Galvanized Steel Substrate
As shown in FIG. 2, using hydrophobins as adhesion promoters on galvanized steel produces values which are in all three cases superior to those without adhesion promoter (lower values for crosscut and creep and higher value for Erichsen cupping). The clearest improvement is achieved in corrosion protection integrity (creep) (grade 1 with adhesion promoter, in contrast to grade 4 without adhesion promoter).
Example 11-3
Aluminum Substrate
As shown in FIG. 3, in the case of an aluminum substrate, crosscut and creep are good even without adhesion promoter. The Erichsen cupping results in a slight improvement still.

## Claims

1. A coated metallic substrate comprising a compound between the coating and the substrate, wherein at least 40% by weight of the compound consists of a polypeptide as an adhesion promoter between the coating and the substrate, wherein the polypeptide is a hydrophobin of the formula


Xn—C1—X5-9—C2—C3—X11-39—C4—X2-23—C5—X5-9—C6—C7—X6-18—C8—Xm  (III),

wherein each amino acid residue of each X independently is a naturally occurring amino acid Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gln, Arg, Ile, Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly; each numerical subscript designates the number of amino acid residues of its adjacent X; subscripts n and m independently designate the number of amino acids from 0 to 500, inclusive, of its adjacent X; and each C independently is Cys, Ala, Ser, Gly, Met or Thr, provided that at least seven of the C residues are Cys, wherein said polypeptide has a property in increasing the contact angle of a water drop on a glass surface coated with said polypeptide by at least 20° as compared to the contact angle of a water drop of similar size on an uncoated glass surface as measured at room temperature, and wherein the substrate comprises steel, steel coated with zinc, zinc alloys, aluminum or aluminum alloys.

2. The coated metallic substrate of claim 1, wherein in the formula (III) the subscripts n and m independently designate the number of amino acids from 15 to 300, inclusive, of the X adjacent each n and m.

3. The coated metallic substrate of claim 1, wherein the coating comprises a paint layer.

4. The coated metallic substrate of claim 1, wherein the substrate-facing surface of the coating consists of a natural or synthetic polymer.

5. The coated metallic substrate of claim 1, wherein the substrate consists of galvanized steel.

6. The coated metallic substrate of claim 1, wherein the polypeptide improves adhesion of the coating and the substrate.

7. A process for preparing the coated metallic substrate of claim 1, comprising:
applying the polypeptide to the substrate as an aqueous solution,
performing a drying procedure, and
applying the coating to the polypeptide-treated substrate.

8. The process of claim 7, wherein the coating is applied as an aqueous solution or dispersion, and the drying procedure is performed after application of the coating.

9. The process of claim 8, wherein the aqueous solution or dispersion is an aqueous dispersion of an emulsion polymer.

10. A process for preparing the coated metallic substrate of claim 1, comprising:
applying the polypeptide to a surface of the substrate or a substrate-facing surface of a coating agent,
performing a drying procedure if the polypeptide is applied as an aqueous solution, and
subsequently applying the coating agent to the substrate.

11. A process for preparing the coated metallic substrate of claim 1, comprising:
applying the polypeptide to a surface of the substrate,
performing a drying procedure if the polypeptide is applied as an aqueous solution, and
preparing the coating by forming a film of a natural or synthetic polymer on the substrate surface.

12. The process of claim 11, wherein the natural or synthetic polymer is in form of an aqueous solution or dispersion, and a drying procedure is carried out after formation of the film.

13. The process of claim 12, wherein the aqueous solution or dispersion is an aqueous dispersion of an emulsion polymer.

14. The process of claim 7, wherein the coating is a paint.