DE102005005737A1 - New hydrophobin polypeptide not connected in normal manner with an external hydrophobin useful for coating of a glass surfaces - Google Patents

Abstract

A new peptide is not connected in a natural manner with an external hydrophobin and has a contact angle of at least 20[deg] during coating of the glass surface. The structural formula of a new polypeptide is: X n-C 1>-X 1-50-C 2>-X 0-50-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, where X : amino acids; n,m : 0 - 500 and; C : cysteine. The proviso is that at least one of the peptide sequences abbreviated by X n or X m is a peptide sequence of at least 20 amino acids in length. The peptide is not connected in a natural manner with an external hydrophobin and has a contact angle of at least 20[deg] during coating of a glass surface. Independent claims are included for: (1) nucleic acid coding for polypeptide; and (2) procedure of polypeptide production by expressing the above nucleic acid in a host organism and isolating the obtained protein.

Description

State of technology

hydrophobins are small proteins of about 100 aa, which are characteristic of filamentous fungi are and do not occur in other organisms. Recently Hydrophobin-like Proteins discovered in Streptomyces coelicolor called "Chapline" and also highly surface active Have properties. At water / air interfaces, Chapline can become amyloid-like Assemble fibrils (Classen et al 2003 Genes Dev 1714-1726; Elliot et al. 2003, Genes Dev. 17, 1727-1740).

hydrophobins are in a water-insoluble Shape on the surface of various fungal structures, e.g. Aerial hyphae, spores, Fruiting bodies, distributed. The genes for Hydrophobins could be obtained from ascomycetes, deuteromycetes and basidiomycetes be isolated. Some mushrooms contain more than one hydrophobing, e.g. Schizophyllum commune, Coprinus cinereus, Aspergillus nidulans. Obviously, different hydrophobins are in different Stages of fungal development involved. The hydrophobins are probably responsible for different functions (van Wetter et al., 2000, Mol. Microbiol., 36, 201-210; Kershaw et al. 1998, Fungal Genet. Biol, 1998, 23, 18-33).

When biological function for Hydrophobine is in addition to reducing the surface tension from water to generate aerial hyphae also the hydrophobization described by spores (Wöst et al. 1999, Curr. Biol., 19, 1985-88; Bell et al. 1992, Genes Dev., 6, 2382-2394). Furthermore, hydrophobins serve for the lining of gas channels in fruiting bodies of lichens and as components in the detection system of plant surfaces by fungal pathogens (Lugones et al., 1999, Mycol. Res., 103, 635-640; Hamer & Talbot 1998, Curr. Opinion Microbiol., Vol. 1, 693-697).

Complementation have shown that hydrophobins are up within a class to a certain degree can replace functionally.

The hitherto known hydrophobins can be mixed with conventional protein-chemical cleaning and isolation methods only in moderate Represent yield and purity. Also, experiments with the help of genetic engineering Procedure, larger To provide amounts of hydrophobins, have not been successful.

task

It The task was novel hydrophobins and their production process to provide an economical production of hydrophobins and their use in various technical fields.

description the invention

The invention relates to polypeptides of the 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 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) and the indices standing at X represent the number of amino acids, the indices n and m being numbers between 0 and 500, preferably between 15 and 300, and C being cysteine, with the proviso that at least one of X _n or X _{m is} abbreviated peptide sequences for a peptide sequence of at least 20 amino acids, which is not naturally associated with a hydrophobin, which cause a contact angle change of at least 20 ° after coating a glass surface.

The cysteines designated C ¹ to C ⁸ may be present either reduced in the proteins according to the invention or form disulfide bridges with one another. Particularly preferred is the intramolecular formation of CC bridges, in particular those having at least one, preferably 2, particularly preferably 3 and very particularly preferably 4 intramolecular disulfide bridges selected from the following group: C ¹ with C ² ; C ³ with C ⁴ , C ⁵ with C ⁶ , C ⁷ with C ⁸ .

If cysteines are also used in the positions marked X, can the numbering of individual cysteine positions in the change according to general formulas.

Particularly advantageous polypeptides are those 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) where X is 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) and the indices standing at X represent the number of amino acids, wherein the indices n and m are numbers between 2 and 300 and C is cysteine, with the proviso that at least one of the X _n or X _m abbreviated peptide sequences for a at least 35 amino acid long peptide sequence that is not naturally linked to a hydrophobin,
which cause a contact angle change of at least 20 ° after coating a glass surface.

Very particularly advantageous are those polypeptides 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 is 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) and the indices standing at X represent the number of amino acids, the indices n and m being numbers between 0 and 200 and C being cysteine, with the proviso that at least one of the peptide sequences abbreviated by X _n or X _{m stands} for a at least 40 amino acid long peptide sequence that is not naturally linked to a hydrophobin,
which cause a contact angle change of at least 20 ° after coating a glass surface.

preferred embodiments of the invention described are polypeptides having the general structural formula (I) (II) or (III), wherein this structural formula is at least one hydrophobin class I, preferably at least one hydrophobin dewA, rodA, hypA, hypB, sc3, basf1, basf2, or parts or derivatives thereof. The hydrophobins mentioned are in the sequence listing below structurally characterized. There may also be several hydrophobins, preferably 2 or 3, identical or different structure linked together and with a corresponding suitable polypeptide sequence naturally not associated with a hydrophobin.

Especially preferred embodiments In the present invention, the novel proteins are as described in SEQ ID NO: 20, 22, 24 represent polypeptide sequences 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. 22, 22 or 24 polypeptide sequences represented by Exchange, insertion or deletion of at least one, all the way through to 10, preferably 5, more preferably 5% of all amino acids, and that the biological property of the source proteins still too at least 50% are particularly preferred embodiments. The biological property of the proteins here is the change the contact angle as described in Example 10 understood.

The proteins according to the invention carry at least one position, which is abbreviated X _n or X _m , a polypeptide sequence of at least 20, preferably at least 35, more preferably at least 50, and especially at least 100 amino acids (hereinafter also referred to as fusion partners) which are not naturally associated with a hydrophobin is linked. This is to express that the proteins according to the invention consist of a hydrophobin part and a fusion partner part which do not occur together in nature in this form.

The fusion partner portion 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, it is also possible, for example, to link two fusion partners with a position (X _n or X _m ) of the protein according to the invention.

When Fusion partners are particularly preferred such polypeptide sequences, that lead to, that the inventive Protein is capable of coating glass surfaces and those with protein treated glass surface becomes resistant a detergent treatment as described in the experimental section (Example 10) in detail for example 1% SDS / 80 ° C / 10 min).

Particularly suitable fusion partners are polypeptides that occur naturally in microorganisms, in particular in E. coli or Bacillus subtilis. Examples of such fusion partners are the sequences yaad (SEQ ID NOs: 15 and 16), yaae (SEQ ID NOs: 17 and 18), and thioredoxin. Also very suitable are 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 are altered relative to said sequence.

The invention Proteins can also be modified in their polypeptide sequence, for example by glycosylation, acetylation or by chemical cross-linking for example with glutaric dialdehyde.

A Property of the inventive Proteins is the change of surface properties, though the surfaces be coated with the proteins. The change of the surface properties can be experimentally determine that the contact angle of a water drop before and after the coating of the surface with the protein according to the invention is measured and the difference between the two measurements is determined.

The exact experimental conditions for measuring the contact angle are laid down in the experimental part in Example 10. Under under these conditions, the proteins according to the invention have the property the contact angle at least 20, preferably 25, more preferably 30 degrees to enlarge.

in the Hydrophobin part of the previously known hydrophobins are the positions of polar and nonpolar amino acids conserved, which manifests itself in a characteristic hydrophobicity plot. differences in biophysical properties and hydrophobicity led to Classification of previously known hydrophobins in two classes, I and II (Wessels et al., 1994, Ann. Rev. Phytopathol., 32, 413-437).

The Assembled membranes from Class I hydrophobins are high grade insoluble (even opposite 1% SDS at elevated Temperature) and can only by concentrated trifluoroacetic acid (TFA), or formic acid again be dissociated. In contrast to this are the assembled forms of class II hydrophobins less stable. You already can by 60% ethanol, or 1% SDS (at room temperature) again disbanded become.

A comparison of the amino acid sequences shows that the length of the region between cysteine C ³ and C ⁴ in class II hydrophobins is significantly shorter than in class I hydrophobins.

class II hydrophobins continue to have more charged amino acids than Class I on.

One Another object of the invention are methods for preparation the inventive Proteins. These polypeptides can be chemically determined by known Methods of peptide synthesis, for example by solid phase synthesis to Merrifield.

Especially However, suitable are genetic engineering techniques, one 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 Protein is generated.

suitable Host organisms (production organisms) can be prokaryotes (including the archaea) or eukaryotes, including bacteria in particular Halobacteria and methanococci, fungi, insect cells, plant cells and mammalian cells, particularly preferred 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 the like.

object The invention are also expression constructs containing the genetic control of regulatory nucleic acid sequences for a polypeptide according to the invention coding nucleic acid sequence, and vectors comprising at least one of these expression constructs.

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

"Operational linkage" is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements such that each of the regulatory ven elements can fulfill its function in the expression of the coding sequence 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 Technology: 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, functional connected with the promoter, which increased one Expression of the nucleic acid sequence enable. Also at the 3'-end of the DNA sequences can additional advantageous sequences are inserted, such as further regulatory Elements or terminators.

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

advantageous Regulatory sequences for the inventive Processes 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 in the lambda P promoter which are advantageously used 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 include ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF, rp28, ADH.

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, apart from plasmids and phages are all others known in the art vectors, ie 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 autonomously replicated in the host organism or replicated chromosomally become. These vectors represent a further embodiment of the invention Suitable plasmids are, 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 make a small selection of possible plasmids Other plasmids are well known to the person skilled in the art and can be used, for example from the book Cloning Vectors (Eds. Pouwels P.H. et al., Elsevier, Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

advantageously, 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 thereby preferably positively influence the gene expression of the introduced genes and thereby increase it. Thus, enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. In addition, however, an enhancement of translation is possible example, by improving the stability of the mRNA.

In In another embodiment of the vector, the nucleic acid construct according to the invention can be used or the inventive nucleic acid vector also advantageously in the form of a linear DNA can be introduced into the microorganisms and via heterologous or homologous recombination integrated into the genome of the host organism become. This linear DNA can be made from a linearized vector such as a plasmid or only from the nucleic acid construct or the nucleic acid according to the invention.

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 inventive Expression cassette is made by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal. For this one uses common recombination and cloning techniques, as described, for example, in T. Maniatis, E.Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as well in T.J. Silhavy, M.L. Berman and L.W. Enquist, Experiments with Gene Fusions, 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) are described.

The recombinant nucleic acid construct or gene construct becomes expression in a suitable host organism advantageously inserted into a host-specific vector, which allows optimal expression of the genes in the host. Vectors are well known to those skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels P.H. et al. Ed., Elsevier, Amsterdam-New York-Oxford, 1985).

With Help the inventive Vectors are recombinant microorganisms to produce, which, for example are transformed with at least one inventive vector and used for the production of the polypeptides according to the invention can be. Advantageously, the above-described inventive recombinant Constructs are introduced and expressed in a suitable host system. there are preferably known in the art known cloning and transfection methods, such as for example co-precipitation, Protoplast fusion, electroporation, retroviral transfection and the like, used to bind 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. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

According to the invention are also homologously recombined microorganisms produced. This will be a vector is prepared which comprises at least a portion of a gene according to the invention or a coding sequence, wherein optionally at least an amino acid deletion, Addition or substitution has been introduced to the sequence according to the invention to change, z. B. functionally disrupted ("knock-out" Vector). The introduced sequence can, for. B. also a homologue be from a related microorganism or from a mammalian, Be derived yeast or insect source. The homologous recombination Alternatively, the vector used may be configured such that mutated or otherwise the endogenous gene upon homologous recombination changed but the functional protein is still encoded (eg upstream regulatory area so changed that the Expression of the endogenous protein is changed). The modified section of the inventive Gene is in the homologous recombination vector. The construction of suitable Vectors for homologous recombination is z. As described in Thomas, K.R. and Capecchi, M.R. (1987) Cell 51: 503.

When recombinant host organisms for the inventive 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. Beneficial are gram-positive or gram-negative bacteria, preferably bacteria of the families 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 inventive Methods used organisms 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, magnesium salts and optionally vitamins Temperatures between 0 C and 100 C, preferably between 10 C to 60 C attracted with oxygen fumigation. In this case, the pH of the nutrient fluid be kept at a fixed value, that is during the Breeding may be regulated or not. The cultivation can be batchwise, semi-batchwise or continuous respectively. nutrient can submitted at the beginning of the fermentation or semicontinuously or continuously refilled become. The enzymes can according to the method described in the examples from the organisms be isolated or used as crude extract for the reaction.

object The invention furthermore relates to processes for recombinant production invention Polypeptides or functional, biologically active fragments thereof, wherein one cultivates a polypeptide-producing microorganism, optionally induces the expression of the polypeptides and these isolated from the culture. The polypeptides can thus also in large-scale Scale are produced, if desired. The recombinant Microorganism can be cultured and fermented by known methods. Bacteria can For example, in TB or LB medium and at a temperature of 20 to 40 ° C and be increased to a pH of 6 to 9. In detail, 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).

The Cells then become, if the polypeptides are not in the culture medium be secreted, open-minded and the product according to known Protein isolation method recovered from the lysate. The cells can optionally by high-frequency ultrasound, by high pressure, such. In a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or digested by combining several of the listed methods become.

A Purification of the polypeptides can be carried out by known, chromatographic Be 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.

Advantageous it may be to isolate the recombinant protein vector systems or oligonucleotides that encode the cDNA for particular nucleotide sequences extend and for that changed Code polypeptides or fusion proteins, the z. B. a simpler Serve cleaning. Such suitable modifications are, for example anchors acting as anchors, such as As the hexa-histidine anchor known modification or epitopes recognized as antigens of antibodies can be (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Further suitable tags are e.g. HA, calmodulin BD, GST, MBD; Chitin-BD, Steptavidin BD Avi Day, Flag Day, T7 etc. These anchors can for attaching the proteins to a solid support, such as. B. a polymer matrix, serve, for example, be filled in a chromatography column can be used, or on a microtiter plate or on another carrier can be. The corresponding cleaning protocols are from the commercial affinity tag providers available.

Lots Hydrophobin-coated fungal surfaces (spores, fruiting bodies, mycelium) show microscopically detectable, characteristic structures, so-called Rodlets. Similar Rodlets with a strength of about 10 nm can also on hydrophobin-coated hydrophilic surfaces (e.g. Glass, mica, etc.) (Wösten et al., 1993, Plant Cell, 5, 1567-1574).

by virtue of the extraordinary Properties of hydrophobins for coating surfaces (e.g. resistant across from Detergents such as e.g. 1% SDS solution) have these proteins high potential for numerous technical applications. In different patents are examples of named such applications, with respect to the hereby Application of hydrophobins is referred to.

The technical use of hydrophobins, especially those of the class So far I failed due to an efficient manufacturing and cleaning method. The methods described so far, starting from natural Sources (spores, fungus mycelium, etc.) only lead to amounts of material in mg scale. (e.g., WO 96/41882).

As well approaches proved to be one Recombinant production in various production organisms as extremely expensive and not very satisfactory.

The invention Hydrophobin proteins, both in their fused form, i.e. together with the fusion partner part, as well as in isolated Shape the desirable ones Properties of hydrophobins. So you can the novel proteins both directly as fusion proteins and after cleavage and separation of the fusion partner as "pure" hydrophobins.

If a separation of the fusion partner is provided, it recommends a potential cleavage site (specific recognition site for proteases) into the fusion protein between hydrophobin part and fusion partner part install. Particularly suitable cleavage sites are those peptide sequences otherwise suitable neither in the hydrophobin part nor in the fusion partner part what can easily be determined with bioinformatic tools leaves. Particularly suitable are, for example, BrCN cleavage on methionine, or protease-mediated cleavage with factor Xa, enterokinase, Thrombin, TEV cleavage (Tobacca etch virus protease).

Experimental part

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 BglII. The resulting 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 digested with the restriction endonuclease BamHI cut. This DNA fragment was used as an insert, and in the previously linearized with the restriction endonuclease BglII Vector pQE60YAAD # 2 cloned.

The resulting 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.

As template DNA, an artificially synthesized DNA sequence - hydrophobin BASF1 - was used (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.

As template DNA, an artificially synthesized DNA sequence - hydrophobin BASF2 - was used (see Appendix).
KaM417: 000GTAGCTAGTGGATCCATTGAAGGCCGCATGAAGTTCTCCGTCTCCGC
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

Inoculation of 3ml LB liquid medium with a yaad-hydrophobin DewA-His ₆ expressing E. coli strain in 15ml 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 each inoculated with 1 ml of preculture and incubated for 9 h at 37 ° C. on a shaker at 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 adding 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) for degradation of 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 is then eluted with 50 mM Tris-Cl pH 8.0 buffer containing 200 mM imidazole. To remove the imidazole, the solution is dialyzed against 50 mM Tris-Cl pH 8.0 buffer.

1 shows the purification of the novel 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 inventive hydrophobin of 1 has a molecular weight of about 53 kD. The smaller bands partially represent degradation products of hydrophobin.

Example 9

Coating / evaluation of surfaces with hydrophobin

The Evaluation of the coating properties of hydrophobin or Hydrophobinfusionprotein is preferably on glass or Teflon as Models for made a hydrophilic or hydrophobic surfaces.

Standard tests for coating

Glass:

• - concentration Hydrophobin: 1-100 μg / mL
• - Incubation of glass slides overnight (Temperature: 80 ° C) in 50 mM Na acetate pH 4 + 0.1% Tween 20
• - to Wash the coating in deionised water
• - after that Incubation 10min / 80 ° C / 1 % SDS
• - to wash in deionised water

Teflon:

• - Concentration: 1-100 μg / mL
• - Incubation Teflon plates overnight (Temperature: 80 ° C) in 10mM Tris pH 8
• - to Wash the coating in deionised water
• - Incubation 10min / 80 ° C / 0.1 % Tween 20
• - to wash in deionised water
• - after that Incubation 10min / 80 ° C / 1% SDS
• - to wash in deionised water

The samples are air dried and the contact angle (in degrees) of a drop of 5 μl of water is determined. For example, the following values result:
Preparation with yaad-DewA fusion protein according to Example 8 (control: without protein, yaad-dewA-his ₆ : 100 μg / ml purified fusion partner):

Example 10

Coating / evaluation of surfaces with hydrophobin

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

• - concentration Hydrophobin: 100 μg / mL
• - Incubation of glass slides overnight (Temperature 80 ° C) in 50 mM Na acetate pH 4 + 0.1% Tween 20
• - to Wash the coating in distilled water
• - after that Incubation 10min / 80 ° C / 1 % 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 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 °; a coating with a functional hydrophobin according to Example 8 (yaad-dewA-his ₆ ) gave contact angles of 75 ± 5 °.

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 (11)

Polypeptides of 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 any of the 20 naturally occurring amino acids, n and m are numbers between 0 and 500, C is cysteine, with the proviso that at least one of the peptide sequences abbreviated by X _n or X _{m represents a} peptide sequence which is at least 20 amino acids in length and is not naturally linked to a hydrophobin, which cause a contact angle change of at least 20 ° after coating a glass surface. Polypeptides according to claim 1, characterized the structural formula (I) comprises a class I hydrophobin. Polypeptides according to claim 1, characterized the structural formula (I) comprises a hydrophobin selected from the group consisting of dewA, rodA, sc3, hypA, hypB, basf1, basf2 is formed. Polypeptides according to claim 1, characterized in that the structural formula (I) has a sequence selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14. Polypeptides according to claim 1, characterized in that X _n or X _m comprises a polypeptide sequence selected from SEQ ID NO: 16, 18. Polypeptides according to claim 1, characterized in that X _n or X _{m stands} for a sequence (His) _4-10 . Polypeptides according to claim 1, characterized the structural formula (I) comprises polypeptides selected from the group SEQ ID NO: 20, 22, 24. Nucleic acid, coding for Polypeptides according to claim 1-7. Process for the preparation of polypeptides according to claim 1-7, by adding a nucleic acid according to Claim 8 expressed in a host organism and the thus obtained Protein possibly isolated after purification. Method according to claim 9, characterized in that E.coli is used as the host organism. Use of hydrophobins according to claims 1-7 for coating of surfaces.