EP1664306A2 - Secretion of proteins from yeasts - Google Patents

Abstract

The invention relates to expression constructs comprising the coding nucleic acid sequence for a shuttle peptide construct that can be processed by yeast cells. Also disclosed are adequate expression vectors containing such constructs, methods for the recombinant production of target proteins, which are carried out with the aid thereof, hosts transformed therewith, shuttle peptides and nucleic acid sequences coding therefor, nucleic acid sequences that code for such shuttle peptides and are fused with a foreign protein, hydrophobin proteins produced by means of such shuttle peptides, and the use of hydrophobins for coating objects such as leather.

Description

Secretion of proteins from yeast

description

The present invention relates to expression constructs comprising the coding nucleic acid sequence for a shuttle peptide construct processable by yeast cells; corresponding expression vectors containing such constructs; processes carried out with the aid thereof for the recombinant production of target proteins; hosts transformed with it; Shuttle peptides and nucleic acid sequences coding therefor; Nucleic acid sequences coding for such shuttle peptides, fused with a foreign protein; Hydrophobin proteins, which were produced using such shuttle peptides, and the use of hydrophobins for coating objects, such as e.g. Leather.

State of the art

a) Expression in yeast

Yeasts as hosts for heterologous protein expression are widely used. The reason for this is that yeast has several advantages as an expression system. Compared to bacteria and other eukaryotic cells, they can grow in higher density and have the ability for protein glycosylation and post-translational modification. In addition, the products produced and secreted by yeasts can be purified in a simple manner because the yeasts have high resistance to cell lysis and small amounts of foreign protein are usually found in the growth medium. In addition, yeasts can grow faster than other eukaryotic cells in high density on inexpensive nutrient media.

There are numerous different approaches in the prior art for the expression and secretion of heterologous proteins in yeasts. For example, US Pat. No. 5,642,487 describes a process for the recombinant production of proteins in yeast, in which yeast is transformed with an expression cassette which codes for a structural element which produces a leader sequence from an animal peptide neurohormone, an adapter sequence, which produces a σ-helix structure , a processing signal and a structural gene are encoded. Furthermore, it is known from the prior art to use regulatory elements from the gene of the σ factor, a pheromone produced by yeasts, for controlling the expression of heterologous proteins in yeasts. For example, factor signal leader peptide sequences were used to express heterologous proteins (see, for example, US Pat. No. 5,010,182).

From the published US patent application US 2003/0077831 an expression vector for the expression of heterologous proteins in yeast is also known which, flanked by suitable transcription and translation start or termination sequences, comprises the coding sequence for a hybrid precursor polypeptide which comprises the signal peptide as elements and the leader peptide of a protein secreted by yeasts and a heterologous protein flanked by N-terminal and C-terminal propeptide sequences of the heterologous protein.

b) hydrophobins

Hydrophobins are small, approximately 100 amino acid residues, cysteine-rich proteins with interesting technical properties. You can make hydrophobic surfaces hydrophilic. They make hydrophilic surfaces hydrophobic.

However, there are a number of property rights to hydrophobins and their application: For example, WO-A-96/41882 hydrophobins from edible mushrooms (cf. SEQ ID NO: 21 and 22). WO-A-00/58342 relates to the purification of hydrophobin-containing fusion proteins by phase extraction. WO-A-01/57066 describes stabilization, solubilization and the associated better use of hydrophobins by sulfite treatment. WO-A-01/57076 describes the purification of hydrophobin by adsorption on Teflon beads and the elution by means of a detergent such as Tween at low temperatures. WO-A-01/57528 describes the fixation of hydrophobins on surfaces by the use of tween and temperatures up to 85 degrees Celsius.

WO-A-01/74864 describes atypical hydrophobins (only one disulfide bridge) with the names RdIA and RdlB (cf. SEQ ID NO: 19 and 20) from filamentous bacteria, in particular Streptomyces sp. The hydrophobin is used for the surface treatment of various objects such as windows, contact lenses and vehicle bodies. It is also proposed to produce the proteins described there in a recombinant host that releases the proteins into the medium. After detachment The hydrophobin-containing medium should be suitable for surface coating of the host. Experimental evidence for actual expression and secretion is not provided.

Brief description of the invention

The object of the present invention is to provide agents which make it possible to secrete expressed homologous or, in particular, heterologous proteins expressed in yeast, in particular Schizosaccharomyces pombe, from the yeast cells into the surrounding medium. In particular, means should be provided which allow the secretion of recombinantly produced hydrophobin from the host cell.

The above object is achieved by providing an expression construct comprising the coding nucleic acid sequence for a shuttle peptide construct of the general formula which can be processed by yeast cells

(Sig-SP),

containing the coding nucleic acid sequences for a) a signal peptide (Sig) in the 5'-3 'direction, processably linked to b) at least one shuttle peptide (SP) that can be secreted by the yeast cells.

The solution to the above problem is modeled on the example of the hydrophobin DewA (protein protein according to SEQ ID NO: 14 with coding sequence according to SEQ ID NO: 13; preprotein with signal sequence: SEQ ID NO: 16 with coding nucleic acid sequence according to SEQ ID NO: 15 ) from Aspergillus nidulans as a heterologous target protein (Targ). This protein is a class I representative of hydrophobins, i.e. of secreted fungal envelope proteins with the ability to self-assemble.

In particular, the DNA sequence coding for the target protein (DewA) (SEQ ID NO: 13) is at the 3'-terminal end for a peptide pheromone from S. pombe (P factor; amino acid sequence according to SEQ ID NO: 6 for mature P-factor) coding DNA sequence (SEQ ID NO: 5 for mature P-factor) fused. The resulting fusion protein contains all the signal sequences necessary for the secretion of the pheromone and the target protein fused to it, in particular the cleavable signal peptide (SEQ ID NO: 4). As part of the secretion, the fusion protein is processed proteolytically. As As a result, the pheromone (P factor) (SEQ ID NO: 6) and the target protein (hydrophobin; SEQ ID NO: 14) are secreted separately into the medium.

The finding according to the invention is surprising in that the regulatory elements of the P-factor preprotein (N-terminal to the mature pheromone) are obviously not sufficient to control the secretion of the target protein by the yeast cells. Only the use of a construct in which an additional, co-secreting protein component (the mature pheromone) is processably connected upstream of the target protein to be secreted enables the desired secretion of the target protein into the culture medium.

Detailed description of the invention:

a) General information

The protein sequences are usually given in the "one letter code" in the description and the figures.

For the purposes of the present invention, "secrete" is a protein which is expressed intracellularly by a host cell, in particular of yeasts, and is excreted from the cell, preferably into the surrounding medium, through the cell membrane via the cell's own mechanisms.

"Processable" in the sense of the present invention is a protein precursor (ie a protein in its originally expressed form, such as a preprotein, with N- and / or C-terminal peptide sequences that are no longer present in the mature processed protein) if it is can be converted into the mature form by proteolytic processes in and / or outside the host cell.

A "processable linkage" is given when individual protein sections in a protein to be processed are linked via peptide bonds which can be cleaved by a proteolytic enzyme of the host cell.

The “processing” can take place N-terminally and optionally also C-terminally to the sequence of the mature, processed protein (target protein). A “homologous” target protein, although originally expressed in the host used according to the invention, is therefore a host's own protein, but is secreted by the host cells due to the transformation of the host with an expression construct according to the invention.

A “heterologous” target protein is not originally expressed in the host used according to the invention, is therefore not a host protein, but is secreted by the host cells due to the transformation of the host with the expression construct according to the invention.

A “shuttle peptide” is part of a “shuttle peptide construct” that can be processed in the host cell used according to the invention. Together with one or more processable regulative C- and / or N-terminal, preferably N-terminal, associated peptide fragments, such as signal sequences, leader sequences, it forms the shuttle peptide construct. In contrast, the shuttle peptide is e.g. to the signal peptide a polypeptide secreted by the host cell. The regulatory elements are preferably processed intracellularly. The shuttle peptide can also be secreted if it is fused, preferably C-terminally, to a target protein in a processable manner. This C-terminal processing is preferably carried out, i.e. Cleavage of the target protein, proteolytic during secretion, e.g. during passage through the host cell envelope or in extracellular space, e.g. in the surrounding culture medium, by the cell's own proteases.

An “expression construct” or an “expression cassette” according to the present invention comprises, operatively linked, to the coding nucleic acid sequence of a processable shuttle peptide construct according to the above definition, the start and termination signals necessary for controlling expression in a special host system, such as in particular yeast cells Transcription and, if necessary, translation. The expression construct in particular comprises binding sites for transcription factors. 5'- Upstream of the coding sequence is a constitutive or inducible, native or heterologous, natural or synthetic promoter operable in the host cell. The expression construct also includes a number of restriction enzyme sites, e.g. B. those for inserting the construct into an expression vector. In addition, the expression construct can include a gene for a selectable marker. An “expression vector” describes a construct that can be obtained by inserting an expression cassette according to the invention into a replicon, such as, for example, into a plasmid, cosmid or a virus. Such a vector is capable of autonomous replication or integration into the host genome and contains the necessary ones Control sequences for controlling transcription and optionally translation of the coding nucleic acid sequences according to the invention for a processable shuttle peptide construct as defined above.

b) Preferred embodiments

A first subject of the invention relates to an expression construct comprising the coding nucleic acid sequence for a shuttle peptide construct of the general formula which can be processed by yeast cells

(Sig-SP),

containing the coding nucleic acid sequences for a) a signal peptide (Sig) in the 5'-3 'direction, processably linked to b) at least one shuttle peptide (SP) that can be secreted by the yeast cells; and optionally one or more nucleic acid sequences which promote processing and / or secretion, 5 'or 3' terminal to the coding signal peptide sequence.

The coding sequences for SP and Sig are in the same reading frame and, in addition, a processable sequence between the C-terminus of Sig and the N-terminus of SP is formed during translation. This processable sequence can be, for example, an artificially introduced, proteolytically cleavable natural or synthetic adapter sequence. However, this is preferably part of the C-terminus of Sig or N-terminus of SP. The adapter sequence can be processed in such a way that the split sequence can be found in whole or in part at the C-terminus of Sig or the N-terminus of SP. The latter is possible as long as this does not significantly impair, in particular not prevent, the ability to secrete SP.

In particular, the invention relates to such expression constructs which code for a processable shuttle peptide construct which is derived from a polypeptide which is processed by yeasts in the broadest sense. In particular, these are yeasts selected from Ascomycetes. Preferred yeasts are selected from those of the Class of the Archiascomycetes, the order of the Schizosaccharomycetales and particularly preferably selected from yeasts of the genus Schizosaccharomyces, such as S. pombe. Although there is data showing that minus cells also secrete the P factor, it is preferred to select the strain that is suitable for the mating factor (pheromone) (i.e. the plus factor (P factor) plus cells and the minus) Factor (M factor) minus cells).

The processable shuttle peptide construct is derived in particular from a pheromone preprotein from a yeast, the pheromone being produced from the preprotein by N- and C-terminal processing. The pheromone N-terminal preferably has a polypeptide which can be split off by processing and which in particular comprises the elements required for processing and / or secretion of the preprotein, such as signal peptide and possibly leader peptide, and the required protease interfaces.

Mushroom pheromones are known and e.g. described both for basidiomycetes such as Ustilago maydis (Urban, M., Kahmann, R. and Bolker, M. (1996) The biallelic a mating type locus of Ustilago maydis: remnants of an additional pheromone gene indicate evolution from a multiallelic ancestor (Mol Gen Genet 250 (4): 414-420)) or Coprinopsis one (Halsall, JR, Milner, MJ and Casselton, LA (2000) Three subfamilies of pheromone and reeeptor genes generate mutiple B mating speeifities in the mushroom Coprineus cinereus (Genetics 154 ( 3): 1115-1123)) and ascomycetes such as Schizosaccharomyces pombe (Imai, Y. and Yamamoto, M. (1995) The fission yeast mating pheromone P-factor: its molecular strueture, gene strueture, and ability to induce gene expression and G1 arrest in the mating partner (Genes Dev 8 (3): 328-338), Davey, J. (1992) Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromones (EMBO J 11 (3): 95 1-960)), Saccharomyces cerevisiae (Michaelis, S. and Herskowitz, I. (1988) The a-factor pheromone of Saccharomyces cerevisiae is essential for mating (Mol Cell Biol

8 (3): 1309-1318), Kurjan, J. and Herskowitz, I. (1982) Strueture of a yeast pheromone gene (MF-alpha): a putative alpha-factor precursor contains four tandem copies of mature alpha-factor ( Cell 30 (3): 933-943)), Kluyveromyces delphensis (Wong, S., Fares, MA, Zimmermann, W., Butler, G. and Wolfe, KH (2003) Evidence from comparative genomics for a complete sexual cycle in the 'asexual' pathogenic yeast Candida glabrata (Genome Biol 4 (2) R10)) and Saccharomyces kluyveri (Egel-Mitani, M. and Hansen, MT (1987) Nucleotide sequence of the gene encoding the Saccharomyces kluyveri alpha mating pheromone (Nucleic Acids Res 15 (15) 6303)).

Pheromones suitable according to the invention are relatively small peptides (such as 5 to 40 or 8 to 30 amino acids). They usually do not show significant homology in the primary sequence. They are formed as preproteins, processed proteolytically and released into the culture medium.

Examples of particularly suitable pheromones or corresponding pre-proteins are the so-called P and M factors or their pre-proteins from S. pombe. (cf.Imai, Y. and Yamamoto, M. (1995) The fission yeast mating pheromone P-factor: its molecular strueture, gene strueture, and ability to induce gene expression and G1 arrest in the mating partner (Genes Dev 8 (3 ): 328-338), Davey, J. (1992) Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone (EMBO J 11 (3): 951 -960), Kjaerulff, S., Davey, J. and Nielsen, O. (1994) Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3 (Mol Cell Biol 14 ( 6) 3895-3905)).

The P-factor preprotein has, for example, a DNA sequence according to SEQ ID NO: 9 and a protein sequence according to SEQ ID NO: 10. The preprotein comprises an N-terminal signal peptide sequence bridged with four successive pheromone peptide sequences which can be separated by processing (cf. FIG. 3).

In preferred constructs according to the invention, the processable shuttle peptide construct is designed such that it contains a signal polypeptide (Sig) which is processably linked to the N-terminal end of a C-terminally processable pheromone polypeptide (Pher).

In particular, the signal polypeptide comprises or is identical to the proteolytically cleavable native signal polypeptide (e.g. SEQ ID NO: 4 encoded by SEQ ID NO: 3) of the pheromone preprotein.

It is further preferred that the C-terminal processed pheromone polypeptide comprises a C-terminal protease interface. The expression construct preferably further comprises the coding nucleic acid sequence for a homologous or heterologous target protein (Targ), processably linked to the C-terminus of the shuttle peptide construct (Sig-SP).

The invention preferably relates to expression constructs of the type described above, comprising the coding nucleic acid sequence for a fusion protein of the general formula which can be processed by yeast cells

Sig-L1 _n -Pher-L2 _m -Targ

wherein

Sig, Pher and Targ are as defined above,

L1 and L2 stand for processable linkers or adapter sequences and n and m stand independently for 0 or 1. However, n is preferably 1 and m is O.

L1 and L2 can be natural or synthetic linkers. They comprise at least one proteolytically processable peptide sequence. If necessary, with L1 and / or L2, e.g. the processing, secreting, transcription and / or translation-promoting effector functions are associated.

Expression constructs are particularly preferred, the coding nucleic acid sequence for the processable shuttle peptide construct being a sequence coding for a signal polypeptide (Sig) according to SEQ ID NO: 3 or a functional equivalent thereof, operatively linked to that for the mature P factor pheromone (Pher) encoding nucleic acid sequence according to SEQ ID NO: 5 or a functional equivalent thereof.

The linker L2 is preferably not present. The linker L1, on the other hand, is preferably provided and comprises the coding sequence for a polypeptide according to amino acid residues 21 to 30 in SEQ ID NO: 10. L1 bridges the signal polypeptide with the first pheromone building block (positions 31 to 57 in SEQ ID NO : 10) the prehormone. The C-terminal end of L1 corresponds to a recognition sequence of the protease required for the proteolytic processing. In a particularly preferred embodiment, the coding nucleic acid sequence for the processable shuttle peptide construct comprises a sequence according to SEQ ID NO: 1.

Basically, the same procedure can also be used with the help of the M factor - the second pheromone occurring in S. pombe - and applicable to the expression of any homo- and heterologous target proteins (target proteins). Genomically there are three genes (mfmf, SEQ ID NO: 42; mfm2 SEQ ID NO: 45; and mfm3 ⁺ , SEQ ID NO: 48) which each encode the M factor, the pheromone of the cells with the minus pairing type. A preprotein (SEQ ID NO: 43, 46 and 49) is first formed from each gene, which is processed as part of the secretion. Ultimately, the M factor (YTPKVPYMC; SEQ ID NO: 51), encoded by SEQ ID NO: 44, 47 and 50) is released into the medium as a mature pheromone (cf. FIG. 9).

Further shuttle peptide constructs suitable according to the invention could therefore be derived from the coding sequences according to SEQ ID NO: 42, 45 or 48, which code for M factor signal peptide, functionally linked to an M factor pheromone. Non-limiting examples of corresponding coding shuttle peptide sequences include e.g. Nucleotide residues 1 to 117 according to SEQ ID NO: 42; Nucleotide residues 1 to 123 according to SEQ ID NO: 45; or nucleotide residues 1 to 114 according to SEQ ID NO: 48; or functionally equivalent constructs derived therefrom which control the secretion and processing of the M-factor pheromone and a homo- or heterologous target protein linked to the pheromone C-terminally and proteolytically cleavable. Functional equivalents can contain the sequence segments located 5 'upstream from the coding sequence of the mature M factor (SEQ ID NO: 44, 47 or 50) unchanged or modified (e.g. by deletion of one or more nucleic acid residues), and thus for an in encode its amino acid sequence modified shuttle peptide, which functionally links the mature M factor peptide sequence with a, eg C-terminal shortened signal sequence section includes.

A target protein (target) expressed according to the invention can be derived from any prokaryotic or eukaryotic organism, in particular humans, animals or yeasts, as long as it can be expressed by the host cell in the manner according to the invention as a component of a fusion protein with the shuttle peptide (SP) and is processable. The secreted and processed product can be therapeutically useful or have other advantageous application properties. Examples of therapeutically useful proteins are immunoglobulins, Peptide hormones, growth factors, lymphokines, protease inhibitors and the like. Hydrophobins are particularly worthy of mention as an example of target proteins with other properties which are of interest in terms of application technology.

In a particularly preferred embodiment of the invention, the target protein is a hydrophobin, in particular a hydrophobin of class I.

Typical hydrophobins are relatively small (100 + 25 amino acids) moderately hydrophobic proteins with a conserved motif of 8 cysteines (X -CX _{2-38 5-} 9-CCX _11-39 -CX _{8- 23} -CX _5-9 -CCX. _{6 18} -CX _2- ι ₃ ). Hydrophobins can assemble at hydrophilic-hydrophobic interfaces to protein films. Such aggregates of class I hydrophobins are insoluble in SDS, while aggregates of class II hydrophobins are soluble in SDS (Wessels, JGH (1997) Hydrophobins: Proteins that change the nature of the fungal surface. Adv Microb Physiol 38: 1-45) ,

Hydrophobins which can be used according to the invention are derived in particular from fungi, e.g. from Ascomycetes, such as those of the genus Aspergillus, in particular A. nidulans.

Useful hydrophobins are also known from the prior art mentioned above and are not restricted to those from fungi.

Non-limiting examples of useful hydrophobins are selected from SEQ ID NO: 14 (DewA), SEQ ID NO: 19 (RdIA) SEQ ID NO: 20 (RdlB) SEQ ID NO: 21 (HYP1) SEQ ID NO: 22 (HYP4) and SEQ ID NO: 56 (RodA).

p52750 (DewA)

MRFIVSLLAF TAAATATALP ASAAKNAKLA TSAAFAKQAE GTTCNVGSIA CCNSPAETNN DSLLSGLLGA GLLNGLSGNT GSACAKASLI DQLGLLALVD HTEEGPVCKN IVACCPEGTT NCVAVDNAGA GTKAE

q9l 190 (RdIA)

MLKKAMVAAA AAASVIGMSA AAAPQALAIG DDNGPAVANG NGAESAFGNS ATKGDMSPQLSLVEGTLNKP CLGVEDVNVA VINLVPIQDI NVLADDLNQQ CADNSTQAKR DGALSHVLED LSVLSANGEG R q934f8 (RdlB)

MIKKWAYAA IAASVMGASA AAAPQAMAIG DDSGPVSANG NGASQYFGNS MTTGNMSPQM ALIQGSFNKP CIAVSDIPVS VIGLVPIQDL NVLGDDMNQQ CAENSTQAKR DGALAHLLED VSILSSNGEG GKG

HYP1_AGAB1 (P49072)

MISRVLVAAL VALPALVTAT PAPGKPKASS QCDVGE1HCC DTQQTPDHTS AAASGLLGVP INLGAFLGFD CTPISVLGVG GNNCAAQPVC CTGNQFTALI NALDCSPVNV NL

HYP4_AGABI (043122)

MVSTFITVAK TLLVALLFVN INIVVGTATT GKHCSTGPIE CCKQVMDSKS PQATELLTKN GLGLGVLAGV KGLVGANCSP ITAIGIGSGS QCSGQTVCCQ NNNFNGWAI CTPINANV

RodA

LPPAHDSQFA GNGVGNKGNS NVKFPVPENV TVKQASDKCG DQAQLSCCNK ATYAGDTTTV DEGLLSGALS GLIGAGSGAE GLGLFDQCSK LDVAVLIGIQ DLVNQKCKQN IACCQNSPSS ADVALLGILGL

The RodA protein together with the DewA protein is part of the outer spore shell of A. nidulans.

The invention also relates to expression vectors comprising, in operative linkage with at least one regulatory nucleic acid sequence, an expression construct as defined above.

The invention also relates to recombinant microorganisms which contain, if appropriate stably integrated into the host genome, at least one expression vector or an expression construct as defined above. A “recombinant microorganism in the sense of the present invention comprises at least one expression vector according to the invention or an expression construct according to the invention and is derived from yeasts in the broadest sense. In particular, the yeasts are derived from Ascomycetes. Preferred yeasts are selected from the class of the Archiascomycetes, the order of the Schizosaccharomycetales, and particularly preferably selected from yeasts of the genus Schizosaccharomyces, such as S. pombe.

Another object of the invention relates to shuttle peptide constructs that can be processed by yeast cells, derived from a pheromone preprotein from a yeast, the pheromone being derivable and secrete from the preprotein by N- and C-terminal processing.

Preferred shuttle peptide constructs containing a signal polypeptide N-terminai are processably linked to the C-terminally processed pheromone polypeptide.

The signal polypeptide is preferably the proteolytically cleavable native signal polypeptide of the pheromone preprotein and the C-terminally processed pheromone polypeptide comprises the C-terminal protease interface.

Preferred shuttle peptide constructs are derived from pheromone preproteins, from yeasts, in particular preproteins of the factors P and M from S. pombe. Particularly preferred shuttle peptides comprise an amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof.

Another object of the invention relates to a method for the recombinant production of a target protein, wherein a recombinant microorganism according to the above definition is cultivated, which expresses the nucleic acid sequence encoding the target protein and the target protein secreted into the culture medium, such as e.g. a hydrophobin as defined above, isolated.

The invention further relates to nucleic acids coding for a shuttle peptide construct as defined above; as well as nucleic acids coding for a fusion protein which can be processed by yeast lines and comprises a target protein and as defined above. The invention also relates to hydrophobins obtainable by a process according to the invention.

Finally, the invention relates to the use of such a hydrophobin for surface treatment, in particular the surface of objects selected from glass, fibers, fabrics, leather, lacquered objects, such as e.g. Motor vehicle bodies, foils, facades treated.

The invention also relates to the use of hydrophobins for the surface treatment of fibers, fabrics and leather.

c) Further refinements of the invention

d) polypeptides / proteins

Also included according to the invention are “functional equivalents” of the specifically disclosed or used polypeptide / proteins. This applies both to the intermediately formed fusion proteins and to their components, ie target proteins (target), shuttle peptides (SP) such as pheromones (Pher) also for signal peptides (Sig) and linkers. In the following, only "polypeptide" is used as a generic term for polypeptide / protein.

“Functional equivalents” or analogs of the specifically disclosed polypeptides are, within the scope of the present invention, different polypeptides thereof which continue to have the desired biological activity. Analog shuttle peptides should continue to be suitable for controlling the secretion and processing of the target protein. The functional equivalents should also correspond accordingly of components of the shuttle peptide, such as signal polypeptide, pheromone, linker, furthermore have the properties required for effective secretion and processing of the fusion protein with release of the target protein.

"Functional equivalents" of polypeptides according to the invention, such as target proteins, shuttle peptides, can in particular contain residues of natural linker or adapter sequences which are formed by proteolytic cleavage at the C- and / or N-terminal. According to the invention, “functional equivalents” are understood to mean, in particular, mutant proteins which have at least one of the sequence positions of the above-mentioned concrete sequences different from the specifically mentioned amino acid, but nevertheless have one of the above-mentioned biological activities. "Functional equivalents" thus include the mutant proteins obtainable by one or more amino acid additions, substitutions (cf. examples in the following table), deletions and / or inversions, the changes mentioned being able to occur in any sequence position as long as they exist a mutant protein with the property profile according to the invention.

Suitable residues for amino acid substitutions include:

he rest examples of substitution

Ala Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn; gin

Hey Leu; Val

Leu He; Val

Lys Arg; Gin; Glu

Met Leu; lle

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val lle; Leu

Functional equivalence is particularly given when the activity pattern between mutant and unchanged polypeptide matches qualitatively. This means, for example, that modified shuttle peptides express or secrete the same target protein with higher or lower efficiency in the same host; or that modified target proteins have an increased or decreased pharmacological effect or modified application properties. “Functional equivalents” in the above sense also include precursors of the polypeptides described and functional derivatives and salts of the polypeptides. The term “salts” means both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules according to the invention. Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts, such as, for example, sodium, calcium, ammonium, iron and zinc salts, and salts with organic bases, such as, for example, amines, such as triethanolamine, arginine , Lysine, piperidine and the like. Acid addition salts, such as, for example, salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and oxalic acid, are also a subject of the invention.

"Functional derivatives" of polypeptides according to the invention can also be prepared on functional amino acid side groups or on their N- or C-terminal end using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtainable by reaction with ammonia or with a primary or secondary amine, N-acyl derivatives of free amino groups, produced by reaction with acyl groups, or O-acyl derivatives of free hydroxyl groups, produced by reaction with acyl groups.

"Functional equivalents" naturally also include polypeptides which are accessible from organisms other than those specifically mentioned, and naturally occurring variants. For example, regions of homologous sequence regions can be determined by sequence comparison and, based on the specific requirements of the invention, equivalent enzymes can be determined.

"Functional equivalents" also include fragments, preferably individual domains or sequence motifs, of the polypeptides according to the invention which, for example, have the desired biological function.

“Functional equivalents” are also fusion proteins which contain one of the abovementioned polypeptide sequences or functional equivalents derived therefrom and at least one further, functionally different, heterologous sequence in functional N- or C-terminal linkage (ie without mutual substantial functional impairment of the fusion protein parts). Examples of such heterologous sequences are signal peptides, enzymes, immunoglobulins, surface antigens, receptors or receptor ligands.

“Functional equivalents” encompassed according to the invention are homologs to the specifically named polypeptides. These have at least 60%, preferably at least 75%, in particular at least 85%, such as 90%, 95% or 99%, homology to one of the concretely disclosed Sequences calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sei. (USA) 85 (8), 1988, 2444-2448. A percentage homology of a homologous polypeptide according to the invention means in particular percentage identity of the amino acid residues based on the total length of one of the amino acid sequences specifically described herein.

In the event of a possible protein glycosylation, equivalents according to the invention include polypeptides in deglycosylated or glycosylated form and also modified forms obtainable by changing the glycosylation pattern.

Homologs of the proteins or polypeptides according to the invention can be generated in a manner known per se by mutagenesis, e.g. by point mutation or shortening of the protein.

c2) Nucleic acid sequences:

Nucleic acid sequences according to the invention, in particular those which code for one of the above polypeptides and their functional equivalents, comprise single and double-stranded DNA and RNA sequences, such as e.g. also cDNA and mRNA.

All nucleic acid sequences mentioned here are either of natural origin or can be prepared in a manner known per se by chemical synthesis from nucleotide components, such as, for example, by fragment condensation of individual overlapping, complementary nucleic acid components.

The chemical synthesis of oligonucleotides can be carried out, for example, in a known manner using the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The attachment of synthetic oligonucleotides and the filling of gaps with the aid of the Klenow fragment of DNA polymerase and ligation reactions as well as general cloning methods are described in Sambrook et al. (1989) Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

The invention also relates to nucleic acid sequences coding for one of the above polypeptides and their functional equivalents, which e.g. are accessible using artificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules which code for polypeptides according to the invention or biologically active sections thereof, and to nucleic acid fragments which e.g. are suitable for use as hybridization probes or primers for identifying or amplifying coding nucleic acids according to the invention.

The nucleic acid molecules according to the invention can also contain untranslated sequences from the 3 'and / or 5' end of the coding gene region.

An "isolated" nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid and, moreover, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals be when it's chemically synthesized.

A nucleic acid molecule according to the invention can be isolated using standard molecular biological techniques and the sequence information provided according to the invention. For example, cDNA can be isolated from a suitable cDNA library by using one of the specifically disclosed complete sequences or a section thereof as a hybridization probe and standard hybridization techniques (as described, for example, in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). In addition, a nucleic acid molecule comprising one of the disclosed sequences or a portion thereof can be isolated by polymerase chain reaction using the oligonucleotide primers which have been created on the basis of this sequence. The nucleic acid amplified in this way can be cloned into a suitable vector and characterized by DNA sequence analysis. The invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a section thereof.

The nucleotide sequences mentioned enable the generation of probes and primers which can be used for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers usually comprise a nucleotide sequence region which, under stringent conditions, can be attached to at least about 12, preferably at least about 25, e.g. about 40, 50 or 75 successive nucleotides of a sense strand of a nucleic acid sequence according to the invention or a corresponding antisense strand are hybridized.

Further nucleic acid sequences according to the invention are derived from the specifically disclosed sequences and differ from them by addition, substitution, insertion or deletion of one or more nucleotides, but continue to code for polypeptides with the desired property profile.

Also included according to the invention are those nucleic acid sequences which comprise so-called silent mutations or which are modified in accordance with the codon usage of a specific source or host organism, in comparison with a specifically named sequence, as well as naturally occurring variants, such as e.g. Splice variants or allele variants, thereof. Sequences obtainable by conservative nucleotide substitutions (i.e. the amino acid in question is replaced by an amino acid of the same charge, size, polarity and / or solubility) are also a subject of the subject.

The invention also relates to the molecules derived from the specifically disclosed nucleic acids by sequence polymorphisms. These genetic polymorphisms can exist between individuals within a population due to natural variation. These natural variations usually cause a 1 to 5% variance in the nucleotide sequence of a gene.

Furthermore, the invention also encompasses nucleic acid sequences which hybridize with the abovementioned coding sequences or are complementary thereto. These polynucleotides can be found when screening genomic or cDNA banks and, if appropriate, can be amplified therefrom using suitable primers by means of PCR and then isolated, for example, using suitable probes. The property of being able to “hybridize” to polynucleotides means the ability of a poly- or oligonucleotide to bind to an almost complementary sequence under stringent conditions, while under these conditions non-specific bindings between non-complementary partners are avoided. For this purpose, the sequences should be closed 70-100%, preferably 90-100%, are complementary The property of complementary sequences to be able to bind specifically to one another is demonstrated, for example, in the Northern or Southern blot technique or in primer binding in PCR or RT-PCR Usually oligonucleotides with a length of 30 base pairs or more are used for this purpose. Stringent conditions mean, for example in Northern blot technology, the use of a washing solution, for example 50-70 ° C., preferably 60-65 ° C. 0.1x SSC buffer with 0.1% SDS (20x SSC: 3M NaCI, 0.3M Na citrate, pH 7.0) for the non-specific hybrid elution cDNA probes or oligonucleotides. As mentioned above, only highly complementary nucleic acids remain bound to one another. The setting of stringent conditions is known to the person skilled in the art and is, for example: in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. described.

c3) Expression constructs and vectors:

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

Such constructs according to the invention preferably comprise a promoter 5'-upstream of the respective 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” is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.

Examples of sequences which can be linked operatively are targeting sequences and enhancers, polyadenylation signals and the like. Other regulatory elements include 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, CA (1990).

The coding nucleic acid sequences can be contained in one or more copies in the gene construct.

Examples of useful promoters are the yeast promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, nmt1, nmt41 and nmt81.

Suitable promoters for the yeast S. pombe are e.g. to name: nmtl, nmt41, nmt81, adhl, fbpl, SV40 or CaMV. Further information at (http://pinqu.salk.edU/~forsburq/vectors.html#exp). The promoters differ in their transcription rate. The selection depends on the desired level of expression. The same applies to other yeasts.

Suitable yeast promoters are described, for example, in published US patent application 2003/0077831, which is hereby expressly incorporated by reference.

The use of inducible promoters, such as e.g. light-inducible and in particular temperature-inducible promoters.

The regulatory sequences mentioned are intended to enable the targeted expression of the nucleic acid sequences. Depending on the host organism, this can mean, for example, that the gene is only expressed or overexpressed after induction, or that it is expressed and / or overexpressed immediately.

The regulatory sequences or factors can preferably have a positive influence on the expression and thereby increase or decrease it. Thus, the regulatory elements can advantageously be strengthened at the transcription level by using strong transcription signals such as promoters and / or "enhancers". In addition, an increase in translation is also possible, for example, by improving the stability of the mRNA. An expression cassette is produced by fusing a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Common recombination and cloning techniques are used, such as those described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausübet, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987).

For expression in a suitable host organism, the recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector which enables 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" (Pouweis P.H. et al., Ed., Elsevier, Amsterdam-New York-Oxford, 1985). In addition to plasmids, vectors are also understood to mean all other vectors known to the person skilled in the art, such as phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be replicated autonomously in the host organism or replicated chromosomally.

Constructs suitable for the yeast S. pombe can be mentioned as examples of expression vectors suitable according to the invention (see, for example: (http://pinqu.salk.edU/~forsburq/vectors.html#exp).

Other examples are: pART1 (McLeod, M., Stein, M., and Beach, D. (1987) The product of the mei3 + gene expressed under control of the mating type locus, induces meiosis and sporulation in fission yeast. EMBO J. 6: 729-736 pCHY21 (Hoffman, CS and Winston, F. (1991). Glucose repression of transcription of the schizosaccharomyces pombe fbpl gene occurs by a camp signaling pathway. Genes Dev. 5: 561-571)

REP1, REP3, REP4 (Maundrell, K. (1990). Nmtl of fission yeast: a highly transcribed gene completely repressed by thiarnine. J. Biol. Chem. 265: 10857-10864) REP41, REP42, REP81, REP82 (Basi, G., Schmid, E. and Maundrell, K. (1993) TATA box mutations in the Schizosaccharomyces pombe nmtl promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123: 131-136)

Yeast expression vectors for expression in yeast S. cerevisiae, such as pYEpSed (Baldari et al., (1987) Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30: 933-943), pJRY88 (Schultz et al. (1987) Gene 54: 113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and methods of constructing vectors suitable for use in other fungi such as filamentous fungi include those described in detail in: van den Hondel, C.A.M.J.J. & Punt, P.J. (1991) "Gene transfer Systems and vector development for filamentous fungi, in: Applied Molecular Genetics of Fungi, J.F. Peberdy et al., Eds., Pp. 1-28, Cambridge University Press: Cambridge.

Further suitable expression systems are described in chapters 16 and 17 by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989

c4) Recombinant microorganisms:

With the aid of the vectors according to the invention, recombinant microorganisms can be produced which, for example, have been transformed with at least one vector according to the invention and can be used to produce the polypeptides according to the invention.

The recombinant constructs according to the invention described above are advantageously introduced and expressed in a suitable host system. Common cloning and transfection methods known to the person skilled in the art, such as, for example, co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, are preferably used to bring the nucleic acids mentioned into expression in the respective expression system. Suitable systems are described, for example, in Current Protocols in Molecular Biology, F. Ausubel et al., Ed., 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. In principle, all organisms which enable expression of the nucleic acids according to the invention, their allele variants, their functional equivalents or derivatives are suitable as host organisms. Preferred host organisms are yeasts.

Methods for introducing exogenous DNA into yeast cells are known from the prior art

Technology known. For example, this can be done by transforming spheroplasts

Hinnen et al. (1978, Proc. Natl. Acad. Sei., USA, 75: 1919-1935).

Chemical transformation methods can be found e.g. for S. pombe in Alfa et al.

(Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) or for S. cerevisiae by Kaiser et al , (Kaiser, C, Michaelis, S. and Mitchell, A. (1994) Methods in Yeast

Genetics. Cold Spring Harbor Laboratory Press, New York).

Auxotrophic markers are often used in yeasts to select transformants.

The strain to be transformed lacks a protein which is necessary for the production of certain metabolic products. The corresponding active protein is introduced into the cell by the vector used. Commonly used markers are

Genes e.g. uracil, leucine, histidine or tryptophan biosynthesis.

Successfully transformed organisms can be selected using marker genes, which are also contained in the vector or in the expression cassette. Examples of such marker genes are genes for antibiotic resistance and for enzymes which catalyze a coloring reaction which stains the transformed cell. These can then be selected using automatic cell sorting. Microorganisms which have been successfully transformed with a vector and carry an appropriate antibiotic resistance gene (e.g. G418 or hygromycin) can be selected using appropriate antibiotic-containing media or nutrient media. Marker proteins that are presented on the cell surface can be used for selection by means of affinity chromatography.

The combination of the host organisms and the vectors which match the organisms, such as plasmids, viruses or phages, such as, for example, plasmids with the RNA polymerase / promoter system, the phages 8 or: or other temperate phages or transposons and / or further advantageous regulatory ones Sequences form an expression system.

c5) Recombinant production of target proteins The invention further relates to methods for the recombinant production of a target protein as defined above.

The recombinant microorganism can be cultivated and fermented by known methods. In particular, suitable cultivation conditions for example for S. pombe in Alfa et al. (Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of Genetics 1, pp 395-446, Plenum Press, New York) or for S. cerevisiae in Kaiser et al. (Kaiser, C, Michaelis, S. and Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, New York).

If the target protein is secreted into the culture supernatant, the cells are separated from this and the target protein is obtained from the supernatant by known protein isolation methods.

Purification of the target protein can be achieved with known chromatographic methods, such as molecular sieve chromatography (gel filtration), ion exchange chromatography, such as Q-Sepharose chromatography, and hydrophobic chromatography, as well as with other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, T.G., Biochemical Working Methods, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

To facilitate the isolation of the recombinant protein, vector systems can also be used which code for modified polypeptides or fusion proteins which serve for easier purification. Such suitable modifications are, for example, so-called "tags" which act as anchors, such as, for example, the modification known as a hexa-histidine anchor or epitopes which can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D ., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press). These anchors can be used to attach the proteins to a solid support, such as, for example, a polymer matrix, which can be filled, for example, in a chromatography column, or can be used on a microtiter plate or on another support. At the same time, these anchors can also be used to recognize the proteins. To recognize the proteins, customary markers, such as fluorescent dyes, enzyme markers which form a detectable reaction product after reaction with a substrate, or radioactive markers, alone or in combination with the anchors, can be used to derivatize the proteins.

c6) surface treatment with hydrophobin

Treatment of surfaces with hydrophobins for modification, e.g. for hydrophobization or hydrophilization, the surface properties are known in principle. According to the invention, it is now considerably simplified in that sufficient raw material is available with the recombinant production of the hydrophobins.

Taking into account the teaching of the prior art (such as, for example, WO-A-01/57066, which describes the stabilization, solubilization and the associated better use of hydrophobins by sulfite treatment; WO-A-01/57076, which describes the purification of Hydrophobin by adsorption on Teflon beads and elution with detergent such as Tween describes at low temperatures; or WO-A-01/57528, which describes the fixation of hydrophobins on surfaces by the use of Tween and temperatures up to 85 degrees Celsius ) a wide variety of solid materials, such as glass, fibers, fabrics, leather, lacquered objects, foils, facades, can be coated with hydrophobin.

The invention is now described in more detail by the following non-limiting examples with reference to the accompanying figures. It shows

Figure 1 different constructs according to the invention for the secretion of the hydrophobins from S. pombe.

FIG. 2A shows the genomic sequence of the DewA gene (SEQ ID NO: 39); the sequences of the two introns are underlined; B) the amino acid sequence and in brackets the corresponding DNA sequence of the DewA protein from Aspergillus nidulans; the signal sequence is printed in bold, the partial sequence following the signal sequence corresponds to the sequence of mature DewA; C) the amino acid sequence and in brackets the corresponding DNA sequence of the HA tag. FIG. 3A) the amino acid sequence and, in parentheses, the corresponding DNA sequence of the P-factor preprotein; the signal sequence is printed in bold; the underlined partial sequences following the signal sequence correspond to the sequences of the four maternal pheromone peptides; the pheromone closest to the signal peptide is called the P factor; B) amino acid sequence and in brackets the corresponding DNA sequence of the cleavable signal peptide and the subsequent 6 amino acids (underlined) of the P-factor preprotein; C) amino acid sequence and in brackets the corresponding DNA sequence for the "P-Shuttle" according to the invention; the signal sequence is printed in bold; the underlined sub-sequence following the signal sequence corresponds to the sequence of the material P factor;

FIG. 4 shows a fusion protein according to the invention consisting of the “P-shuttle” sequence (signal sequence bold; mature P protein underlined), the mature DewA (double underlined) and the C-terminally fused HA tag (SEQ ID NO: 18; coded) from SEQ ID NO: 17);

FIG. 5 shows the immunological detection of hydrophobins in S. pombe ^*

The hydrophobic genes DewA and RodA from A. nidulans became immunological

Detection fused with an HA tag, cloned into the expression vector pJR1-3XL and transformed into S. pombe. "Membrane fraction" and "cytosolic proteins" were separated by SDS-PAGE. The detection in the Westem analysis was carried out with HA antibodies. The size standard in kDa is given on the left. A: samples of a culture with the insert-free vector (pJR1-3XL, negative control), with an HA-tagged control protein (positive control) and with a vector which contains the HA-tagged DewA gene with introns (DewA-HA (+ Introns)) contains. B: Samples of a culture with the vector (pJR1-3XL, negative control) are applied, with a vector which contains the HA-tagged DewA gene without introns (DewA-HA (intron)) or the HA-tagged RodA gene Contains introns (RodA-HA (-Hntrons)).

Figure 6 shows the immunological detection of the expression of hydrophobins in S. pombe. The PDewAHA protein was expressed in S \ pombe. The cells were harvested, the culture supernatant was aliquoted and part of the TCA was precipitated. The protein was detected by SDS-PAGE and Western blot with the help of HA antibodies. The bands marked with * correspond to the precursor protein (approx. 18 kD, upper band) and the mature form (approx. 17 kD lower band).

7 shows the detection of the expression of hydrophobins in S. pombe. S. pombe cells were transformed with plasmids which express P + 6DewA by a strong promoter (pJR1-3XL) or weaker promoter (pJR1-81XL). The cells carry a version of the prp7 gene chromosomally with a c-myc tag. This serves as a control to rule out that the culture supernatant has been contaminated by lysed cells. Cells were harvested (pellet), the culture supernatant TCA- precipitated (supernatant). The proteins were detected by SDS-PAGE and Western blot with the help of antibodies against HA (A) or against c-myc (B).

Figure 8 shows the detection of secretion using the "P-Shuttle" method. S. pombe cells were transformed with plasmids which express PfakDewA through a weaker promoter (pJR1-81XL). The cells were harvested (pellet), the culture supernatant TCA-precipitated (US). The protein was detected according to SDS-PAGE and Westem blot with the help of antibodies against HA.

FIG. 9 the three genes which each encode the M factor (SEQ ID NO: 51 for mature factor) from S. pombe: A) sequences for the mfm1 ^{+ '} gene; B) sequences for the tr7t / 772 ^{+ "} gene; and C) sequences for the mfm3 * ^' gene.

Figure 10 shows the RodA gene. The genomic sequence (SEQ ID NO: 52) of the RodA gene contains two introns (underlined), which are not present in the corresponding coding ORF (SEQ ID NO: 53). The preprotein (SEQ ID NO: 54) contains a cleavable signal sequence (printed in bold) which is missing in the mature protein (SEQ ID NO: 56; encoded by SEQ ID NO: 55).

Experimental part

General experimental information:

a) General cloning procedures

The cloning steps carried out in the context of the present invention, e.g. Restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of

Bacteria, multiplication of phages and sequence analysis of recombinant DNA were if no other information is given, as in Sambrook et al. (1989) described above.

DNA was purified from reaction mixtures or after gel electrophoresis using the NucleoSpin Extract Kit (Machery-Nagel, Düren) and plasmid DNA from E. coli was isolated using the NucleoSpin Plasmid Quick Pure Kit (Machery-Nagel, Düren) the manufacturer's instructions.

Restriction enzymes (Invitrogen) were used according to the manufacturer. DNA was ligated using the T4 ligase (Promega, Mannheim) according to the manufacturer.

Transformations in E. coli were carried out by electroporation using the Gene Pulser II device (BIO-RAD, Munich) using 2 mm electroporation cuvettes (Biozym Diagnostik, Hess. Oldendorf) according to the manufacturer. Transformants were selected on LB medium (150 mg / l) containing ampicillin (Lennox, 1955, Virology, 1: 190).

b) Polymerase chain reaction (PCR)

PCR amplifications were carried out using the Combizyme DNA polymerase (Invitek, Berlin, Germany) according to the manufacturer's instructions. 100 μl of reaction volume per 1 pmol of the corresponding primers were used.

c) cultivation

The cultivation of S. pombe was carried out as in Alfa et al. (Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) and Gutz et al. (Gutz, H., Heslot, H., Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of Genetics 1, pp 395-446, Plenum Press, New York).

The cultivation of recombinant strains was carried out as in Alfa et al. (Alfa, C, Fantes, P., Hyams, J., McLeod, M. and Warbrick, E. (1993) Experiments with fission yeast. Cold Spring Harbor Laboratory Press, New York) and Gutz et. al. (Gutz, H., Heslot, H., Leupold, U. and Loprieno, U. (1974) Schizosaccharomyces pombe. In: Handbook of Genetics 1, pp 395-446, Plenum Press, New York). d) cell disruption

For rapid expression controls, cells were centrifuged (5 min, 3,500xg) and the cell pellet directly in Laemmli buffer (Laernmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (Nature 227: 680-685)) added for gel electrophoresis.

For cell disruption, cells were harvested by centrifugation at 3,500xg for 5 min. The cell pellets were resuspended in 1 ml of 1xPBS and 1 volume of glass beads was added. The mixture was vortexed for 5 min, the supernatant removed over the glass beads.

e) Organisms used

The strains DH5α (Invitrogen), XL10-Gold (Stratagene) or BL21 (BioLabs) were used to work with E. coli.

S. pombe strains used are from the gap yeast strain collection of the working group of Prof. Dr. G. Rodel taken from the Institute of Genetics at the Technical University of Dresden.

Example 1: Production of the expression construct DewA and DewAHA and cloning into the vector pJR1-3XL

a) Isolation of the genomic DNA sequence of the DewA gene and removal of the introns

Chromosomal DNA, which, as in Kaiser ef al. (Kaiser, C, Michaelis, S. and Mitchell, A. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, New York) from A. nidulans was kindly supervised by Prof. Dr. A. Brakhage (Hanover).

The approximately 550 bp genomic DNA fragment PCR was amplified using chromosomal DNA as a template using the primers SpDewBamrev and ScDewBamfor. ScDewBamfor:

5 '. TAA TAA GGA TCC ATG CGC TTC ATC GTC TCT CTC C - 3 '(SEQ ID NO: 41)

SpDewBamrev:

5 '. TAA TAA GGA TCC TTA CTC AGC CTT GGT ACC GGC - 3 '(SEQ ID NO: 28)

The reaction mixture was separated by gel electrophoresis and the corresponding DNA band eluted as described above. The fragment, which is flanked on both sides by a Sa / πHI site, which was inserted through the primer, was cut with the restriction endonuclease ßamHI (Invitrogen) according to the manufacturer's instructions and purified from the reaction mixture (see above).

The vector pUC18 (Yanisch-Pron, C, Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of M13mp18 and pUC19 vectors. Gene 33: 103) was also cut with ßamHI, separated by gel electrophoresis and then eluted from the gel (see above).

Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli. Recombinant plasmids were identified after plasmid preparation and subsequent restriction digestion. After cloning, the correct DNA sequence of the cloned PCR products was - as with all constructs produced below - verified by sequencing. Sequencing reactions were carried out according to Sanger et al. (Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA sequencing with chain terminating inhibitors. Proc NatI Acad Sei USA 74: 5463-5467). The sequencing reactions were carried out using the "Thermo-Sequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP" (Amesham Pharamacia Biotech, Freiburg) and S'-side IRD800 labeled primers (MWG Biotech AG, Ebersberg). The products were separated and the sequence evaluated using the automatic LI-COR 4000/4200 (MWG Biotech AG, Ebersberg) sequencing system.

A construct that contains the intron-containing genomic DewA gene cloned into the ßamHI site of the vector pUC18 was called pDewAgen.

The two introns in the genomic DNA of DewA (see genomic Dew> A sequence SEQ ID NO: 39) were analyzed using "Overlap Extension PCR" (OEP) (cf. Pogulis, RJ, Vallejo, AN and Pease, LR In vitro recombination and mutagenesis by overlap extension PCR. Methods Mol Biol. 1996; 57: 167-76) removed.

First, using DNA of the construct pDewAgen as templates with the aid of the primer pairs ScDewBamfor / DewUrev, Dewl1for / Dewl2rev and Dewl2for / SpDewBamrev, subfragments of the open reading frame (ORF) of DewA PCR were amplified.

ScDewBamfor: 5 '- TAA TAA GGA TCC ATG CGC TTC ATC GTC TCT CTC C - 3' (SEQ ID NO: 41)

DewUrev:

5 '- GT GTG GTC GAC GAG AGC GAG CAG ACC CAG CTG - 3' (SEQ ID NO: 24)

DewHfor:

5 '- CAG CTG GGT CTG CTC GCT CTC GTC GAC CAC AC - 3' (SEQ ID NO: 23)

Dewl2rev:

5 '- GTC GAC GGC AAC ACA GTT GGT GGT TCC CTC - 3' (SEQ ID NO: 26)

Dewl2for:

5 "- GAG GGA ACC ACC AAC TGT GTT GCC GTC GAC - 3 '(SEQ ID NO: 25)

SpDewBamrev: 5 '- TAA TAA GGA TCC TTA CTC AGC CTT GGT ACC GGC - 3' (SEQ ID NO: 28)

These subfragments were separated and purified by gel electrophoresis. In the final PCR, the intronless ORF was amplified with the subfragments as a template and the distal primers ScDewBamfor and SpDewBamrev. The approximately 410 bp long PCR product was separated by gel electrophoresis, purified and cut with the restriction endonuclease ßamHI. Appropriate interfaces had been inserted through the distal primers. The cut fragment was purified and cloned into the vector pUC18, also cut with ßamHI. Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli. Recombinant plasmids were identified after plasmid mini-preparation and the correct sequence of the cloned ORF was verified by sequencing. The con- strukt (pDewAORF) served as a template for the construction of corresponding expression plamides.

b) Preparation of the expression constructs of DewHA (+ introns) and DewHA (-trons) and introduction of a C-terminal hemagglutinin tag

Since no specific antibodies against DewA are available, DEPA was fused with the HA epitope by OEP to detect heterologous expression. First, the primer pairs SpDewXhofor / DewAHArev and DewAHAfor / DewAHANcorev were used in the primary PCRs.

SpDewXhofor:

5 '- TAA TAA CTC GAG ATG CGC TTC ATC GTC TCT CTC C - 3' (SEQ ID NO: 27) De AHArev:

5 '- TCC ACG CGG AAC CAG CTC AGC CTT GGT ACC - 3' (SEQ ID NO: 30)

DewAHAfor:

5 '- GGT ACC AAG GCT GAG CTG GTT CCG CGT GGA - 3' (SEQ ID NO: 29) DewAHANcorev:

5 '- ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC - 3' (SEQ ID NO: 31)

DNA of the construct pDewAgen was used as a template for the production of DewHA (+ introns) and DNA of the construct pDewA-ORF for the production of DewHA (-ntrons). The vector yEP351 HA (Kettner, K., Friederichs, S., Schlapp, T. and Rodel G (2001) Expression of a VEGF-like) carrying the DNA sequence of the HA tag was used as a template for the PCR with DewAHAfor / DewAHANcorev protein from Parapoxvirus ovis in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Protein Expr Purif Aug; 22 (3): 479-83). In the final PCRs using the primer pair

SpDewXhofor / DewAHANcorev and the respective subfragments, the DNA coding for the HA epitope was fused to the respective DewA DNA. The fragments amplified in this way are flanked on the 5 'side by an Xho \ restriction site and on the 3' side by a Λ / col restriction site which were introduced with the aid of the distal primers. The fragments were separated by gel electrophoresis, purified and cut with the restriction endonucleases Xho \ and Ncol and purified from the reaction mixture. The vector pJR1-3XL (Moreno, MB, Duran, A. and Ribas, JC Afamily of multifunctional thiamine-repressible expression vectors for fission yeast. Yeast. 2000 Jun 30; 16 (9): 861-72) was also used with the restriction enzymes Xho \ and Λ / col cut, separated and purified by gel electrophoresis. Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli by electroporation. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing. In the expression plamids DewA-HA (+ introns) and DewA-HA (introns) thus obtained, the expression of the fusion constructs in S. pombe is under the control of the strong nmt7 promoter.

c) Expression of DewA-HA (+ Intron) and DewA-HA (-Intron)

The vectors DewA-HA (+ introns) and DewA-HA (introns) obtained according to a) and b) were in the S. pombe host strain K0103 (t? ^S ade6-M210 leu1-32 his7-366) as by Schiestl and Gietz (Schiestl, RH and Gietz, RD (1989) High efficency transformation of intact yeast cells using Single stranded nucleic acids as a carrier. Curr Genet 16: 339-346), which are described by the feι / 7-32 mutation Conditional leucine auxotrophy of the S. pombe strain is complemented by the LE1 / 2 gene from S. cerevisiae present on the expression vectors. Transformants can thus be selected on minimal medium without leucine. The expression of the fusion proteins in corresponding yeast transformants was determined by means of Western blotting. Analyzes examined.

The antibodies anti-HA (article 1 583816, anti-HA (12CA5) -mouse monoclonal antibody) and anti-c-myc (article 1 667 149, anti-c-myc antibody) were obtained from Röche Diagnostics (Mannheim).

After cultivation, yeast transformants were harvested, digested with glass beads and the 3,500xg supernatant from the centrifugation was removed. 50 μg each of the 20,000 × g pellet (“membrane fraction”) and the supernatant (“cytosolic proteins”) were applied and separated in SDS-PAGE. The detection in the Western analysis was carried out with HA antibodies. The result is shown in FIG. 5. The size standard in kDa is given on the left. FIG. 5A shows samples of a culture with the insert-free vector (pJR1-3XL, negative control), with an HA-tagged control protein (positive control) and with a vector which contains the HA-tagged DewA gene with introns (DewA -HA (+ introns)). In FIG. 5B, samples are culture with the vector (pJR1-3XL, negative control), with a vector which contains the HA-tagged DewA gene without introns (DewA-HA (introns)) or the HA-tagged RodA gene with introns (Rod-AHA ( + lntrons)) contains, applied.

RodAHA (+ introns) was produced in analogy to the information in Examples 1a) and 1b). RodA is another hydrophobin from A. nidulans.

Example 2: Production of expression vectors for the secretion of the expressed DewA vector containing the construct PDewAHA

a) Preparation of the construct PDewAHA, containing the coding sequence for the P-factor signal peptide

In order to optimize the secretion of the protein from S. pombe cells, the authentic secretion signal of the A. nidulans protein, which is not effective in the split yeast, was first replaced by the cleavable signal peptide of the P factor from S. pombe. The P factor is secreted by the cells into the medium as a peptide pheromone. It is synthesized in the cell as a precursor protein (preprotein) consisting of a cleavable N-terminal signal sequence and four P-factor copies, each separated by short spacer sequences, and matures during secretion, including the cleavage of the signal sequence and the proteolytic release of the four P-factor peptides.

First, the P-factor signal sequence was amplified by means of PCR and genomic DNA from S. pombe as a template using the primer pair SigPXhofor / PDewArev and the corresponding PCR product was purified.

SigPXhofor: 5 '- TAA TTT CTC GAG ATG AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA C - 3' (SEQ ID NO: 34) PDewArev: 5 '- GGC AGA GGC CGG GAG TGG AAT AGG TGA GGC - 3' (SEQ ID NO: 33)

The PCR product of the primer pair PDewfor / DewAHANcorev and DNA of the construct DewA-HA (-Introns) were also separated and purified as a template by gel electrophoresis. PDewfor:

5 '- GCC TCA CCT ATT CCA CTC CCG GCC TCT GCC - 3' (SEQ ID NO: 32) DewAHANcorev: 5 '- ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC - 3' (SEQ ID NO: 31)

These two primary PCR products were used in the final PCR with the distal primers SigPXhofor / DewAHANcorev as a template. The PDewAHA fragment amplified in this way is flanked on the 5 'side by an Xho \ restriction site and at its 3' end by an Nco \ restriction site which was introduced with the aid of the above primers. In the fusion protein (PDewAHA) encoded by this fragment, the cleavable signal sequence of the DewA from A. nidulans is replaced by the cleavable signal peptide of the P-factor precursor protein.

The PDewAHA fragment was cut with the restriction endonucleases Xho \ and Λ / col, separated by gel electrophoresis and ligated into the vector pJR1-3XL (see above) cut with the same restriction endonucleases. Vector and fragment were ligated (see above) and the ligation mixture was transformed into E. coli by electroporation. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing. The construct obtained was called PDewAHA.

b) Expression

The experiments were carried out in analogy to Example 1c).

The PDewAHA protein was expressed in S. pombe. The cells were harvested, the culture supernatant was aliquoted and part of the TCA was precipitated. The TCA precipitate was taken up in Laemmli buffer (Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4 (Nature 227: 680-685)). Cell pellet, supernatant and TCA-precipitated supernatant were examined. The protein was detected by SDS-PAGE and Westem blot with the help of HA antibodies. The result is shown in FIG. 6. The bands marked with * correspond the precursor protein (approx. 18 kD, upper band) and the mature form (approx. 17 kD lower band).

The analysis showed that no effective secretion was observed. The N-terminal fusion of the cleavable signal peptide of the P factor is not sufficient for secretion of the fused hydrophobin.

Example 3: Production of expression vectors for the secretion of the expressed DewA vector containing the construct P + 6DewAHA

a) Preparation of the construct P + 6DewAHA, containing the coding sequence for the P-factor signal peptide, C-terminally extended by 6 amino acids

In order to ensure an authentic sequence environment of the signal peptide, which may be important for secretion, the sequence of the signal peptide in the fusion protein was determined using OEP using the primer pairs SigPXhofor / P + 6DewArev and P + 6DewAfor / DewAHANcorev with DNA of the construct PDewAHA as template in the primary PCR reactions

SigPXhofor:

5 '- TAA TTT CTC GAG ATG AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA

C - 3 '(SEQ ID NO: 34)

P + 6DewArev: 5 '- CAC ACC AGG ATC GGC AAC TGG AAT AGG TGA GGC - 3' (SEQ ID NO: 36)

P + 6DewAfor:

5 '- GTT GCC GAT CCT GGT GTG CTC CCG GCC TCT GCC - 3' (SEQ ID NO: 35) DewAHANcorev: 5 '- ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC - 3' (SEQ ID NO: 31 )

and the primer pair SigPXhofor / DewAHANcorev in the final PCR reaction with the 6 C-terminal amino acids (P + 6DewA).

The P + 6DewA fragment was cut with the restriction endonucleases Xho \ and Λ / col, separated by gel electrophoresis and in those with the same restriction Sendonucleases cut vector pJR1-3XL (see above) and the ligation mixture transformed by electroporation in E. coli. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing. The P + 6DewA fragment was also cloned into the vector pJR-81XL. Here, the transcription of the fusion gene is under the control of the weak nmtδ promoter. With this construct, a negative influence of the very high transcription in pJR1-3XL constants on the secretion should be tested.

The corresponding constructs were named P + 6DewA / pJR1-3XL and P + 6DewA / pJR1- 81XL.

The experiment was carried out in analogy to Example 2a. The amplified sequences are cloned in pJR1-3XI analogously to Example 2a.

b) Expression

The expression was carried out in analogy to Example 2b).

S. pombe cells were transformed with the two plasmids, which express P + 6DewA by a strong promoter (pJR1-3XL) or weaker promoter (pJR1-81XL). The cells carry a version of the prpl gene chromosomally with a c-myc tag. This serves as a control to rule out that the culture supernatant has been contaminated by lysed cells. Cells were harvested (pellet), the culture supernatant TCA-precipitated (supernatant). The precipitate was taken up in Laemmli buffer and also analyzed. The proteins were detected by SDS-PAGE and Western blot with the aid of antibodies against HA (FIG. 7A) or against c-myc (Röche Diagnostics) (FIG. 7B).

As can be seen in FIG. 7, this construction is also unsuitable for effective secretion.

Example 4: Production of expression vectors for the secretion of the expressed DewA vector containing the construct PfakDewAHA

a) Preparation of the construct PfakDewAHA, containing the coding sequence for the mature first P-factor including the P-factor signal peptide DewAHA was fused to the carboxyl terminal end of the mature P factor sequence using OEP. The in the primary PCR reactions using the primer pairs SigPXhofor / PfakDewArev and genomic DNA from S. pombe as a template

SigPXhofor:

5 '. TAA TTT CTC GAG ATG AAG ATC ACC GCT GTC ATT GCC CTT TTA TTC TCA C - 3 '(SEQ ID NO: 34) PfakDewArev: 5' - GGC AGA GGC CGG GAG GCG CTT TTT CAA GTT GGG TC - 3 '(SEQ ID NO: 38)

and PfakDewAfor / DewAHANcorev and DNA of the construct P + 6DewA / pJR1-81XL as a template

PfakDewAfor:

5 '- AAC TTG AAA AAG CGC CTC CCG GCC TCT GCC - 3' (SEQ ID NO: 37)

DewAHANcorev:

5 '- ATT ATT CCA TGG CTA TTA GCG GCC GCA CTG AGC AGC - 3' (SEQ ID NO: 31)

PCR fragments obtained were separated by gel electrophoresis, purified and used as a template for the final PCR using the primer pair SigPXhofor / DewAHANcorev. The PfakDewA fragment thus obtained was cut with the restriction endonucleases Xho \ and Λ / col, separated by gel electrophoresis and ligated into the vector pJR1-81XL cut with the same restriction endonucleases (see above). The ligation mixture was transformed into E. coli by electroporation. Recombinant plasmids were identified after plasmid minipreparation and the correct sequence of the cloned ORF was verified by sequencing. Such a construct was called PfakDewA / pJR1-81XL. In this construct, the P-factor preprotein including the first amino terminal pheromone and the fused sequence encoding the hydrophobin are under the control of the nmt81 promoter.

The experiment was carried out in analogy to Example 2a, but using the expression vector pJR1-81XL. The amplified sequences were cloned into pJR1 -81XL analogously to Example 2a. The amplified and cut with the restriction endonucleases Xho \ and Λ / col DNA was cloned into the Xho \ and Λ / col sites of the expression vector pJR1-81XL.

b) Expression

The expression was carried out in analogy to Example 3b)

The cells were pelleted and the culture supernatant was TCA-precipitated. The precipitate was taken up in Laemmli buffer and also analyzed. The protein was detected by SDS-PAGE and Western blot with the help of antibodies against HA. The result is shown in Figure 8. As can be seen, an effective secretion of the hydrophobin into the medium is achieved with the help of this construction. In the corresponding fusion protein, all sequences of the P-factor preprotein necessary for secretion are thus present in their authentic context. The P-factor itself is split off proteolytically.

Example 5: Microscopic detection of the adsorption of expressed hydrophobin on Teflon

A fluorescence-labeled HA antibody (Molecular Probes, Cat. No. A-21287) is used for the microscopic detection of the adsorption of expressed hydrophobin on Teflon.

Transformed host cells, produced according to one of Examples 1 to 4, are cultivated. Cells and any supernatant are harvested separately. Cells which have been transformed and cultured with a corresponding vector without hydrophobic genes or corresponding culture supernatants serve as reference sample.

The cells are opened mechanically (vibrating mill). Teflon plates are incubated at room temperature for 18 h in cell disruption or supernatant, rinsed with water (3 x 10 min). The treated teflon is then incubated in PBS with fluorescence-labeled antibody. Then again with PBS (3 x 15 min) rinsed and dried in an N ₂ jet. Finally, the fluorescence microscopic evaluation is carried out.

No fluorescence is observed on the reference sample (results not shown), but spot-shaped fluorescence after incubation in cell homogenate or culture supernatant (when the hydrophobin is secreted by the expressing cells).

Claims

claims

1. Expression construct comprising the coding nucleic acid sequence for a shuttle peptide construct of the general formula which can be processed by yeast cells

(Sig-SP),

containing the coding nucleic acid sequences for a) a signal peptide (Sig) in the 5'-3 'direction, processably linked to b) at least one shuttle peptide that can be secreted by the yeast cells

(SP).

2. Expression construct according to claim 1, wherein the shuttle peptide construct (Sig-SP) is derived from a polypeptide processed by yeasts of the genus Schizosaccharomyces, in particular from S. pombe.

3. Expression construct according to one of the preceding claims, wherein the shuttle peptide construct (Sig-SP) is derived from a pheromone preprotein from a yeast, wherein the pheromone (Pher) can be derived and secreted from the preprotein by N- and C-terminal processing.

4. Expression construct according to claim 3, wherein the signal polypeptide (Sig) is the proteolytically cleavable native signal polypeptide of the pheromone preprotein

5. Expression construct according to claim 4, wherein the C-terminal processed pheromone (Pher) comprises a C-terminal protease interface.

6. Expression construct according to one of the preceding claims, further comprising the coding nucleic acid sequence for a homologous or heterologous target protein (Targ), processably linked to the C-terminus of the shuttle peptide construct (Sig-SP).

7. Expression construct according to one of the preceding claims, comprising the coding nucleic acid sequence for a fusion protein processable by yeast cells of the general formula

Sig-L1 _n -Pher-L2 _m -Targ

wherein Sig, Pher and Targ are as defined above,

L1 and L2 stand for processable linkers and n and m stand independently for 0 or 1.

8. Expression construct according to one of the preceding claims, wherein the coding nucleic acid sequence for the shuttle peptide construct (Sig-SP) is a coding sequence for a signal polypeptide according to SEQ ID NO: 3 or a functional equivalent thereof, operatively linked to that for a mature phenomone Protein (P factor) encoding nucleic acid sequence according to SEQ ID NO: 5 or a functional equivalent thereof.

9. Expression construct according to one of the preceding claims, wherein the coding nucleic acid sequence for the shuttle peptide construct comprises a sequence according to SEQ ID NO: 1, optionally extended at the 3 'end by the coding sequence for a target protein (target)

10. Expression construct according to one of the preceding claims, wherein the target protein is a hydrophobin, in particular a hydrophobin of class I.

11. Expression construct according to claim 10, wherein the hydrophobin is selected from SEQ ID NO: 14 (DewA), SEQ ID NO: 19 (RdIA) SEQ ID NO: 20 (RdlB) SEQ ID NO: 21 (HYP1) and SEQ ID NO : 22 (HYP4) or from a nucleic acid sequence according to SEQ ID NO: 13.

12. Expression vector comprising, in operative linkage with at least one regulatory nucleic acid sequence, an expression construct according to one of the preceding claims.

13. Recombinant microorganism containing, optionally stably integrated into the host genome, at least one expression vector according to claim

12 or an expression construct according to one of claims 1 to 11.

14. Microorganism according to claim 13, selected from yeast.

15. Microorganism according to claim 14, selected from yeasts of the genus

Schizosaccharomyces, especially S. pombe.

16. Shuttle peptide construct (Sig-SP) processable from yeast cells, derived from a pheromone preprotein from a yeast, the pheromone passing through N- and C-terminal processing can be derived and secreted from the preprotein.

17. Shuttle peptide construct according to claim 16, comprising a signal polypeptide N-terminai processably linked to the C-terminal processed pheromone polypeptide.

18. Shuttle peptide construct according to claim 17, wherein the signal polypeptide is the proteolytically cleavable native signal polypeptide of the pheromone preprotein

19. Shuttle peptide construct according to claim 17, wherein the C-terminal processed pheromone polypeptide comprises the C-terminal protease interface.

20. Shuttle peptide construct according to one of claims 16 to 19, comprising a

Amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof.

21. A method for the recombinant production of a target protein, wherein one cultivates a microorganism according to one of claims 13 to 15, which the

Nucleic acid sequence encoding target protein is expressed and the target protein secreted into the culture medium is isolated.

22. The method of claim 21, wherein the target protein is a hydrophobin as defined in claim 10 or 11.

23. Nucleic acid coding for a shuttle peptide construct according to one of claims 16 to 20.

24. Nucleic acid as defined in one of claims 1 to 11.

25. Hydrophobin obtainable by a process according to claim 22.

26. Use of a hydrophobin according to claim 25 for surface treatment.

27. Use according to claim 26, wherein the surface of objects selected from glass, fibers, fabrics, leather, lacquered objects, films and facades is treated.

8. Use of a hydrophobin as defined in claim 10 or 11 for the surface treatment of fibers, fabrics and leather.

1.9

Fig.1

Constructs for the secretion of the hydrophobins from S. pombe

construct

Authentic signal sequence

Hydrophobin HA protein

P-factor signal peptide

PDe A

P-factor signal peptide + 6 amino acids

P-factor signal peptide including P-factor

PfakDewA

= postulated protease interfaces 9.2

Fig. 2

A) Genomic sequence of the DewA gene:

(The sequences of the two introns are underlined)

ATGCGCTTCA TCGTCTCTCT CCTCGCCTTC ACTG-CCGCGG CCACCGCAAC CGCCCTCCCG GCCTCTGCCG CAAAGAACGC GAAGCTGGCC ACCTCGGCGG CCTTCGCCAA GCAGGCTGAA GGCACCACCT GCAATGTCGG CTCGATCGCT TGCTGCAACT CCCCCGCTGA GACCAACAAC GACAGTCTGT TGAGCGGTCT GCTCGGTGCT GGCCTTCTCA ACGGGCTCTC GGGCAACACT GGCAGCGCCT GCGCCAAGGC GAGCTTGATT GACCAGCTGG GTCTGCTCGG TACGTGATCC CCACTCAGTC GCTCCCGGAG AGGCTGAGGG AAGACGAGCG ACGGTCTAGA AATGGTGTGC TAATAGATGC ATGTGTGCAG CTCTCGTCGA CCACACTGAG GAAGGCCCCG TCTGCAAGAA CATCGTCGCT TGCTGCCCTG AGGGAACCAC CAACGTACGT CTTTCAGATC TGCTACAAGT GAGGCGATCA AAACTAACAT ATTCCAGTGT GTTG-CCGTCG ACAACGCTGG CGCCGGTACC AAGGCTGAGT AA

B) Sequence of the DewA protein from Aspergill s nidulans:

MRFIVSLLAF TAAATATALP ASAAKNAKLA TSAAFAKQAE GTTCNVGSIA CCNSPAETNN DSLLSGLLGA GLLNGLSGNT GSACAKASLI DQLGLLALVD HTEEGPVCKN IVACCPEGTT NCVAVDNAGA GTKAE

(ATGCGCTTCA TCGTCTCTCT CCTCGCCTTC ACTGCCGCGG CCACCGCAAC CGCCCTCCCG

GCCTCTGCCG CAAAGAACGC GAAGCTGGCC ACCTCGGCGG CCTTCGCCAA GCAGGCTGAA

GGCACCACCT GCAATGTCGG CTCGATCGCT TGCTGCAACT CCCCCGCTGA GACCAACAAC

GACAGTCTGT TGAGCGGTCT GCTCGGTGCT GGCCTTCTCA ACGGGCTCTC GGGCAACACT

GGCAGCGCCT GCGCCAAGGC GAGCTTGATT GACCAGCTGG GTCTGCTCGC TCTCGTCGAC

CACACTGAGG AAGGCCCCGT CTGCAAGAAC AT7CGTCGCTT GCTGCCCTGA GGGAACCACC AACTGTGTTG CCGTCGACAA CGCTGGCGCC GGTACCAAGG CTGAGTAA)

C) Sequence of the HA tag:

LVPRGSIEGR GGRIFYPYDV PDYAGYPYDV PDYAGSYPYD VPDYAAQCGR

(CTGGT TCCGCGTGGA TCCATCGAAG GTCGTGGCGG CCGCATCTTT TACCCATACG

ATGTTCCTGA CTATGCGGGC TATCCCTATG ACGTCCCGGA CTATGCAGGA TCCTATCCAT ATGACGTTCC AGATTACGCT GCTCAGTGCG GCCGCTAATA G) 3.9

Fϊg.3

A) Sequence of the P-factor preprotein:

MKITAVIALL FSLAAASPIP VADPGWSVS KSYADFLRVY QS NTFANPD RPNLKKREFE

AAPAKTYADF LRAYQS NTF VNPDRPNLKK. RE FEAAPEKS YADFLRAYHS NTFVNPDRP

NLKKREFEAA PAKTYADFLR AYQSWNTFVM PDRPNLKKRT EEDEENEEED EEYYRFLQFY I TVPENSTI TDVNITAKFE S

(ATGAAGATCA CCGCTGTCAT TGCCCTTTTA TTCTCACTTG CTGCTGCCTC ACCTATTCCA

GTTGCCGATC CTGGTGTGGT TTCAGTTAGC AAGTCATATG CTGATTTCCT TCGTGTTTAC

CAAAGTTGGA ACACTTTTGC TAATCCTGAT AGACCCAACT TGAAAAAGCG CGAATTCGAA

GCTGCTCCCG CAAAAACTTA TGCTGATTTC CTTCGTGCTT ATCAAAGTTG GAACACTTTT

GTTAATCCTG ACAGACCCAA TTTGAAAAAG CGTGAGTTTG AAGCTGCCCC AGAGAAGAGT

TATGCTGATT TCCTTCGTGC TTACCATAGT TGGAACACTT TTGTTAATCC TGACAGACCC

AACTTGAAAA AGCGCGAATT CGAAGCTGCT CCCGCAAAAA CTTATGCTGA TTTCCTTCGT

GCTTACCAAA GTTGGAACAC TTTTGTTAAT CCTGACAGAC CCAACTTGAA AAAGCGCACT

GAAGAAGATG AAGAGAATGA GGAAGAGGAT GAAGAATACT ATCGCTTTCT TCAGTTTTAT ATCATGACTG TCCCAGAGAA TTCCACTATT ACAGATGTCA ATATTACTGC CAAATTTGAG AGCTAA)

B) Sequence of the cleavable signal peptide and the subsequent 6 amino acids of the P-factor preprotein:

MKITAVIALL FSLAAASPIP VADPGV

(ATGAAGATCA CCGCTGTCAT TGCCCTTTTA TTCTCACTTG CTGCTGCCTC ACCTATTCCA GTTGCCGATC CTGGTGTG)

C) Sequence used for the "P-Shuttle":

MKITAVIALL FSLAAASPIP VADPGWSVS KSYADFLRVY QSWNTFANPD RPNLKKR (ATGAAGATCA CCGCTGTCAT TGCCCTTTTA TTCTCACTTG CTGCTGCCTC ACCTATTCCA

GTTGCCGATC CTGGTGTGGT TTCAGTTAGC AAGTCATATG CTGATTTCCT TCGTGTTTAC CAAAGTTGGA ACACTTTTGC TAATCCTGAT AGACCCAACT TGAAAAAGCG C) Fig. 4

Fusion protein consisting of the "P-Shuttle" sequence, the mature DewA and the C-terminally fused HA tag:

MKITAVIALL FSLAAASPIP VADPGWSVS KSYADFLRVY QSWNTFANPD RPNLKKRLPA

SAAKNAKLAT SAAFAKQAEG TTCNVGSIAC CNSPAETNND SLLSGLLGAG LLNGLSGNTG

SACAKASLID OLGLLALVDH TEEGPVCKNI VACCPEGTTN CVAVDNAGAG TKAELVPRGS

IEGRGGRIFY PYDVPDYAGY PYDVPDYAGS YPYDVPDYAA QCGR

(ATGAAGATCA CCGCTGTCAT TGCCCTTTTA TTCTCACTTG CTGCTGCCTC ACCTATTCCA GTTGCCGATC CTGGTGTGGT TTCAGTTAGC AAGTCATATG CTGATTTCCT TCGTGTTTAC CAAAGTTGGA ACACTTTTGC TAATCCTGAT AGACCCAACT TGAAAAAGCG CCTCCCGGCC TCTGCCGCAA AGAACGCGAA GCTGGCCACC TCGGCGGCCT TCGCCAAGCA GGCTGAAGGC ACCACCTGCA ATGTCGGCTC GATCGCTTGC TGCAACTCCC CCGCTGAGAC CAACAACGAC AGTCTGTTGA GCGGTCTGCT CGGTGCTGGC CTTCTCAACG GGCTCTCGGG CAACACTGGC AGCGCCTGCG CCAAGGCGAG CTTGATTGAC CAGCTGGGTC TGCTCGCTCT CGTCGACCAC ACTGAGGAAG GCCCCGTCTG CAAGAACATC GTCGCTTGCT GCCCTGAGGG AACCACCAAC TGTGTTGCCG TCGACAACGC TGGCGCCGGT ACCAAGGCTG AGCTGGTTCC GCGTGGATCC ATCGAAGGTC GTGGCGGCCG CATCTTTTAC CCATACGATG TTCCTGACTA TGCGGGCTAT CCCTATGACG TCCCGGACTA TGCAGGATCC TACGGTAGGAGCC TATGGCTGATG CAC

7.9

Figure 8

ÜS pellet

8.9

Fig. 9

A) mfm1 ⁺ gen

Sequence of the mfm1 preprotein

MDS ANSVSSSSWNAGNKPAETLNKTVKNYTPKVPYMCVIA

Sequence of the mfm1 * gene atggactcaa tggctaactc cgtttcttcc tcctctgtcg tcaacgctgg caacaagcct gctgaaactc ttaacaagac cgttaagaat tataccccca aggttcctta catgtgtgtc attgcataa mfml matures M-pheromone

YTPKVPYMC

DNA sequence of the mature mfml M-pheromone tataccccca aggttcctta catgtgt

B) mfm2 ⁺ gene

Sequence of the mfm2 preprotein DSIATNTHSSSIVNAYNNNPTDWKTQNIKNYTPKVPYMCVIA

Sequence of the mfm2 ⁺ gene atggactcca ttgcaactaa cactcattct tcatccattg tcaatgccta caacaacaat cctaccgatg ttgtaaaaac tcaaaacatt aaaaattata ctccaaaggt tccttatatg tgtgtaattg cttaa Mfheromon matures

YTPKVPYMC

DNA sequence of the mature mfm2 M pheromone tata ctccaaaggt tccttatatg tgt

C) mfm3 ⁺ gene

Sequence of the mfm3 preprotein

MDSMANTVSSSWNTGNKPSΞTLNKTVKNYTPKVPYMCVIA

Sequence of the mfm3 * gene atggactcaa tggctaacac tgtttcttcc tccgtcgtta acactggcaa caagccttct gaaactctta acaagactgt taagaattat acccccaagg ttccttacat gtgtgtcatt gcataa mfm3 matures M-pheromone

YTPKVPYMC

DNA sequence of the mature mfm3 M pheromone tat acccccaagg ttccttacat gtgt 9.9

Fig. 10

Genomic sequence of the RodA gene

ATGAAGTTCT CCATTGCTGC CGCTGTCGTT GCTTTCGCCG CCTCCGTCGC GGCCCTCCCT CCTGCCCATG ATTCCCAGTT CGCTGGCAAT GGTGTTGGCA ACAAGGGCAA CAGCAACGTC AAGTTCCCTG TCCCCGAAAA CGTGACCGTC AAGCAGGCCT CCGACAAGTG CGGTGACCAG GCCCAGCTCT CTTGCTGCAA CAAGGCCACG TACGCCGGTG ACACCACAAC CGTTGATGAG GGTCTTCTGT CTGGTGCCCT CAGCGGCCTC ATCGGCGCCG GGTCTGGTGC CGAAGGTCTT GGTCTCTTCG ATCAGTGCTC CAAGCTTGAT GTTGCTGGTC AGTTCTTCGA AAATCACTTT CGTGATGCCC CAATGCTAAC AATTACCA.GT CCTCATTGGC ATCCAAGATC TTGTCAACCA

GAAGTGCAAG CAAAACATTG CCTGCTGCCA GAACTCCCCC TCCAGCGCGG TATGTTCCCT TGTTTTACAG CTTATTCACT TAAACCGATT AATCTAACAA CGCTCACA.GG ATGGCAACCT TATTGGTGTC GGTCTCCCTT

GCGTTGCCCT TGGCTCCATC CTCTAA

DNA sequence of the open reading frame (ORF) of the RodA gene

ATGAAGTTCT CCATTGCTGC CGCTGTCGTT GCTTTCGCCG CCTCCGTCGC GGCCCTCCCT CCTGCCCATG

ATTCCCAGTT CGCTGGCAAT GGTGTTGGCA ACAAGGGCAA CAGCAACGTC AAGTTCCCTG TCCCCGAAAA

CGTGACCGTC AAGCAGGCCT CCGACAAGTG CGGTGACCAG GCCCAGCTCT CTTGCTGCAA CAAGGCCACG

TACGCCGGTG ACACCACAAC CGTTGATGAG GGTCTTCTGT CTGGTGCCCT CAGCGGCCTC ATCGGCGCCG

GGTCTGGTGC CGAAGGTCTT GGTCTCTTCG ATCAGTGCTC CAAGCTTGAT GTTGCTGTCC TCATTGGCAT

CCAAGATCTT GTCAACCAGA AGTGCAAGCA AAACATTGCC TGCTGCCAGA ACTCCCCCTC CAGCGCGGAT GGCAACCTTA TTGGTGTCGG TCTCCCTTGC GTTGCCCTTG GCTCCATCCT CTAA

Sequence of the RodA protein

MKFSIAAAW AFAASVAALP PAHDSQFAGN GVGNKGNSNV KPPVPENVTV KQASDKCGDQ AQLSCCNKAT YAGDTTTVDE GLLSGALSGL IGAGSGAEGL GLFDQCSKLD VAVLIGIQDL VNQKCGLPCQNIAGQILVK