WO2017055587A1

WO2017055587A1 - Recombinant production of peptides

Info

Publication number: WO2017055587A1
Application number: PCT/EP2016/073479
Authority: WO
Inventors: Max Fabian FELLE; Carsten Schwalb; Daniel HÜMMERICH; Markus Fehr
Original assignee: Basf Se; Basf S.A. Condominio Rochaverá, Torre Crystal
Priority date: 2015-10-01
Filing date: 2016-09-30
Publication date: 2017-04-06

Abstract

The present invention relates to novel repetitive self-assembling precursor proteins, nucleic acid sequences and expression constructs encoding the same, and to methods for the recombinant production of peptides using such precursor proteins.

Description

Recombinant production of peptides

The present invention relates to novel repetitive self-assembling precursor proteins, nucleic acid sequences and expression constructs coding therefor, and to methods for the recombinant production of peptides using such precursor proteins.

Background of the Invention Various methods for the biotechnological production of peptides are known. Since the stability of short polypeptide chains in microbial host cells is usually low and the free peptides may possibly have a toxic effect on the host organism (eg antimicrobial peptides), in most methods larger precursor proteins are formed, from which the peptide after the purification of the precursor protein is cut out.

One way to obtain a stable precursor protein is to express a peptide together with a stable protein as a fusion protein. The properties of the fusion protein, which have a great influence on subsequent processing steps, are determined largely independently of the peptide sequence by the fusion partner and are thus readily controllable and suitable for the production of peptides with different sequences.

WO 2008/085543 describes a special process for the preparation of proteins and peptides with the aid of a fusion protein. In addition to the desired peptide sequence, this fusion protein contains a fusion partner which ensures that the fusion protein displays an inverse phase transition behavior. On the one hand, this behavior allows a simple and cost-effective purification of the fusion protein from the cellular context. On the other hand, the separation of the fusion partner after the proteolytic cleavage of the peptide can also be carried out easily and inexpensively. While a fusion protein can often be obtained in good yields, the proportion of peptide on the precursor protein is usually low and thus the efficiency of the process is not optimal. In another approach, repetitive precursor proteins containing multiple copies of the desired peptide are produced recombinantly. WO 03/089455 describes the preparation of multimeric precursor proteins from which the desired peptide sequences which have antimicrobial properties are excised by acid cleavage.

There are a number of other published approaches (examples: Metlitskaya et al., Biotechnol Appl. Biochem 39: 339-345 (2004); Wang & Cai Appl. Biochem and Biotechnol. 141; 203-213 (2007)) which have been shown in that peptide sequences or families of peptide sequences can be prepared by a specific method with the aid of repetitive precursor proteins. In part, the use of special auxiliary sequences located between repeats of the desired peptide sequences has been described. In particular, anionic auxiliary sequences have been proposed which are said to reduce the deleterious effect of cationic antimicrobial peptide sequences within a repetitive precursor protein on the host cell (see, for example, WO 00/31279 and US 2003/0219854). While in this repetitive approach the proportion of the desired peptide sequence on the precursor protein is higher than on fusion proteins, the properties of the repetitive precursor protein are strongly influenced by the sequence of the desired cationic peptide.

The inventors have so far no method is known in which any peptide sequences can be prepared by a simple, inexpensive and efficiently feasible protocol using repetitive precursor proteins. Various antimicrobial peptides have been previously described and summarized in reviews (Hancock, REW, and Lehrer, R., 1998 Trends in Biotechnology, 16: 82-88, Hancock, REW, and Sahl, HG, 2006, Nature Biotechnology, 24: 1551-1557). Fusion peptides that combine two effective peptides are also described in the literature. Wade et al. report the antibacterial activity of various fusions of Cecropin A from Hyalophora cecropia and the bee venom melittin (Wade, D. et al., 1992, International Journal of Peptide and Protein Research, 40: 429-436). Shin et al. describe the antibacterial activity of a fusion peptide of cecropin A from Hyalophora cecropia and magainin 2 from Xenopus laevis, consisting of 20 amino acids. acids. Cecropin A consists of 37 amino acids and shows activity against Gram-negative bacteria, but lower activity against Gram-positive bacteria. Magainin 2 consists of 23 amino acids and is active against bacteria as well as tumor cell lines. Compared to the fusion of cecropin A and melittin, this fusion shows markedly lower hemolytic activity with comparable antibacterial activity (Shin, SY Kang, JH, Lee, MK, Kim, SY, Kim, Y., Hahm, KS, 1998, Biochemistry and Molecular Biology International, 44: 1 1 19-1 126). US 2003/0096745 A1 and US Pat. No. 6,800,727 B2 claim these fusion peptides, consisting of 20 amino acids and variants of this fusion, which are more positively charged and more hydrophobic due to the exchange of amino acids, in particular of positively charged amino acids and hydrophobic amino acids.

Further developments of this cecropin A magainin-2 fusion peptide are described by Shin et al. 1999. It was found that the peptide with SEQ ID NO: 6 had a lower hemolytic activity compared to the starting fusion, but the antibacterial activity against Escherichia coli and Bacillus subtilis was not impaired (Shin et al., 1999 Journal of Peptides Research, 53: 82-90).

From WO2010 / 139736 precursor proteins of the general formula (Pep-Aux) _x or (Aux-Pep) _{x are} known, wherein x> 1, and wherein the aux elements are the same or different and comprise amino acid sequence elements, the self-assembling properties of the precursor protein to lend; and the Pep elements are the same or different and comprise the amino acid sequence of identical or different peptide molecules. The self-assembling sequence motifs (SA elements) are, in particular, the motifs A _n , (GA) _m , V _n , (VA) _m , and (VVAA) ₀ , where n stands for an integer value of 2 to 12, m for a integer value from 2 to 10, and o stands for an integer value from 1 to 6.

Summary of the invention

It was therefore an object of the present invention to provide a versatile, further improved process for the production of peptides with the aid of repetitive precursor proteins. This object has been achieved by providing specific repetitive precursor proteins as further defined in the appended claims.

Surprisingly, it has been found that an advantageous production of valuable peptides is also possible if a modular precursor protein is provided which contains no sequence motifs (SA elements) with self-assembling properties known from the prior art.

Description of the Figures In the accompanying figures shows

Figure 1 is a helical wheel representation of amino acid sequences, as a projection of an alpha helical structure. The amino acid sequence A1-A7 (A) contained in the repetitive precursor protein is represented on a circle (B). From this arrangement, the location of the amino acids in an alpha helix becomes visible;

FIG. 2 a and b show, on the basis of SDS gels, the results of the expression of the peptide P18 after 0, 3, 5 hours of induction by IPTG using precursor epitems according to SEQ ID NO: 1 to 6.

Detailed Description of Individual Aspects of the Invention 1. General Definitions Unless otherwise indicated, the first amino acid of the sequences disclosed herein represents the particular N-terminus.

"Helical wheel representations are a type of plot or visual representation used to illustrate the properties of α-helices in proteins or peptides. The amino acid sequence, which can form a helical region of a secondary structure of the protein or peptide, is applied in a rotating manner about a virtual axis of rotation, the angle of rotation between successive amino acids being 100 °, so that the final representation is viewed along the helical axis of rotation , The plot shows whether hydrophobic, polar and hydrophilic amino acids are evenly distributed or helix halves with more hydrophobic or polar / hydrophilic character can form. "Amphiphilic peptides" include both hydrophilic and hydrophobic and / or polar amino acid residues. "Cationic amphiphilic peptides" are those in which the hydrophilic amino acid residues are formed wholly or predominantly of basic amino acid residues.

"Anionic amphiphilic peptides" are those in which the hydrophilic amino acid residues are formed wholly or predominantly of acidic amino acid residues.

"Polar amino acid residues" are selected from the residues serine (S), threonine (T) cysteine (C), tyrosine (Y), asparagine (N), and glutamine (Q).

"Hydrophobic amino acid residues" are selected from alanine (A), valine (V), leucine (L), isoleucine (I), phenylalanine (F), proline (P) methionine (M) and tryptophan (W).

"Acidic amino acid residues" or "anionic amino acid residues" are selected from aspartic acid (D) and glutamic acid (E); "Basic amino acid residues" or "cationic amino acid residues" are selected from lysine (K), arginine (R) and histidine (H).

"Amphipathic" and "amphiphilic" are used interchangeably herein. Under a sequence "derived" or "homologous" from a concretely disclosed sequence, e.g. a deduced amino acid or nucleic acid sequence, according to the invention, unless otherwise stated, is understood to mean a sequence having an identity to the starting sequence of at least 80% or at least 90%, in particular 91%, 92%, 93%, 94%, 95% %, 96%, 97%, 98% and 99%.

"Self-assembling" properties of the precursor protein are characterized in that the precursor protein already during expression "spontaneously", ie by itself without additionally required action forms stable associates, or the formation of such stable associates of soluble precursor proteins "inducible", ie by Precursor proteins having self-assembling properties have the advantage over other precursor proteins that they can be purified in a simple and efficient manner. Such associates include, usually exclusively or substantially, the formation of non-covalent bonds, such as hydrogen bonds, ionic and / or hydrophobic interactions. Solutions of cosmotropic salts can be used as "trigger." Cosmotropic salts containing at least one ion species which, according to the so-called Hofmeister series, have more cosmotropic properties than sodium or chloride ions are examples of such salts Examples of such salt solutions are 0.5 M potassium phosphate or 0.8 M ammonium sulfate.

2. Preferred Embodiments The invention particularly relates to the following embodiments:

1 . A synthetic, in particular recombinantly produced, precursor protein comprising an enzymatically and / or chemically cleavable repetitive sequence (also referred to as repetitive modules) of peptide-linked amino acid partial sequences, the repetitive sequence having the following general formula (1):

- [- L-SU-L-Sp-Pep-Sp -] - _n (see synonym for - [- Sp-Pep-Sp-L-SU-L -] - _n ) (1) (spelling N-terminus - > C-terminus) wherein in these modules

n is an integer value of more than 1, e.g. up to 100 or up to 50, preferably 2 to 32, in particular 4 to 16, stands

Pep is in each case identical or different, in particular the same, a cationic amphiphilic value peptide,

Sp is the same or different, in particular the same, chemically or enzymatically cleavable cleavage peptide sequences,

SU stands for the same or different, in particular the same, anionic amphiphilic protective peptides, wherein in particular each SU in its helical-helical projection has an amphiphilic distribution of hydrophobic ben / polar, in particular hydrophobic, amino acid residues and anionic amino acid residues, and

L is the same or different, especially the same, linker. The elements Pep and SU independently have a sequence length of 5 to 100, in particular 6 to 50, preferably 7 to 40, particularly preferably 7 to 30 contiguous amino acid residues.

Preferably, however, the sequence lengths of Pep and SU are coordinated and differ by less than ± 70, in particular less than ± 60, preferably less than ± 40, in particular less than ± 25%.

Most preferred are constructs in which Pep and SU are approximately equal in length and less than ± 5, especially less than ± 4, most preferably less than ± 3, preferably less than ± 2, such as ± 1 or ± 0, amino acid residues in differ in length.

The optimal sequence lengths of SU and Pep can be determined by simple expression experiments to determine the expression efficiency. For example, with a sequence length of Pep of about 18 or 19

Amino acid residues observe optimal expression when using SU element with approximately the same sequence length, wherein the expression deteriorates with decreasing SU sequence length, but even at SU sequence lengths of about 7 or 8 amino acid residues still obtain useful expression results.

The above statements on the selection of suitable sequence lengths for SU and Pep apply analogously to the modules -L-SU-L and -sp-Pep-Sp-. On the one hand, the elements L and Sp are relatively short in comparison to the elements SU and Pep (and comprise 1 to 10, in particular 1 to 5 amino acids, as described in more detail below) and thus have a similar size to one another.

Preferably, the sequence lengths of -L-SU-L and - Sp-Pep-Sp- are coordinated and differ by less than ± 70, in particular less than ± 60, preferably less than ± 40, in particular less than ± 25%. Most preferred are constructs in which -L-SU-L and - Sp-Pep-Sp- are about the same length and less than ± 5, in particular less than ± 4, especially less than ± 3, preferably less than ± 2, such as ± 1 or ± 0, amino acid residues differ in length.

In a further embodiment, the anionic amphiphilic SU elements according to the invention are chosen such that, at pH = 7, they have a total charge of less than 0, such as e.g. -1 to -20. or -1 to -10 or -1 to -5

Preferably, the SU elements in the precursor protein are capable of forming an amphiphilic helical structure.

Preferably, the Pep element is a (at pH = 7) cationic, a positive total charge (greater than 0, such as +1 to +20 or +1 to +10 or +1 to +5) containing peptide, such as an antimicrobial peptide.

The individual structural elements L, Sp, SU and Pep are peptidically linked to one another;

The Sp elements allow Pep to be specific, i. exclusively or essentially at the defined Sp-sequence from the precursor protein can be cleaved by chemical or enzymatic means. Advantageously, the amino acid composition of the Sp- and L-

Elements should be chosen so that the formation of secondary structures (α-helix and β-sheet) is largely avoided within these elements. The use of pattern breaking amino acid residues within these Sp and L elements is therefore preferred. On the basis of the values known from the literature (see "Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding" (Alan Fersht, I BN: 0-7167-3268-8, 1999) for the Relative Helix Stability ( RHS) and relative β-sheet stability (RSS) of individual amino acid residues (with alanine as a reference and a value of 0 kcal / mol), the skilled person can tailor the concrete elements. Preferred structure-breaking amino acid residues are G and P.

In a further preferred embodiment, the proportion of G and / or P in the total number of amino acid residues of the L elements is at least 20%, e.g. at least 30, 40, 50, 60, 70, 80, 90 or 100%.

In a further preferred embodiment, the proportion of G and / or P in the total number of amino acid residues of the Sp elements is at least 20%, e.g. at least 30, 40, 50, 60, 70, 80, or 90%.

In a further preferred embodiment, the elements L and / or the elements Sp, in particular L and Sp, themselves have no self-assembling properties and essentially do not contribute to the self-assembly of the precursor protein according to the invention. In particular, the elements L and Sp do not comprise sequence motifs known from the prior art (in particular WO2010 / 139736), self-assembling, or self-assembling sequences (so-called SA elements), in particular the SA sequence motifs A _n , (GA _m , V _n , (VA) _m , and (VVAA) ₀ , where n is an integer from 2 to 12, m is an integer from 2 to 10, and o is an integer from 1 to 6 and in particular n = 7 - 9, m = 6 - 7 and o = 2-3.

In particular, the precursor proteins are chosen so that the Pep element is not derived from spider silk proteins. In particular, those elements are not included which contain glycine-rich elements, in particular repetitive glycine-rich elements, in particular GGX or GPGXX (as described, for example, in WO2006 / 008163). Precursor protein according to embodiment 1, wherein n has an integer value of 2 to 10, in particular 3 to 8, especially 4 to 6.

Precursor protein according to one of the preceding embodiments, wherein Sp has the amino acid sequence motif DX, wherein X is an arbitrary amino acid residue, and Sp has in particular a sequence length of 2 to 6, in particular 2 to 4. Preferably, the remaining amino acid residues in the Sp element are chosen such that no stable secondary structures are formed (see above provisos). In particular, the proportion of residues G and / or P should be at least 20% relative to the total number of amino acid residues of the Sp element except for the sequence length of the Sp element.

A precursor protein according to embodiment 3, wherein the Sp elements are independently selected from the sequences of the general formula

XADPXB

wherein X _A and X _B independently of one another represent a chemical bond or one of the amino acid residues G, S, A, I, L, or Q, and in particular G or S.

A precursor protein according to embodiment 4, wherein the Sp elements are independently selected from the sequences of GDPG, GDPS, GDP, DPG, DPS, SDP and DP. 6. Precursor protein according to one of the preceding embodiments, wherein L has a length of 1 to 10, in particular 2 to 10, preferably 2 to 5 contiguous amino acid residues.

Preferably, the amino acid residues in the L element are chosen so that no stable secondary structures form (compare the above specifications). In particular, the proportion of residues G and / or P in the total number of amino acid residues of the L elements should be at least 20%, based on the mean length of the L element. A precursor protein according to Embodiment 6, wherein the L elements are independently selected from GSGPG, GGPG, SGPG, GSPG, GGP, GPG, GPS, GAG, GAA, GGA, GQQ, GGQ, GSS, SGA, GGY, GGL, GS (and the sequence analogs derivable by permutation of the amino acid residues thereof) and G, in particular the L-elements containing a GS (or SG) and / or GP (or PG) motif thereof. Precursor protein according to one of the preceding embodiments, in which the cationic amphiphilic value peptide Pep represents identical or different amino acid sequences according to SEQ ID NO: 7 Xi X ₂ KX ₃ X ₄ X ₅ KIP X ₁₀ KFX ₆ X ₇ X ₈ AX ₉ KF (SEQ ID NO: 7), in which

X _{10 represents} a peptide bond or one or any two or more basic or hydrophobic amino acid residues or one or two proline residues; and

Xi to X ₉ denote any basic or hydrophobic amino acid residues other than proline;

and cationic amphiphilic mutants or derivatives thereof. Precursor protein according to one of the preceding embodiments, wherein the cationic amphiphilic value peptide Pep represents identical or different amino acid sequences according to SEQ ID NO: 8

SEQ ID NO Xi X ₂ KX ₃ X ₄ X ₅ KIP Xu X ₁₂ KFX ₆ X ₇ X ₈ AX ₉ KF (SEQ ID NO: 8), in which

Xi for lysine, arginine or phenylalanine,

X ₂ for lysine or tryptophan,

X ₃ for leucine or lysine,

X ₄ for phenylalanine or leucine,

X ₅ for leucine or lysine,

X ₆ for leucine or lysine,

X ₇ for histidine or lysine,

X ₈ for alanine, leucine, valine or serine,

X ₉ for leucine or lysine,

Xu for proline or a chemical bond, and

Xi _{2 is} proline or a chemical bond,

and cationic amphiphilic mutants or derivatives thereof. Precursor protein according to one of the preceding embodiments, wherein the cationic amphiphilic value peptide Pep for an amino acid sequence according to SEQ ID NO: 9

KWKLFKKIPKFLHLAKKF (SEQ ID NO: 9)

stands. Precursor protein according to one of the preceding embodiments, wherein the SU elements are identical or different, in particular identical and each SU element is an amphiphilic, in particular amphiphilic anionic peptide comprising a sequence section of at least seven peptidisch linked

Amino acids capable of forming an amphiphilic α-helix, the helix having in its vertical projection (helical wheel projection) a separation of the amino acid residues into a hydrophobic and a hydrophilic half of the helix, the hydrophobic half of the helix having at least 3 adjacent ones in the vertical projection has different hydrophobic amino acid residues and the hydrophilic helix half at least 3 in the vertical projection adjacent same or different hydrophilic (ie, acidic, basic and possibly polar) amino acid residues.

In particular, the SU elements of the present invention which protect the host cell from the deleterious effects of the repetitive precursor protein, particularly the cationic value peptide, therein

In a specific embodiment, the precursor protein contains anionic auxiliary sequences SU which protect the host cell from the deleterious effects of cationic antimicrobial peptide sequences Pep contained in the repetitive precursor protein. In particular, these protective sequences contain negatively charged amino acids (Asp (D), Glu (E)). In particular, the SU sequence contains a number of negatively charged amino acids glutamate and / or aspartate such that the total net charge at pH = 7 within the repetitive precursor protein is greater than -10 and less than +10, especially greater than -5 and less than +5 , such as is greater than -2 and less than +2.

In another specific embodiment, the negatively charged protective sequence (SU) forms an amphipathic helix. An amphipathic helix according to The present invention is then formed when, in the circular array (ie, in its axial (along the helical axis) projection, helical wheel projection) of a sequence of 7 amino acids (A1-A7) sequential in the primary structure in the following order : A1 - A5 - A2 - A6 - A3 - A7 - A4 (Figure 1) at least 3 amino acids adjacent to each other on the circle are hydrophobic amino acids (Ala, Met, Phe, Leu, Val, Ile) or glycine and 3 on the circle next to each other hydrophilic (acidic, basic and polar) amino acids (Cys, Thr, Ser, Trp, Tyr, His, Glu, Gin, Asp, Asn, Lys, Arg) or glycine. This circular arrangement is also referred to as "helical wheel projection".

Precursor protein according to one of the preceding embodiments, wherein the proportion of charged amino acid residues of the SU element is selected such that the total net charge of the precursor protein at pH = 7 is greater than -10 and less than +10. Precursor protein according to one of the preceding embodiments, wherein the SU elements of the precursor protein are identical or different, in particular identical and have an amino acid sequence according to SEQ ID NO: 10:

Ex _a Ex _b XcEEx _d Xe EXfXgX _h XiXkEExi (SEQ ID NO: 22) where x _a to X | represent an amino acid residue other than E selected from hydrophobic and polar amino acid residues

As an example:

E (W, F, L, I or A) E (W, F, L, I or A) (W, F, L, I or A) EE (W, F, L, I or A) (S, Q or A) E (W, F, L, I or A) (W, F, L, I or A) (Q, S or A) (S, A or Q) (W, F, L, I or A ) EE (W, F, L, I or A) (SEQ ID NO: 23), or

E (W, F, L, or I) E (L, F, or I) (F, L, or Ι) ΕΕ (I, A, L, or F) (S, Q, or A) E (F, L, I, or A) (L, F, I, or A) (Q, S, or A) (S, A, or Q) (L, F, I, or A) EE (F, L, I , or A) (SEQ ID NO: 24), Precursor protein according to one of the preceding embodiments, wherein the SU elements are selected from the sequences:

EWELFEEISEFLQSLEEF (SEQ ID NO: 18),

EWEFLEEISELFQSLEEF (SEQ ID NO: 19),

EWEFLEEISELFQALEEF (SEQ ID NO: 20) and

EWEFLEEASELFQALEEF (SEQ ID NO: 21)

Precursor protein according to one of the preceding embodiments, which additionally has an N-terminal or C-terminal, in particular N-terminal TAG sequence motif.

Precursor protein according to one of the preceding embodiments, wherein the additional N-terminal or C-terminal, in particular N-terminal, TAG sequence motif is selected from a methionine residue and an auxiliary sequence. A precursor protein according to embodiment 16, wherein the TAG-auxiliary sequence favors the expression, stability and / or work-up of the precursor protein. A precursor protein according to embodiment 17, wherein the TAG-auxiliary sequence is selected from a His-tag, T7-tag, S-tag, c-Myc-10-tag, Strep-tag or HA-tag and optionally via a further linker sequence L1 1 to 6, in particular 3 to 5, any contiguous amino acid residues N-terminal or C-terminal, in particular N-terminal, to the repetitive sequence motif of the above formula (1) is bound.

For example, L1 may be defined as L (see above). However, conventional linkers may also be employed, comprising a series of glycine and serine residues, such as glycine and serine residues. GGSGGS (SEQ ID NO: 39), GSGGGS (SEQ ID NO: 40), and variations thereof.

Precursor protein according to embodiment 18, of general formula (2) TAG-L1 - [- L-SU-L-Sp-Pep-Sp] _n (2)

wherein

TAG, L, SU, Sp, Pep and n are as defined above and

L1 for a chemical bond or other linker sequence comprising 1 to 5 contiguous amino acid residues, e.g. RGSM, GGGS,

GSGGM, QGSG, SGGGR, GGSGG, GGGGS (SEQ ID NOs: 25 to 31) (as well as the sequence analogs derivable by permutation of the amino acid residues thereof). A precursor protein according to any one of the preceding embodiments, selected from the amino acid sequences SEQ ID NO: 2 to SEQ ID NO: 6, and analogs derived therefrom having a sequence identity of at least 80, 85, 90, 95, 96, 97, 98 or 99 %. 21. Nucleic acid sequence encoding at least one precursor protein according to one of the preceding embodiments

22. An expression cassette comprising at least one nucleic acid sequence according to embodiment 21, operatively linked to at least one regulatory nucleic acid sequence.

23. A recombinant vector for transforming a eukaryotic or prokaryotic, in particular prokaryotic, host, comprising a nucleic acid sequence according to embodiment 21 or an expression cassette according to embodiment 22.

24. Recombinant eukaryotic or prokaryotic, in particular prokaryotic, host, preferably a recombinant. co // strain comprising a nucleic acid sequence according to one of embodiment 21 or an expression cassette according to embodiment 22 or a vector according to embodiment 23.

25. A process for the preparation of a peptide of value Pep, which comprises preparing a precursor protein according to any of embodiments 1 to 20 and cleaving the pep peptides from the precursor protein. 26. The method of embodiment 25, wherein the precursor protein is produced in a recombinant microorganism carrying at least one vector according to embodiment 19.

27. Method according to embodiment 26, wherein the precursor protein is produced in a recombinant E. coli strain, preferably in the form of inclusion bodies. 28. The method according to any one of embodiments 25 to 27, wherein the expressed precursor protein, after it has optionally been converted into a stably associated form, to release the desired peptide (Pep) cleaved chemically or enzymatically.

Method according to one of embodiments 25 to 28, comprising the following steps:

a) fermentation of a recombinant host according to embodiment 24 with expression of a precursor protein according to formula (1)

b) optionally separation of the cell mass,

c) digestion of the cells and simultaneous or subsequent chemical or enzymatic cleavage of the precursor protein according to formula (1) on the Sp elements

d) optionally separating cell fragments, e.g. by centrifugation, and e) purification of the cationic, amphiphilic Wertpeptids, in particular by adsorption, chromatography, extraction, crystallization precipitation, optionally followed by solubilization or combinations of such measures.

Further preferred embodiments

30. Inventive precursor proteins according to any one of the preceding embodiments are further characterized by having self-assembling properties such that they spontaneously, ie by themselves or inducibly, form stable, non-covalent associates which under standard conditions, such as in particular by 0.2 M NaOH within one hour or in particular 30 minutes or of 2 M urea or 1 M guanidinium hydrochloride in each case within 10 min or especially 5 min at room temperature are not resolvable. If at least one of these three mentioned criteria is fulfilled, then a stable associate according to the invention is present. The invention further provides a process for the preparation of a desired peptide (Pep), wherein

a) preparing a precursor protein according to any of the above embodiments, b) cleaving the pep peptides from the precursor protein chemically or enzymatically; and

c) if appropriate, the peptide enzymatically or chemically modified, such as amidated, esterified, oxidized, alkylated or linked (eg, by native chemical ligation or via a Michael addition) with another molecule; wherein, for example, the peptide is modified with a molecule that increases the hydrophobicity of the peptide, such as modified with a molecule containing an alkyl radical; wherein the modification may be before or after optional purification of the peptide. Suitable alkyl radicals are, for example, C ₂ -C ₆ -alkyl radicals, such as ethyl, isopropyl or n-propyl, n-, i-, sec- or tert-butyl, n- or i-pentyl; also n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl n-tridecyl, n-tetradecyl, n-pentadecyl and n-hexadecyl, and the one or more times branched analogs thereof, and optionally substituted modifications thereof, which include one or more, such as 1, 2 or 3 halogen (such as F, Cl, Br), hydroxy, mercapto amino, CC ₄ -

Alkylamino substituents, or may be interrupted by one or more, such as 1, 2 or 3 heteroatoms, such as O or N, in the alkyl chain. Ci-C ₄ - alkyl is especially methyl, ethyl, i- or n-propyl, n-, i-, sec- or tert-butyl.

The invention also relates to a process for the preparation of a desired peptide (Pep), wherein the expressed precursor protein, after it has optionally been converted into a stably associated form, is purified and cleaved chemically or enzymatically to liberate the desired peptide (Pep) , 33. Another object of the invention is a process for the preparation of a desired peptide (Pep) which comprises the following work-up steps:

Washing the associates of the precursor protein with a solvent which does not or substantially not dissolve contaminating proteins, but does not dissolve the associates, e.g. 0.1M to 1.0M NaOH.

Cleavage of the precursor proteins, e.g. with an acid when the desired peptide, e.g. P18, is integrated into the precursor protein via acid cleavable groups.

34. Where appropriate, such a process shall include at least one of the following additional processing steps:

Treatment of the Associates of the Precursor Protein After Cell Disruption with a Precipitation Aid Such as e.g. phosphoric acid

Purification of the peptide from the cleavage batch by a chromatographic method;

Wash the purified and dried peptide with an acidic solvent or solvent mixture.

35. The object of the invention is also a process for the preparation of a desired peptide (Pep) which comprises the following work-up steps:

Treatment of the associates of the precursor protein after cell disruption is achieved by adding 85% strength phosphoric acid to pH = 3,

Washing the associates of the precursor protein with a sodium hydroxide solution, e.g. 0.4 M NaOH,

Cleavage of the precursor protein with phosphoric acid or formic acid, e.g. 2% phosphoric acid,

optionally washing the dried peptide with hexanoic acid or a mixture of 99 parts of hexane and one part of acetic acid

36. Gegensand is also a process which includes the following workup steps:

Hydrolyzing or splitting the pellets, for example by means of 5% H ₃ P0 ₄ ;

centrifugation;

Adjusting the pH of the supernatant to about 4.0, eg with 25% NaOH Purify the supernatant by cation exchange chromatography

Precipitation of the desired peptide e.g. by adding NaOH to the eluate centrifugation;

Resuspension of the pellet in water

Dissolution of the peptide, e.g. by adding acetic acid

37. The object of the invention is also a process for the preparation of a desired peptide (Pep) which comprises the following work-up steps:

Treatment of the associates of the precursor protein after cell disruption or simultaneous cell disruption by adding 85% phosphoric acid or formic acid between 0.5%% 25% (w / v) to pH = 3 is reached, wherein the volume ratio of the associates or biomass and the added volume of acid may be between 1: 1 and 1:20.

Cleavage of the associates and release of the peptides between 85 ° C-1 10 ° C for a period of 1 h -24 h,

Solids separation by centrifugation, filtration or the like by optionally adding flocculants (salts, polymer, sugar),

If necessary rewashing the filtrate / residue to increase the peptide yield,

Adjusting the pH of the supernatant to about 4.0, e.g. with 25% NaOH

Purify the supernatant by cation exchange chromatography, precipitate the desired peptide e.g. by adding NaOH to the eluate centrifugation,

Resuspension of the pellet in water,

Dissolution of the peptide, e.g. by adding acetic acid.

3. Peptides

Peptides (Pep) according to the present invention, also referred to as "desired peptides", "value peptides" or "target peptides", are amino acid chains in which 2 to 100, such as 5 to 70 and especially 7 to 50, such as 10 to 40, 12 to 35 or 15 to 25, peptides can be composed of any α-amino acids, in particular of the proteinogenic amino acids, In particular, the peptides (Pep) are cationic amphiphilic peptides. The peptides may possess certain desired biological or chemical and in particular also pharmacologically usable properties. Examples of such properties are: antimicrobial activity, specific binding to specific surfaces, nucleating properties in crystallization processes and particle formation, control of crystal structures, bonding of metals or metal ions, surface-active properties, emulsifying properties, foam-stabilizing properties, influencing cellular adsorption. The peptides may have one or more of these properties.

In one embodiment, the invention relates to a method for producing antimicrobial peptides. Such "antimicrobial peptides" are characterized in that in the presence of concentrations <100 μM of the antimicrobial peptide, the growth and / or multiplication of at least one type of gram-positive or gram-negative bacteria and / or at least one kind of yeasts and / or at least one kind of In one embodiment, the invention relates to the provision of cationic antimicrobial peptides Cationic antimicrobial peptides are characterized in that they have an antimicrobial effect in accordance with the invention having the above-mentioned definition as well as a net charge at pH 7 greater than 0. Such cationic peptides contain, for example, the following sequence:

Xi X ₂ KX ₃ X ₄ X ₅ KIP X ₁₀ KFX ₆ X ₇ X ₈ AX ₉ KF (SEQ ID NO: 7)

wherein

and / or mutants or derivatives thereof; wherein the repetitive sequence motifs contained in the precursor protein may be the same or different.

In a further particular embodiment, the invention relates to the preparation of peptides which contain the following sequence:

Xi X ₂ KX ₃ X ₄ X ₅ KIP Xu X ₁₂ KFX ₆ X ₇ X ₈ AX ₉ KF (SEQ ID NO: 8) wherein

Xi for lysine, arginine or phenylalanine,

X ₂ for lysine or tryptophan,

X ₃ for leucine or lysine,

X ₄ for phenylalanine or leucine,

X ₅ for leucine or lysine,

X ₆ for leucine or lysine,

X ₇ for histidine or lysine,

X ₈ for alanine, leucine, valine or serine,

X ₉ for leucine or lysine,

Xu for proline or a chemical bond, and

X _{12 is} proline or a chemical bond,

and / or mutants or derivatives thereof;

wherein the repetitive sequence motifs contained in the precursor protein are the same or different;

Further suitable peptides are e.g. describe in International Patent Application of the present Applicant, PCT / EP2008 / 010912, filing date 19.12.2008, which is incorporated herein by reference.

4. Repetitive Precursor Proteins Repetitive precursor proteins according to the present invention are characterized in that at least 60%, in particular at least 80% of their amino acid sequence, such as 60- 99%, 70-95%, 75-85%, in each case based on the total sequence length peptidic repeat units (as defined below). Of the The remaining portion may include, for example, non-repeating peptides such as signal peptides, tags, and the like.

5. Repeat units

Peptidic repeating units (modules) contain at least one peptide (Pep), which is advantageously prepared according to the present invention, and at least one protective peptide SU, and are constructed in principle as follows - [- L-SU-L-Sp-Pep-Sp -] - _n (see synonym for - [- Sp-Pep-Sp-L-SU-L -] - _n ) (1) where n> 1, and where Pep is the above-identified peptide and L, SU and Sp is as defined above. A repeating unit (module) according to the present invention is in particular an amino acid sequence with a length of 10-200, such as 20-130 and or 30-80 amino acids, within a precursor protein several times as an identical sequence or as a variation of a particular sequence with at least 70%, such as at least 80% and especially at least about 90% identity, such as 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity. Repetitive precursor proteins according to the present invention may thus contain, for example, identical copies or variations of a single or multiple different amino acid sequences, such as the Pep building block, or the SU building block. In addition, in a repetitive precursor protein, any number of above repeating units, such as 1-100, 1-50, or 2-10, and especially 4-6, may be joined together.

The proportion of the peptide of the present invention to the repeating unit in terms of molecular weight is 20% -80%, such as 30% -70% or 40 to 60%. The remaining portion of the repeat unit is occupied by the L, Sp and SU sequences.

6. Auxiliary sequences Auxiliary sequences in the broadest sense are amino acid sequences in a precursor protein according to the invention which influence the properties of the precursor protein in such a way that the expression, the stability and / or the workup of the precursor protein is improved. Auxiliary sequences are in particular aminoterminal or carboxy-terminal to be added to the precursor protein, such as 6 x His-tag (HHHHHH) (SEQ ID NO: 32), T7 tag (MASMTGGQQMG) (SEQ ID NO: 33), S-tag ( KETAAAK FERQHMDS) (SEQ ID NO: 34), c-Myc tag (EQKLISEEDL) (SEQ ID NO: 35), Strep tag (WSHPQFEK) (SEQ ID NO: 36) or HA tag (YPYDVPDYA) (SEQ ID NO: 37), glutathione S-transferase, maltose-binding protein, cellulose binding protein. These and other auxiliary sequences are in Terpe; Appl Microbiol Biotechnol; 60 (5): 523-33 (2003). Furthermore, the auxiliary sequences CanA (May, "In Vitro Studies on the Extracellular Network of Pyrodictium abyssi TAG1 1" Dissertation, University of Regensburg (1998)) and yaaD (Wohlleben Eur Biophys J, (2009) online publication), aminoterminal or carboxyterminal to be added to the precursor protein.

7. Cleavage sequences

Cleavage sequences (Sp) are amino acid sequences which are arranged before and after the desired peptide sequences (Pep) according to the invention. These sequences allow for the "specific" cleavage of the Pep building blocks from the repetitive precursor protein. "Specifically" in this context means that the cleavage in the precursor protein is essentially, in particular, exclusively at one or more defined positions, thereby producing the desired peptide or amino acid Precursor thereof, is separated.

A "precursor" may be, for example, that at one or both ends of the peptide chain are contained amino acid residues which are not part of the native, original peptide sequence, but whose further use and functionality do not interfere, or if necessary cleavable with conventional chemical or biochemical methods are.

Cleavage sequences can function as a specific recognition sequence for proteolytically active enzymes that bind to this sequence and can be split between two specific amino acids. acids cleave the peptide bond. Examples are recognition sequences for Arg-C proteinase, Asp-N-endopeptidase, caspases, chymotrypsin, clostripain, enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, LysC lysyl endopeptidase (achromobacter proteinase I) LysN peptidyl-Lys metalloendopeptidase, pepsin, proline endopeptidase, proteinase K , Staphylococcal peptidase I, thermolysin, thrombin, trypsin. The corresponding recognition sequences are described in the literature, for example in Keil, "Specificity of proteolysis" p.335 Springer-Verlag (1992).

Alternatively, certain amino acid sequences permit the selective cleavage of the polypeptide backbone with certain chemicals, such as BNPS skatoles (2- (2'-nitrophenylsulfenyl) -3-methyl-3-bromoinolenines), cyanogen bromide, acids, hydroxylamine, iodosobenzoic acid, NTCB (2-nitro -5-thiocyanobenzoic acid).

In particular, the cleavage sequences used make it possible to cleave the repetitive precursor proteins with chemicals. Particularly suitable cleavage sequences include the sequence motifs Asn-Gly which cleave with hydroxylamine or Asp-Pro or Asp-Xxx, which allows cleavage with acid, wherein Xxx represents any proteinogenic amino acid. 8. Further embodiments of sequences according to the invention

8.1 amino acid sequences

In addition to the sequences for peptides (Pep) and auxiliary sequences (L, L1, Sp, SU, TAG), repetitive sequence moieties and sequences for repetitive precursor proteins concretely disclosed herein, functional equivalents, functional derivatives and salts of this sequence are also provided by the invention.

According to the invention, "functional equivalents" are in particular also understood as meaning mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned, but nevertheless possess the same properties of the originally unchanged peptides or multiple mutations available from amino acid additions, substitutions, deletions, and / or inversions, wherein said changes may occur in any sequence position, as long as they lead to a mutant with the property profile according to the invention. Functional equivalence is especially given when the reactivity patterns between mutant and unchanged polypeptide are qualitatively consistent. "Functional equivalents" in the above sense are also "precursors" of the described polypeptides as well as "functional derivatives" and "salts" of the polypeptides.

"Precursors" are natural or synthetic precursors of the polypeptides with or without the desired biological activity.

Examples of suitable amino acid substitutions are shown in the following table:

Original rest Examples of substitution

Ala Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn; gin

all leuu; Val

Hell! Val

Lys Arg; Gin; Glu

Met Leu; lle

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val lle; Leu By the term "salts" is meant both salts of carboxyl groups and acid addition salts of amino groups of the peptide molecules of the present invention. "Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts such as sodium, calcium, ammonium, egg salts and salts with organic bases, such as, for example, amines, such as triethanolamine, arginine, lysine, piperidine, etc. Acid addition salts, 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 the subject of the invention.

"Functional derivatives" (or "derivatives") of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by 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 prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups. Furthermore, N- and / or C-terminal additionally 1 to 5, such as. For example, 2, 3 or 4, any D or L amino acid residues may be covalently (peptidyl) bound.

8.2 Nucleic acids, expression constructs, vectors and microorganisms containing them

nucleic Acids:

The invention furthermore comprises the nucleic acid molecules coding for the peptide and protein sequences used according to the invention.

All nucleic acid sequences mentioned herein (single- and double-stranded DNA and RNA sequences, such as cDNA and mRNA) can be prepared in a manner known per se by chemical synthesis from the nucleotide units, for example by fragment condensation of individual overlapping, complementary nucleic acid units of the double helix , The chemical synthesis of oligonucleotides can be For example, in a known manner, by the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press New York, pages 896-897). The attachment of synthetic oligonucleotides and filling of gaps with the aid of the Klenow fragment of the DNA polymerase and ligation reactions and general cloning methods are described in Sambrook et al. (1989), Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

The invention relates both to isolated nucleic acid molecules which code for polypeptides or proteins or biologically active portions thereof according to the invention, as well as nucleic acid fragments which, for. B. can be used as hybridization probes or primers for the identification or amplification of coding nucleic acids according to the invention.

The nucleic acid molecules according to the invention may additionally 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 present in the natural source of the nucleic acid and, moreover, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free from chemical precursors or others Chemicals when chemically synthesized.

A nucleic acid molecule according to the invention can be isolated by means of 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 portion thereof as a hybridization probe and standard hybridization techniques (such as described in Sambrook, J., Fritsch, EF and Maniatis, T., et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989). Moreover, 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 prepared on the basis of this sequence. The thus amplified nucleic acid can be cloned into a suitable vector and characterized by DNA sequence analysis. The oligonucleotides according to the invention nen further by standard synthesis methods, for. B. with an automatic DNA synthesizer manufactured.

The invention further comprises the nucleic acid molecules complementary to the specifically described nucleotide sequences or a portion thereof.

The nucleotide sequences of the invention enable the generation of probes and primers useful for the identification and / or cloning of homologous sequences in other cell types and organisms. Such probes or primers usually include a nucleotide sequence region which, under stringent conditions, is at least about 12, preferably at least about 25, e.g. B. about 40, 50 or 75 consecutive nucleotides of a sense strand of a nucleic acid sequence of the invention or a corresponding antisense strand hybridized. Also included according to the invention are those nucleic acid sequences which comprise so-called silent mutations or are altered according to the codon usage of a specific source or host organism in comparison to a specifically mentioned sequence, as well as naturally occurring variants, such as, for example, B. splice variants or allelic variants thereof. The subject is also afforded 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).

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

Furthermore, the invention also encompasses nucleic acid sequences which hybridize with or are complementary to the above-mentioned coding sequences. These polynucleotides can be found by screening genomic or cDNA libraries and optionally multiply therefrom with suitable primers by means of PCR and then isolate, for example, with suitable probes. Another possibility is the transformation of suitable microorganisms with polynucleotides or vectors according to the invention, the multiplication of the microorganisms and thus the Polynucleotides and their subsequent isolation. In addition, polynucleotides of the invention can also be chemically synthesized.

The ability to "hybridize" to polynucleotides means the ability of a poly- or oligonucleotide to bind under stringent conditions to a nearly complementary sequence, while under these conditions, non-specific binding between non-complementary partners is avoided The property of complementary sequences to be able to specifically bind to one another is utilized, for example, in the Northern or Southern blot technique or in the primer binding in PCR or RT-PCR Usually, oligonucleotides starting from a length of 30 base pairs are used for this purpose. Stringent conditions are, for example, the use of a washing solution at 50-70 ° C., preferably 60-65 ° C., for example in the Northern blot technique x SSC buffer with 0.1% SDS (20 × SSC: 3 M NaCl, 0.3 M Na citrate, pH 7.0) for elution nonspecifically hybridi siert cDNA probes or oligonucleotides. As mentioned above, only highly complementary nucleic acids remain bound to each other. The setting of stringent conditions is known in the art and is z. In Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

"Identity" between two nucleic acids is understood to mean the identity of the nucleotides over the entire nucleic acid length, in particular the identity which is determined by comparison with the Vector NTI Suite 7.1 software from Informax (USA) using the Clustal method (Higgins DG, Sharp Computing Appl. Biosci, 1989 Apr; 5 (2): 151 -1) is calculated using the following parameters:

Multiple alignment parameters:

Gap opening penalty 10

Gap extension penalty 10

Gap Separation penalty ranks 8

Gap separation penalty off

% identity for alignment delay 40

Residue specific gaps off Hydrophilic residues gap off

Transition weighing 0

Pairwise alignment parameter:

FAST algorithm on

K-tuple size 1

Gap penalty 3

Window size 5

Number of best diagonals 5

Expression constructs and vectors:

The invention also relates to expression constructs comprising, under the genetic control of regulatory nucleic acid sequences, a nucleic acid sequence coding for a peptide or precursor protein according to the invention, as well as 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 optionally further customary regulatory elements, in each case operatively linked to the coding sequence. "Operational linkage" is understood to mean the sequential arrangement of promoter, coding sequence, terminator and optionally further regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence as intended Enhancers, polyadenylation signals, etc. Other regulatory elements include selectable markers, amplification signals, origins of replication, etc. Suitable regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). ,

In addition to the artificial regulatory sequences, the natural regulatory sequence may still be present before the actual structural gene. By genetic modification, this natural regulation can optionally be switched off and the expression of the genes increased or decreased. The gene construct can also be constructed simpler, that is, there are no additional regulatory signals is inserted in front of the structural gene and the natural promoter with its regulation is not removed. Instead, the natural regulatory sequence is mutated so that regulation stops and gene expression is increased or decreased. The nucleic acid sequences may be contained in one or more copies in the gene construct.

Examples of useful promoters are: cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, laclq, T7, T5, T3, gal, trc , ara, SP6, lambda PR or in the lambda PL promoter, which are advantageously used in Gram-negative bacteria; and the gram-positive promoters amy and SP02, the yeast promoters ADC1, MFa, AC, P-60, CYC1, GAPDH or the plant promoters CaMV / 35S, SSU, OCS, Iib4, usp, STLS1, B33, not or the ubiquitin or phaseolin promoter. Particularly preferred is the use of inducible promoters, such as. B. light and in particular temperature-inducible promoters, such as the P _r P _r promoter. In principle, all natural promoters can be used with their regulatory sequences. In addition, synthetic promoters can also be used to advantage.

The regulatory sequences mentioned are intended to enable targeted expression of the nucleic acid sequences and protein expression. Depending on the host organism, this may 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 thereby preferably positively influence the expression and thereby increase or decrease. Thus, enhancement of the regulatory elements can advantageously be done at the transcriptional level by using strong transcription signals such as promoters and / or enhancers. In addition, however, an enhancement of the translation is possible by, for example, the stability of the mRNA is improved. The production of an expression cassette is carried out by fusion of a suitable promoter with a suitable coding nucleotide sequence and a terminator or polyadenylation signal. Common recombinant and cloning techniques are used, for example as described in T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) and in TJ Silhavy, ML Berman and LW Enquist, Experiments with Gene Fusion, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience (1987). The recombinant nucleic acid construct or gene construct is advantageously inserted into a host-specific vector for expression in a suitable host organism, which enables optimal expression of the genes in the host. Vectors are well known to the person skilled in the art and can be found, for example, in "Cloning Vectors" (Pouwels PH et al., Eds. Elsevier, Amsterdam-New York-Oxford, 1985). Vectors other than plasmids are also to be understood as meaning all other vectors known to the person skilled in the art, such as, for example, phages, viruses such as SV40, CMV, baculovirus and adenovirus, transposons, IS elements, phasmids, cosmids, and linear or circular DNA. These vectors can be autonomously replicated in the host organism or replicated chromosomally.

As examples of suitable expression vectors may be mentioned:

Common fusion expression vectors such as pGEX (Pharmacia Biotech, Smith, DB and Johnson, KS (1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT 5 (Pharmacia, Piscataway, NJ) which glutathione-S-transferase (GST), maltose E binding protein or protein A is fused to the recombinant target protein.

Non-fusion protein expression vectors such as pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11 d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California ( 1990) 60-89).

Yeast expression vector for expression in the 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: 1 13-123) and pYES2 (Invitro Corporation, San Diego, CA). Vectors and methods for constructing vectors suitable for use in other fungi, such as filamentous fungi, include those described in detail in: van den Hondel, CAMJJ & Punt, PJ (1991) Gene transfer systems and vector development for filamentous fun gi, in: Applied Molecular Genetics of Fungi, JF Peberdy et al., Eds., pp. 1-28, Cambridge University Press: Cambridge.

Baculovirus vectors available for expression of proteins in cultured insect cells (for example, Sf9 cells) include the pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL Series (Lucklow and Summers, (1989) Virology 170: 31-39).

Plant expression vectors, such as those described in detail in: Becker, D., Kemper, E., Schell, J. and Masterson, R. (1992) "New plant binary vectors with selectable markers located proximal to the left Biol. 20: 1 195-1 197; and Bevan, M.W. (1984) "Binary Agrobacterium vectors for plant transformation", Nucl. Acids Res. 12: 871 1 -8721. Mammalian expression vectors such as pCDM8 (Seed, B. (1987) Nature 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195).

Other suitable expression systems for prokaryotic and eukaryotic cells are described in Sections 16 and 17 of Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Recombinant microorganisms: With the aid of the vectors according to the invention, recombinant microorganisms can be produced, which are transformed, for example, with at least one vector according to the invention and can be used to produce the polypeptides according to the invention. Advantageously, the above-described recombinant constructs according to the invention are introduced into a suitable host system and expressed. In this case, it is preferable to use familiar cloning and transfection methods known to the person skilled in the art, for example co-precipitation, protoplast fusion, electroporation, retroviral transfection and the like, in order to express the stated nucleic acids 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. ing: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

Homologously recombined microorganisms can also be produced according to the invention. For this purpose, a vector is produced which contains at least a portion of a gene according to the invention or of a coding sequence in which optionally at least one amino acid deletion, addition or substitution has been introduced in order to modify the sequence according to the invention, e.g. The introduced sequence may, for example, also be a homologue from a related microorganism or be derived from a mammalian, yeast or insect source The vector used for homologous recombination may be alternatively, be designed so that the endogenous gene is mutated or otherwise altered upon homologous recombination but still encodes the functional protein (eg, the upstream regulatory region may be altered to alter expression of the endogenous protein). The modified section of the gene according to the invention is in the homologous recombination vector The construction of suitable vectors for homologous recombination is described, for example, in Thomas, KR and Capecchi, MR (1987) Cell 51: 503. As host organisms, in principle all organisms are suitable which have a Expression of the nucleic acids according to the invention, their allelic variants, their functi allow for electronic equivalents or derivatives. Host organisms are understood as meaning, for example, bacteria, fungi, yeasts, plant or animal cells. Non-limiting examples of prokaryotic expression organisms are E. coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum etc. Nonlimiting examples of eukaryotic expression organisms are yeasts such as Saccharomyces cerevisiae, Pichia pastoris and others, filamentous fungi such as Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum and others, mammalian cells, such as Heia cells, COS cells, CHO cells and others, insect cells, such as Sf9 cells, MEL cells, among others, plants or plant cells, such as Solanum tuberosum, Nicotiana et al.

The selection of successfully transformed organisms can be carried out by marker genes, which are also contained in the vector or in the expression cassette. examples for such marker genes are genes for antibiotic resistance and for enzymes that catalyze a coloring reaction that causes staining of the transformed cell. These can then be selected by means of automatic cell sorting. Microorganisms successfully transformed with a vector carrying a corresponding antibiotic resistance gene (eg G418 or hygromycin) can be selected by means of appropriate antibiotics-containing media or nutrient media. Marker proteins presented on the cell surface can be used for selection by affinity chromatography. 9. Recombinant Production of Precursor Proteins and Peptides

The peptides and precursor proteins used according to the invention can in principle be prepared recombinantly in a manner known per se, whereby a peptide / precursor protein-producing microorganism is cultured, optionally the expression of the polypeptides is induced and these are isolated from the culture. The peptides and precursor proteins can thus also be produced on an industrial scale, if desired.

The recombinant microorganism can be cultured and fermented by known methods. Bacteria can be propagated, for example, in TB or LB medium and at a temperature of 20 to 40 ° C and a pH of 6 to 9. Specifically, suitable cultivation conditions are described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).

The cells are then disrupted if the peptides or precursor proteins are not secreted into the culture medium and the product recovered from the lysate by known protein isolation techniques. The cells can optionally by high-frequency ultrasound, by high pressure, such as. B. in a French pressure cell, by osmolysis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by combining several of the listed methods are digested.

Purification of the peptides or precursor proteins can be achieved by known chromatographic methods, such as molecular sieve chromatography (gel filtration). on), such as Q-sepharose chromatography, ion exchange chromatography and hydrophobic chromatography, as well as other conventional methods such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, FG, Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, New York or in Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

Furthermore, to isolate the recombinant peptide or precursor protein, vector systems or oligonucleotides can be used which extend the cDNA by certain nucleotide sequences and thus encode altered polypeptides or fusion proteins, e.g. B. serve a simpler cleaning. Such suitable modifications are, for example, acting as an anchor so-called "tags" such. The modification known as hexa-histidine anchors, or epitopes that can be recognized as antigens of antibodies (described, for example, in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor (US Pat. NY) Press). These anchors can be used to attach the proteins to a solid support, such as. As a polymer matrix serve, which may be filled for example in a chromatography column, or may be used on a microtiter plate or other carrier.

At the same time, these anchors can also be used to detect the proteins. In addition, conventional markers such as fluorescent dyes, enzyme labels which form a detectable reaction product upon reaction with a substrate, or radioactive labels alone or in combination with the anchors may be used to derivatize the proteins to recognize the proteins.

In particular, the preparation of the repetitive precursor proteins is effected by expression of synthetically produced gene sequences which code for the inventive repetitive precursor proteins. One possibility for the production of synthetic gene sequences is described in Hummerich et al. Biochemistry 43; 13604-13612 (2004).

The repetitive precursor proteins may be soluble or insoluble in the host cell. In both cases, the cells are disrupted. In particular, the digestion is carried out by means of a high pressure homogenizer at 1000-1500 bar. In soluble repetitive precursor proteins, much of the cellular protein is by heating the lysate at 60-100 ° C, such as 70-90 ° C or 75-85 ° C and separated by a suitable separation method (eg sedimentation or filtration) of the soluble repetitive precursor protein. The repetitive precursor protein is then precipitated by the addition of a cosmotropic salt (as described above). The re- pective precursor proteins form stable associates. The final concentrations of the cosmotropic salts added can vary depending on the associate and are approximately in the range of about 0.2-3 M, or eg 0.8-2 M. Optimal concentrations are to be determined in a simple manner familiar to the protein chemist. The assembly of the repetitive precursor proteins can also take place without an external trigger. Then the repetitive precursor proteins already assemble in the host cell to corresponding stable associates. The associates are separated after digestion of the cells by a suitable separation method (eg sedimentation or filtration) of soluble components.

The separation of the associates can be improved by the addition of precipitation aids after digestion of the cells. The precipitation aids cause further clumping of the associates, e.g. in sedimentation lower accelerations must be used to separate the associates from the aqueous medium. Suitable precipitation aids are acids, bases, polymer solutions, in particular aqueous solutions of charged polymers. Examples of precipitation aids are phosphoric acid or solutions of polyethyleneimine.

The stable associates of repetitive precursor proteins can be further purified. For this purpose, solutions are used for this purification, in which the stable associates are insoluble, but other impurities are solved. In particular, aqueous solutions of bases, acids, urea, salts and detergents are used. Solutions of alkali hydroxides, urea, guanidinium salts or charged detergents, such as alkyltrimethylammonium salts or alkyl sulfates, are particularly suitable. In particular, solutions of> 0.2 M sodium hydroxide,> 2 M urea,> 1 M guanidinium hydrochloride,> 1 M guanidinium thiocyanate or> 0.1% sodium dodecyl sulfate or> 0.1% cetyltrimethylammonium bromide are used. For purification, the stable associates are resuspended in the appropriate solutions and then separated from the solution by a suitable separation process (eg sedimentation or filtration). Subsequently, washed the repetitive precursor proteins with water and dried by the skilled person methods.

In order to recover the peptides from the repetitive precursor proteins, these sequences must be cleaved from the precursor proteins and separated from the auxiliary sequences. The cleavage takes place on the cleavage sequences contained in the repetitive precursor proteins. Methods for the specific cleavage of amino acid chains are described in the literature. Repetitive precursor proteins can be cleaved enzymatically or chemically. Examples of enzymes with which amino acid chains can be specifically cleaved are Arg-C proteinase, Asp-N-endopeptidase, caspases, chymotrypsin, clostripain, enterokinase, factor Xa, glutamyl endopepidase, Granzyme B, LysC lysylendopeptidase (Achromobacter proteinase I) LysN peptidyl Lys metalloendopeptidase, pepsin, proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin, trypsin. Examples of chemicals with which amino acid chains can be specifically cleaved are BNPS skatoles (2- (2'-nitrophenylsulfenyl) -3-methyl-3-bromoinolenines), cyanogen bromide, acids, hydroxylamine, iodosobenzoic acid, NTCB (2-nitrobenzene). 5-thiocyanobenzoic acid).

In particular, repetitive precursor proteins are chemically cleaved, such as by cleavage with hydroxylamine or acid. For the acid cleavage, any inorganic or organic acid is suitable with a pK _s value of less than 5 and greater than 0, preferably less than 4 and greater than 1. In particular, 1 to 5% of phosphoric acid or 1 to 5% of formic acid is used for the cleavage. Depending on the acid cleavage conditions, there is a simple cleavage between the amino acids Asp and Pro or Asp and Xxx, where Xxx represents any proteinogenic amino acid or there is first a cleavage between the amino acids Asp and Pro or Asp and Xxx and then a complete cleavage of the aspartate of the amino acid sequence located in the amino acid sequence of the peptide N-terminal before the aspartate.

The cleavage may be with the purified repetitive precursor protein or with a cell fraction containing the repetitive precursor protein (eg, soluble components of the host cell or insoluble components of a host cell) or with intact host cells containing the repetitive precursor protein. After cleavage, the cleaving substance must be inactivated. Methods for this are known to the person skilled in the art. After inactivation, the cleavage mixture contains, inter alia, the desired peptide, cleaved auxiliary sequences and inactivated cleavage substances. The peptides may already have their desired activity in this solution. If a greater purity is required, then after the cleavage, the peptides released from the repetitive precursor proteins can be separated from the auxiliary sequences. An advantage of the self-assembling auxiliary sequences is that the auxiliary sequences assemble during or after cleavage. This assembly can be done spontaneously during cleavage under the selected cleavage conditions, or by addition of materials that assist in the assembly of the auxiliary sequences. Such assemblage-promoting substances are, for example, cosmotropic salts which contain at least one type of ion which, according to the Hofmeister series, has more cosmotropic properties than sodium or chloride ions. Other materials that promote assembly are acids or alkalis or organic solvents that are miscible with water, such as alcohols. The assembled auxiliary sequences can be separated from the soluble released peptide by sedimentation or filtration. Further purification steps may be required to separate residual protein or peptide contaminants or salts or other substances added during or after cleavage from the desired peptide. For this purpose, for example, chromatographic methods, precipitations, dialysis, 2-phase extractions and other methods known in the art can be used.

Thereafter, the peptide-containing solution may be used directly for the desired application or the solution may be dried by methods known to those skilled in the art (e.g., spray-drying or freeze-drying) and the corresponding dry product used.

After drying, it is possible to remove impurities which can not be separated from the peptide, as long as it is dissolved in water, by washing with solvents in which the peptide is insoluble. Suitable solvents are organic solvents such as n-hexane N-methylpyrrolidone or mixtures of solvents and acids such as mixtures of n-hexane and acetic acid or organic acids such as acetic acid or hexanoic acid. For this purification step, the dried peptide is resuspended in the appropriate solvent / mixture and then separated again by sedimentation or filtration. res- te of the solvent / solvent mixture can be removed by drying.

The desired peptides may have the desired activity in the form obtained by the cleavage. However, it may also be necessary to further modify the peptides after cleavage. For example, the peptide can be amidated, esterified, oxidized, alkylated or chemically linked to any molecule. Examples of molecules which can be used for such modifications are alcohols, alcohol cysteine esters, carboxylic acids, thioesters or maleimides. In particular, molecules which increase the hydrophobicity of the peptide are used for such modifications. Such molecules may contain modified or unmodified alkyl radicals as defined above. Such molecules preferably contain C ₂ -C ₆ -alkyl radicals, in particular C ₆ -C ₄ -alkyl radicals. Corresponding methods are known to the person skilled in the art. The modification can be carried out at any time: for example directly after cell disruption, after purification of the precursor protein, after cleavage of the precursor protein or after purification of the peptide.

Peptide solutions having the desired degree of purity can be used directly. Alternatively, different preservation methods can be used for longer-term storage. Examples of preservation methods are cooling, freezing, addition of preservatives. Alternatively, the peptides can be dried. Examples of drying methods are lyophilization or spray drying. Dried peptides can then be stored. For the use of the peptides, the dried substance is dissolved in a suitable solvent, preferably an aqueous solution. This aqueous solution may contain salts or buffer substances or no further additives.

The present invention will be further illustrated by the following nonlimiting examples.

EXPERIMENTAL PART The universal applicability of the method described in the present invention for the preparation of any peptide sequences (Pep) is shown by the preparation of the peptide P18. Unless otherwise specified, standard methods of organic and biochemical analysis as well as the recombinant production of proteins and cultivation of microorganisms are used.

Example 1: Preparation of the peptide P18

Peptide P18 is a peptide derived from a potent antimicrobial peptide sequence reported by Shin et al. J. Peptide Res. 58: 504-14 (2001).

The following synthetic expression constructs according to SEQ ID NO: 1 to 6 are prepared:

Expression construct SEQ ID NO: 1 (not according to the invention): repetitive (tetrameric) arrangement of aux-pep elements with N-terminally peptidically linked T7-tag. The aux element according to WO 2010/139736 comprises a peptide-linked self-assembling SA element and a and SU element.

Expression construct SEQ ID NO: 2: repetitive arrangement of (SU-Pep) with N-terminally peptidically linked T7-tag

Expression construct SEQ ID NO 3: SEQ ID NO 2 without T7 tag.

Expression construct SEQ ID NO 4: SEQ ID NO 2 with modified SU sequence. Expression construct SEQ ID NO 5: SEQ ID NO 2 with modified SU sequence. Expression construct SEQ ID NO 6: SEQ ID NO 2 with modified SU sequence. 1. Cloning of the coding precursor protein sequences

Synthetic genes encoding dimers of the precursor proteins (corresponding to the formula: [-L-SU-L-Sp-Pep-Sp-] 2 were synthesized using the restriction endonucleases BamHI and HindIII in the methods described in Hummerich et al., Biochemistry 43, 13604- 13612 (2004) described vector pAZI and cloned according to the protocol described therein dimerized and cloned into the vector pET21 (Novagen). The sequences subsequently present in this vector (optionally containing N- or C-terminal tags) encode the repetitive precursor proteins according to SEQ ID-7. The repetitive precursor proteins contain 4 repeat units, each containing one copy of the peptide P18 and one SU element.

The expression took place in the strain E. coli BI21 [DE3] (Novagen).

2. Expression

The E. coli BL21 DE [DE3] (Novagen) strain is made competent by electroporation and the respective expression plasmid transformed into the cells. The selection of successful transformants is carried out on LB / agar plates with antibiotic, which corresponds to the selection marker, which is encoded on the expression plasmid.

The culture and expression of the precursor genes is carried out in 200 ml Erlenmeyer shake flasks in PM medium (see below). At an optical density (OD 600nm) of 1.5 to 2.0, induction of gene expression occurs by addition of 1 mM I PTG. a. Preparative scale

The expression took place in the strain E. coli BL21 [DE3] (Novagen).

Cultivation and protein synthesis were carried out at pO ₂ > 20% and pH = 6.8 in the fed-batch process.

Medium:

8 liters of water

25 g of citric acid monohydrate

40 g of glycerol (99%)

125 g potassium dihydrogen phosphate (KH ₂ P0 ₄ )

62.5 g of ammonium sulfate ((NH ₄ ) ₂ S0 ₄ )

18.8 g of magnesium sulfate heptahydrate (MgS0 ₄ * 7H ₂ O) 1, 3 g of calcium chloride dihydrate (CaCl ₂ ^* 2 H ₂ 0)

155 ml trace saline

fill with water to 9.8 liters

Adjust the pH to 6.3 with 25% NaOH

3 ml Tego KS 91 1 (Antifoam, Goldschmidt products)

1 g of ampicillin

190 mg of thiamine hydrochloride

20 mg of vitamin B12

Trace salt solution:

5 liters of water

200.00 g of citric acid monohydrate

55.00 g ZnSO ₄ ^* 7H ₂ 0

42.50 g (NH ₄ ) ₂ Fe (S0 ₄ ) ₂ ^* 6 H ₂ 0

15.00 g MnSO ₄ ^* H ₂ 0

4.00 g CuS0 ₄ ^* 5 H ₂ 0

1, 25 g cos 0 ₄ ^* 7 H ₂ 0

Feeding solution:

1 125 g of water

41, 3 g of citric acid monohydrate

81, 6 g sodium sulfate (Na ₂ S0 ₄ )

6.3 g (NH ₄ ) ₂ Fe (S0 ₄ ) ₂ ^* 6 H ₂ 0

4734 g of glycerol 99.5% After exhaustion of the glycerol contained in the basic medium, a constant feed was started at a rate of 100 ml / h.

Protein synthesis was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside after the bacterial culture reached an optical density of OD ₆ oo = 60. 10 After induction, the cells were harvested by sedimentation at 5000 xg for 30 min. The organic moisture amounted to 1932g.

The biomass was cleaned according to the following protocol:

Resuspension of the cell pellet: 6 g of 20 mM sodium phosphate buffer (pH 7.5) per g of biomass were added and mixed.

Digestion of the cells in the high-pressure homogenizer at 1500 bar

Addition of phosphoric acid to pH = 3 ± 0.5

10 min incubation at 23 ° C with stirring

- Centrifugation: 20 min, min. 5000 xg

Resuspension of the pellet: Add 25 ml of 0.2 M NaOH per g of wet mass, homogenize and incubate for 4 hours at 23 ° C. with stirring

Neutralization: Adjust the pH value of 8.5 ± 0.5 with 85% H ₃ P0 ₄

Centrifugation: 20 min, min. 5000 xg

The pellet consisting of washed inclusion bodies containing the P18 precursor protein was hydrolyzed or cleaved by means of 2% H ₃ PO ₄ .

Cleavage conditions:

i. Insert 5 ml_ H ₃ P0 ₄ per g of pellet

ii. Homogenize

iii. Incubate for 16 hours at 90 ° C with shaking.

Allow cleavage approach to cool.

Centrifugation: 20 min, min. 5000 xg

Neutralization: Adjust the pH to 5.5 ± 0.5 with 10 M NaOH

- Centrifugation: 20 min, min. 5000 xg

Dilute the supernatant with water until the conductivity is less than 10 mS / cm

P18 from supernatant via cation exchange chromatography (Fractogel COO;

Merck); Elution with 450 mM NaCl

Pool peptide-containing fractions and dilute with water until conductivity is less than 10 mS / cm

Purify P18 from pooled fractions via cation exchange chromatography (Fractogel COO; Merck); Elution with 50 mM HCl

The eluate was neutralized with 2 M NaOH.

The neutralized solution was lyophilized. The lyophilized product was analyzed by HPLC: For this, the product was dissolved in water at a concentration of 1 mg / ml and analyzed with a reversed phase chromatography column (Jupiter Proteo 4.6 x 250 mm, Phenomenex). The eluent used was 0.1% trifluoroacetic acid in water, which was replaced with a linear gradient by 0.1% trifluoroacetic acid in acetonitrile. The detection was carried out at 280 nm. For a further analysis, the fractions of the main peak were collected and the substance contained therein further investigated. N-terminal sequencing confirmed that this component is the peptide P18.

To study the activity of the P18 peptide, E. coli B cultures were measured in LB medium (5 g / L yeast extract, 10 g / L tryptone 5 g / L sodium chloride), which had an optical density of 0.1 measured at 600 nm with different concentrations of the P18 peptide with shaking at 37 ° C incubated. Bacterial growth was monitored by measuring the optical density after 24 hours. Complete inhibition of growth (optical density at 600 nm after 24 h <0.15) was achieved at a peptide concentration of 31 ppm or more. Further improvement in antimicrobial activity could be achieved by amidation of the C-terminal carboxyl group. For this purpose, the carboxyl groups were activated with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxy-sulfosuccinimide and amidated by subsequent addition of ammonia. b. Shake flask experiments Expression comparison of six different constructs in BL21 DE3 E. coli cells 200 ml shake flasks with 50 ml PM medium (+ antibiotic) are inoculated with a clone of the desired expression plasmid-carrying E. coli cell (BL21 DE3) from LB / agar plate. The cultures are incubated at 220rpm at 28 ° C. The cell density is measured by means of optical density (OD) at a wavelength of 600 nm. At an OD (600) of about 1.5- 2.0, expression of the precursor construct is induced by addition of 1 mM IPTG.

After addition of the inductor, the shake flasks are removed for 0 h, 3 h, 5 h, 7 h, 9 h samples for the analysis. The optical density of the samples is determined (OD-600 measurement). After the formula 12.5 ml / OD600 value, the volume (ml) of the recovery sample is determined and then centrifuged for 10 min at 4000 rpm. The supernatant will discarded and the cell pellet homogenized with 0.5 ml of Bugbuster (Novagen) and incubated for 20 min at room temperature. The samples are centrifuged at 4000 rpm for 10 minutes, the oil is recovered and the pellet is taken up in 8M urea and heated at 50 ° C. for 60 minutes. The samples of oil and pellet are then analyzed in equal concentrations by SDS-PAGE and Coomassie Blue staining.

The results of expression after 0, 3, 5 hours of induction by IPTG are shown in attached Figures 2a and 2b.

The 4-module precursor protein is located in inclusion bodies. Therefore, both supernatant (soluble proteins) and pellet (insoluble proteins) are applied on the SDS PAGE.

All constructs show a very good expression and formation of inclusion bodies. M: molecular weight ladder

Ü: supernatant

P: pellet

Overview of sequences according to the invention

SEQ ID Sequence Name NO: Type

1 MASMTGGQQMGRGSM T7- (Aux-Pep) ₄ GAAAAAAAASGPG -Aux

EWELFEEISEFLQSLEEFGGPGSSAAAAAAGPGDPG SA-SU-SA

KWKLFKKIPKFLHLAKKFGDPGSSAAAAAAAASGPG

EWELFEEISEFLQSLEEFGGPGAAAAAAAGPGDPG pHüm 285

KWKLFKKIPKFLHLAKKFGDPGAAAAAAAASGPG

EWELFEEISEFLQSLEEFGGPGSSAAAAAAAAGPGDPG

KWKLFKKIPKFLHLAKKFGDPGSSAAAAAAAASGPG

EWELFEEISEFLQSLEEFGGPGAAAAAAAAGPGDPG

KWKLFKKIPKFLHLAKKFGDPG

2 MASMTGGQQMGRGSM T7- (SU-Pep) ₄

Preferred GSGPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS form GPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG pHüm 353 SGPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS GPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG

3 MGSGPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS (SU-Pep) ₄ GPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG APPLES 047 SGPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS GPGEWELFEEISEFLQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG

4 MASMTGGQQMGRGSM T7- (SU-Pep) ₄

GSGPGEWEFLEEISELFQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS APPLES 054 GPGEWEFLEEISELFQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG SGPGEWEFLEEISELFQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS GPGEWEFLEEISELFQSLEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG

5 MASMTGGQQMGRGSM T7- (SU-Pep) ₄

GSGPGEWEFLEEISELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS pFel 055 GPGEWEFLEEISELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG SGPGEWEFLEEISELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS GPGEWEFLEEISELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG

6 MASMTGGQQMGRGSM T7- (SU-Pep) ₄

GSGPGEWEFLEEASELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS pFel 056 GPGEWEFLEEASELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG SGPGEWEFLEEASELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPS GPGEWEFLEEASELFQALEEFGGPGDPGKWKLFKKIPKFLHLAKKFGDPG

7 XXKXXXKIPX KFXXXAXKF Pep Motif

8 XXKXXXKIPX XKFXXXAXKF Pep Motif

9 KWKLFKKIPP KFLHLAKKF Pep

10 RWKLFKKIPK FLHLAKKF RP18

1 1 FKKLFKKIPK FLHAAKKF KKFP18

12 KWKLLKKIPK FKKLALKF KKLP18

13 KWKLFKKIPK FLHAAKKF AP18

14 KWKKFLKIPK FLHAAKKF KFLP18

15 KWKKLLKIPK FLHAAKKF KLLP18

16 PGKWKLFKKI PKFLHLAKKF G P18_21AA

17 KWKLFKKIPK FLHLAKKF P18J 8AA 18 EWELFEEISE FLQSLEEF SU1

19 EWEFLEEISE LFQSLEEF SU2

20 EWEFLEEISE LFQALEEF SU3

21 EWEFLEEASE LFQALEEF SU4

22 EXEXXEEXXE XXXXXEEX SU Motif

23 EXEXXEEXXE XXXXXEEX SU Motif

24 EXEXXEEXXE XXXXXEEX SU Motif

25 RGSM linker

26 GGGS linker

27 GSGGM linker

28 QGSG linker

29 SGGGR linker

30 GGSGG linker

31 GGGGS linker

32 HHHHHH His day

33 MASMTGGQQM G T7 day

34 KETAAAKFER QHMDS S day

35 EQKLISEEDL cMyc day

36 WSHPQFEK Strep day

37 YPYDVPDYA HA day

38 GSGPG linker

39 GGSGGS linker

40 GSGGGS linker

SU = protective peptide

Pep = peptide to be prepared In addition to the specific Pep amino acid sequences described above, these listed sequences can be modified C-terminally and / or N-terminally by addition of specific cleavage sequences, in particular the sequences Asn-Gly (ie NG) or Asp-Xxx defined above (ie, DX) (such as, in particular, the motifs suitable for acid cleavage between the residues "DP" or a hydroxylamine cleavage between the residues "NG") (ie, sequences of the "- Sp-Pep-Sp-" type as in claim 1) Are defined); or modified by the remaining amino acid residues resulting from such cleavage. Optionally, additionally between the cleavage sequence or the residue remaining after cleavage and the Pep sequence, a spacer residue, such as e.g. a G-remainder, to be inserted. In particular, the following modifications of the above Pep sequences are to be mentioned, which may result from the acid or hydroxylamine cleavage of Pep sequences prepared according to the invention:

N-terminal: addition of a PG, P or G residue;

C-terminal: addition of a residue -GD; -G N; -G; -N or -D Such modifications apply in particular to each of the above Pep sequences, in particular according to SEQ ID NO: 7 to 17.

Reference is made expressly to the disclosure of the references cited herein.

Claims

claims

1 . Precursor protein comprising a cleavable, repetitive sequence of peptide-linked amino acid partial sequences, the repetitive sequence having the following general formula (1):

- [- L-SU-L-Sp-Pep-Sp -] - _n (1) wherein

n is an integer value of more than 1,

Pep stands for a cationic amphiphilic value peptide,

Sp stands for identical or different chemically or enzymatically cleavable cleavage peptide sequences,

SU stands for an anionic amphiphilic protective peptide, and

L stands for the same or different linkers.

2. Precursor protein according to claim 1, wherein n has an integer value of 2 to 10, in particular 3 to 8, especially 4 to 6.

Precursor protein according to one of the preceding claims, wherein the elements L and / or the elements Sp have no self-assembling properties.

Precursor protein according to one of the preceding claims, wherein Sp has the amino acid sequence motif DX, wherein X is an arbitrary amino acid residue, and Sp has in particular a sequence length of 2 to 6, in particular 2 to 4.

A precursor protein according to claim 4, wherein the Sp elements are independently selected from the sequences of the general formula

XADPXB

A precursor protein according to claim 5, wherein the Sp elements are independently selected from the sequences of GDPG, GDPS, GDP, DPG, DPS, SDP and DP.

A precursor protein according to any one of the preceding claims wherein L is from 1 to 10, more preferably from 1 to 5, of any contiguous amino acid residues.

The precursor protein of claim 7, wherein the L elements are independently selected from GSGPG (SEQ ID NO: 38), GGPG, SGPG, GSPG, GGP, GPG, GPS, GAG, GAA, GGA, GQQ, GGQ, GSS, SGA, GGY, GGL, GS and G.

Precursor protein according to one of the preceding claims, in which the cationic amphiphilic value peptide Pep represents identical or different amino acid sequences according to SEQ ID NO: 7

Xi X ₂ KX ₃ X ₄ X ₅ KIP X1 ₀ KFX ₆ X ₇ X ₈ AX ₉ KF (SEQ ID NO: 7), in which

X1 is ₀ and a peptide bond or one or two any basic or hydrophobic amino acid residues or one or two proline residues

and cationic amphiphilic mutants or derivatives thereof.

0. A precursor protein according to any one of the preceding claims, wherein the cationic amphiphilic peptide value Pep for identical or different amino acid sequences according to SEQ ID NO: 8

Xi for lysine, arginine or phenylalanine,

X ₂ for lysine or tryptophan,

X ₃ for leucine or lysine, X ₄ for phenylalanine or leucine,

X ₅ for leucine or lysine,

X ₆ for leucine or lysine,

X ₇ for histidine or lysine,

X ₈ for alanine, leucine, valine or serine,

X ₉ for leucine or lysine,

Xu for proline or a chemical bond, and

X is proline or a chemical bond,

and cationic amphiphilic mutants or derivatives thereof.

1 1. Precursor protein according to one of the preceding claims, wherein the cationic amphiphilic value peptide Pep for an amino acid sequence according to SEQ ID NO: 9

KWKLFKKIPKFLHLAKKF (SEQ ID NO: 9).

A precursor protein according to any one of the preceding claims, wherein the SU elements are the same or different and each SU element is an amphiphilic peptide comprising a sequence portion of at least seven peptidically linked amino acids capable of forming an amphiphilic alpha helix, said helix in its vertical projection has a separation of the amino acid residues in a hydrophobic and a hydrophilic helical half, the hydrophobic helix half at least 3 in the vertical projection adjacent same or different hydrophobic amino acid residues and the hydrophilic helix half at least 3 in the vertical projection) adjacent same or different hydrophilic amino acid residues having.

A precursor protein according to any one of the preceding claims, wherein the proportion of charged amino acid residues of the SU element is selected so that the total net charge of the precursor protein at pH = 7 is greater than -10 and less than +10.

14. A precursor protein according to any one of the preceding claims, wherein the SU elements of the precursor protein are the same or different and have an amino acid sequence according to SEQ ID NO: 10: Ex _a Ex _b XcEEx _d Xe EXfXgX _h XiXkEExi (SEQ ID NO: 22) where x _a to X | represent an amino acid residue other than E selected from hydrophobic and polar amino acid residues.

A precursor protein according to any one of the preceding claims, wherein the SU elements are selected from the sequences:

EWELFEEISEFLQSLEEF (SEQ ID NO: 18),

EWEFLEEISELFQSLEEF (SEQ ID NO: 19),

EWEFLEEISELFQALEEF (SEQ ID NO: 20) and

EWEFLEEASELFQALEEF (SEQ ID NO: 21)

Precursor protein according to one of the preceding claims, which additionally has an N-terminal or C-terminal TAG sequence motif.

A precursor protein according to any one of the preceding claims wherein the additional N-terminal or C-terminal TAG sequence motif is selected from a methionine residue and an auxiliary sequence.

The precursor protein of claim 17, wherein the TAG helper sequence promotes expression, stability, and / or work-up of the precursor protein.

The precursor protein of claim 18, wherein the TAG helper sequence is selected from a His tag, T7 tag, S tag, c-Myc 10 tag, Strep tag or HA tag, and optionally another linker sequence comprising 1 to 5 any contiguous amino acid residues N-terminal or C-terminal to the repetitive sequence motif of the above formula (1) is bound.

20. The precursor protein according to claim 19, the general formula (2)

TAG-L1 - [- L-SU-L-Sp-Pep-Sp] _n (2)

wherein

TAG, L, SU, Sp, Pep and n are as defined above and L1 for a chemical bond or another linker sequence comprising 1 to 5 contiguous amino acid residues, in particular RGSM, GGGS, GSGGM, QGSG, SGGGR, GGSGG, GGGGS (SEQ ID NO: 25 to 31).

21. Precursor protein according to one of the preceding claims, selected from the amino acid sequences SEQ ID NO: 2 to SEQ ID NO: 6.

22. Nucleic acid sequence coding for at least one precursor protein according to one of the preceding claims.

23. An expression cassette comprising at least one nucleic acid sequence according to claim 2 operatively linked to at least one regulatory nucleic acid sequence.

24. A recombinant vector for transforming a eukaryotic or prokaryotic host, comprising a nucleic acid sequence according to claim 22 or an expression cassette according to claim 23. A recombinant eukaryotic or prokaryotic host comprising a nucleic acid sequence according to claim 22 or an expression cassette according to claim 23 or a vector according to claim 24.

26. A method for producing a peptide of value Pep, which comprises preparing a precursor protein according to any one of claims 1 to 21, and cleaving the pep peptides from the precursor protein.

27. The method of claim 26, wherein the precursor protein is produced in a recombinant microorganism carrying at least one vector according to claim 19.

28. The method of claim 27, wherein the precursor protein is produced in a recombinant E. coli strain.

29. The method according to any one of claims 26 to 28, wherein the expressed precursor protein, after it has optionally converted into a stably associated form, to release the desired peptide (Pep) cleaved chemically or enzymatically enzymatically.

30. The method according to any one of claims 26 to 29, comprising the following steps: a) fermentation of a recombinant host according to claim 25 with expression of a precursor protein according to formula (1)

b) optional separation of the cell mass,

c) digestion of the cells and simultaneous or subsequent cleavage of the

Precursor protein according to formula (1)

d) optional separation of cell fragments

e) Purification of the cationic, amphiphilic Wertpeptids, in particular by adsorption, chromatography, extraction, crystallization or precipitation, optionally followed by solubilization or combinations of such measures.