RU2403260C2 - Polypeptides, which have antimicrobial action, and polynucleotides coding them - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: to obtain microbe-resistant plants plant cells are transformed with vector, which contains polynucleotide structure, which codes defensin polypeptide, which has amino-acid sequence C-x (3)-C-x (7, 9)-C-C, C-C-(8)-C-x-C and C-x-C-x (8, 11)- C.

EFFECT: obtaining polypeptide, which can be also applied as drug or veterinary medication, as well as in forage for animals.

23 cl, 4 tbl, 6 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to isolated polypeptides having antimicrobial activity and isolated polynucleotides encoding these polypeptides. The invention also relates to nucleic acid constructs, vectors and host cells containing polynucleotides, as well as to methods for producing and using polypeptides.

BACKGROUND OF THE INVENTION

An object of the present invention is to provide polypeptides having an antimicrobial effect and polynucleotides encoding them.

SUMMARY OF THE INVENTION

The present invention relates to antimicrobial polypeptides that contain the amino acid sequence represented by: Cx (3) -Cx (7.9) -C-C; C-C-x (8) -C-x-C; or Cx-Cx (8.11) -C.

In an embodiment of the invention, the polypeptides are defensins.

In yet another embodiment, the polypeptides are selected from the group consisting of:

(a) a polypeptide having an amino acid sequence that has at least 60% identity with:

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

(b) a polypeptide that is encoded by a nucleotide sequence that hybridizes under at least average conditions with (i)

nucleotides 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27; or (ii) a complementary chain (i); and

(c) a variant comprising a conservative substitution, deletion and / or insertion of one or more amino acids from:

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

The present invention also relates to isolated polynucleotides encoding polypeptides having an antimicrobial effect selected from the group consisting of:

(a) a polynucleotide encoding a polypeptide having an amino acid sequence that has at least 60% identity with:

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

(b) a polynucleotide having at least 60% identity with nucleotides 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27; and

(c) a polynucleotide that hybridizes under at least moderate conditions with (i):

nucleotides 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27; or (ii) a complementary chain (i).

The present invention also relates to nucleic acid constructs, recombinant expression vectors, and recombinant host cells containing polynucleotides.

The present invention also relates to methods for producing such polypeptides having an antimicrobial effect, comprising (a) culturing recombinant host cells containing a nucleic acid construct comprising a polynucleotide encoding a polypeptide under conditions conducive to obtaining a polypeptide; (b) isolation of the polypeptide.

The present invention also relates to methods of using the polypeptides and polynucleotides of the invention.

DEFINITIONS

Antimicrobial action: The term "antimicrobial action" is defined here as an action that can lyse cells or inhibit the growth of microbial cells. In relation to the present invention, the term "antimicrobial" means that there is a bactericidal and / or bacteriostatic and / or fungicidal and / or fungistatic effect and / or virucidal effect, where the term "bactericidal" is understood to be capable of lysing bacterial cells. The term “bacteriostatic” should be understood as being capable of inhibiting the growth of bacteria, i.e. inhibit the growth of bacterial cells. The term "fungicidal" should be understood as being able to lyse fungal cells. The term "fungistatic" should be understood as being capable of inhibiting the growth of fungi, i.e. inhibit the growth of fungal cells. The term "virucidal" should be understood as capable of inactivating the virus. The term "microbial cells" means bacterial or fungal cells (including yeast).

In relation to the present invention, the term "inhibition of microbial cell growth" means that the cells are in the stationary phase, i.e. they are not capable of reproduction.

For the purposes of the present invention, an antimicrobial effect can be determined by the method described by Lehrer et al., Journal of Immunological Methods, Vol. 137 (2) p. 167-174 (1991). Alternatively, antimicrobial activity may be determined according to the NCCLS (National Committee for Clinical and Laboratory Standards) guidelines from CLSI (Institute for Clinical and Laboratory Standards; formerly known as the National Committee for Clinical and Laboratory Standards).

Antimicrobial polypeptides may be able to reduce the number of living cells of Escherichia coli (DSM 1576) to 1/100 after 8 hours (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and, in features, after 30 minutes) incubation at 20 ° C in 25% (by weight) aqueous solution; preferably in a 10% (by weight) aqueous solution; more preferably in a 5% (w / w) aqueous solution; even more preferably, in a 1% (by weight) aqueous solution; most preferably in a 0.5% (w / w) aqueous solution; and, in particular, in a 0.1% (by weight) aqueous solution of a polypeptide having an antimicrobial effect.

Antimicrobial polypeptides may also be able to inhibit the growth of Escherichia coli (DSM 1576) for 24 hours at 25 ° C in a microorganism growth substrate when added at a concentration of 1000 ppm (ppm); preferably when added at a concentration of 500 ppm (ppm); more preferably when added at a concentration of 250 ppm; even more preferably, when added at a concentration of 100 ppm; most preferably when added at a concentration of 50 ppm; and especially when added at a concentration of 25 ppm.

Antimicrobial polypeptides may be able to reduce the number of living Bacillus subtilis cells (ATCC 6633) to 1/100 after 8 hours (preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and, in features, after 30 minutes) incubation at 20 ° C in 25% (by weight) aqueous solution; preferably in a 10% (by weight) aqueous solution; more preferably in a 5% (w / w) aqueous solution; even more preferably, in a 1% (by weight) aqueous solution; most preferably in a 0.5% (w / w) aqueous solution; and, in particular, in a 0.1% (by weight) aqueous solution of a polypeptide having an antimicrobial effect.

Antimicrobial polypeptides may also be able to inhibit the growth of Bacillus subtilis (ATCC 6633) for 24 hours at 25 ° C in a microorganism growth substrate when added at a concentration of 1000 ppm (ppm); preferably when added at a concentration of 500 ppm; more preferably when added at a concentration of 250 ppm; even more preferably, when added at a concentration of 100 ppm; most preferably when added at a concentration of 50 ppm; and especially when added at a concentration of 25 ppm.

The polypeptides of the present invention possess at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least at least 80%, even more preferably at least 90%, most preferably at least 95% and, even more preferably at least 100% of the antimicrobial activity of the polypeptide consisting of the amino acid sequence represented by

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

Defensin: The term “defensin”, as used herein, refers to polypeptides recognized by those skilled in the art as belonging to the class of defensins of antimicrobial peptides. To determine whether the polypeptide is the defensin of the invention, the amino acid sequence is preferably compared with the hidden Markov model profiles (HMM profiles) of the database. PFAM by using the freely available NMMER software package (see Example 6).

PFAM defensin families include Defensin_1 or Mammalian Defensin (accession number PF00323), Defensin_2, or Arthropod Defensin (accession number PF01097), Beta defensin or Beta defensin (accession number PF00711). Defensin propep or “Defensin propeptide” (access number PF00879) and Gamma-thionine or “Gamma-thionine family” (access number PF00304).

Defensins may belong to the alpha-defensins class, the beta-defensins class, the theta-defensins class, the insect or arthropod classes defensins, or the plant defensins class.

In an embodiment of the invention, the defensin amino acid sequence of the invention contains 4, 5, 6, 7, 8, 9, or 10 cysteine residues, preferably 6, 7, 8, 9, or 10 cysteine residues, more preferably 6, 8 or 10 cysteine , and most preferably 6 or 8 cysteine residues.

Defensins can also be synthetic defensins, combining the characteristic properties of any class of defensins.

Examples of defensins include, but are not limited to, α-Defensin HNP-1 (human neutrophil peptide), HNP-2, and HNP-3; β-Defensin-12, Drosomycin, Heliomycin, γ1-purothionine, Insect Defensin A and defensins disclosed in PCT applications WO 99/53053, WO 02/06324, WO 02/085934, WO 03/044049, PCT / DK2005 / 000725 and PCT / DK2005 / 000735.

Isolated Polypeptide: The term “isolated polypeptide” as used herein refers to a polypeptide that has at least 20% purity, preferably at least 40% purity, more preferably at least 60% purity, even more preferably at least at least 80% purity, most preferably at least 90% purity, and even more preferably at least 95% purity determined by SDS-PAGE.

Almost pure polypeptide: The term "substantially pure polypeptide" means here a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, at most 3%, even more preferably at most 2%, most preferably at most 1% and, even more preferably at most 0.5% by weight of the other polypeptide material to which it is bound crude. Thus, it is preferable that the substantially pure polypeptide has at least 92% purity, preferably at least 94% purity, more preferably at least 95% purity, more preferably at least 96% purity, more preferably at least 97% purity, more preferably at least 98% purity, even more preferably at least 99% purity, most preferably at least 99.5% purity, and even more preferably at least 100% purity by weight of the total polypept the same material present in the preparation.

The polypeptides of the present invention are preferably in substantially pure form. In particular, it is preferred that the polypeptides are predominantly pure, i.e., that the polypeptide preparation is predominantly free of another polypeptide material to which it is bound crude. This can be achieved, for example, by preparing a polypeptide by widely known recombinant methods or classical purification methods.

Here, the term “substantially pure polypeptide” is synonymous with the terms “isolated polypeptide” or “polypeptide isolated”.

Identity: The relationship between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

For the purposes of the present invention, the degree of identity between two amino acid sequences is determined by using the FASTA program included in version 2.0x of the FASTA software package (see WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85: 2444 -2448; and WR Pearson (1990) "Rapid and Sensitive Sequence Comparison with FASTP and FASTA", Methods in Enzymology 183: 63-98). The weight matrix used was BLOSUM50, the penalty for skipping was -12, and the penalty for continuing the gap was -2.

The degree of identity between two nucleotide sequences was determined using the same algorithm and the software package described above. The weight matrix used was the similarity matrix, the penalty for skipping was -16, and the penalty for continuing the gap was -4.

Alternatively, alignment of two amino acid sequences was determined using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. Needle runs the general alignment algorithm described in Needleman, S. B. and Wunsch, C. D. (1970) J. MoI. Biol. 48, 443-453. The substitution matrix used is BLOSUM62, the penalty for the first amino acid of the gap is 10, and the penalty for continuing the gap is 0.5.

The degree of identity between the amino acid sequence of the present invention (“the sequence of the invention”; for example, amino acids 1 to 49 of SEQ ID NO: 2) and the different amino acid sequence (“foreign sequence”) is calculated as the number of exact matches in overlapping alignment of the two sequences, divided by the length of the "sequence of the invention" or the length of the "foreign sequence", any shortest. The result is presented in percent identity.

A complete match occurs when the “sequence of the invention” or “foreign sequence” have identical amino acid residues at the same overlapping positions. The length of the sequence is the number of amino acid residues in the sequence (for example, the length of amino acids 1 to 49 of SEQ ID NO: 2 is 49).

Polypeptide fragment: The term "polypeptide fragment" is defined here as a polypeptide having one or more amino acids deleted from the amino and / or carboxy terminus of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or sequences homologous to them, where the fragment has an antimicrobial effect.

Subsequence: The term “subsequence” is defined herein as a nucleotide sequence having one or more nucleotides deleted from the 5 ′ and / or 3 ′ end of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or sequences homologous to them, where the subsequence encodes a polypeptide fragment having an antimicrobial effect.

Allelic variant: The term "allelic variant" refers here to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic diversity arises in nature through mutations and can lead to polymorphism within populations. Mutations of genes can be silent (there are no substitutions in the encoded polypeptide) or they can encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Nearly pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other external or unwanted nucleotides and in a form suitable for use in systems for producing proteins genetically engineered. Therefore, a substantially pure nucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3 %, even more preferably, at most 2%, most preferably at most 1%, and even more preferably at most 0.5% by weight of another polynucleotide material to which it is bound crude. A substantially pure polynucleotide may, however, include naturally occurring 5 'and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide has at least 90% purity, preferably at least 92% purity, more preferably at least 94% purity, more preferably at least 95% purity, more preferably at least 96% purity, more preferably at least 97% purity, even more preferably at least 98% purity, most preferably at least 99% purity, and even more preferably at least 99.5% purity by weight. The polynucleotides of the present invention are preferably in substantially pure form. In particular, it is preferable that the polynucleotides disclosed herein are in a "predominantly pure state", i.e. so that the polynucleotide preparation is predominantly free from another polynucleotide material with which it is bound crude. Here, the term “substantially pure polynucleotide” is synonymous with the terms “isolated polynucleotide” or “polynucleotide in isolated form”. Polynucleotides can be genomic, cDNA, RNA, semisynthetic, synthetic, or any combination thereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can be obtained by reverse transcription from a mature spliced mRNA molecule derived from a eukaryotic cell. The cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial primary RNA transcript is an mRNA precursor that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences through a process called splicing. cDNA derived from mRNA thus has no intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a single or double chain nucleic acid molecule that is isolated from a naturally occurring gene or that is modified to contain nucleic acid segments so that it doesn’t otherwise would exist in nature. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of the coding sequence of the present invention.

Control sequence: The term "control sequence" is defined here to include all components that are necessary or preferable for the expression of a polynucleotide encoding a polypeptide according to the present invention. Each control sequence may be its own or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. The smallest control sequences include a promoter and transcriptional and translational stop signals. Control sequences can be provided with linkers for the purpose of introducing specific restriction sites that facilitate ligation of the control sequences with the coding region of the nucleotide sequence encoding the polypeptide.

Functionally Attached: The term “functionally attached” means here a configuration in which the control sequence is positioned relative to the coding sequence of the polypeptide sequence so that the control sequence controls the expression of the coding sequence of the polypeptide.

Coding sequence: As used herein, the term "coding sequence" means a nucleotide sequence that directly defines the amino acid sequence of its protein product. The boundaries of the coding sequence are usually defined by an open reading frame, which usually starts with an ATG start codon or alternative start codons such as GTG or TTG. The coding sequence may be DNA, cDNA, or a recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in the preparation of a polypeptide, including, but not limited to, transcription, post-transcriptional modifications, translation, post-translational modifications, and secretion.

Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide of the invention and that is operably linked to additional nucleotides that provide for its expression.

Host cell: The term "host cell", as used herein, includes any type of cell that is susceptible to transformation, transfection, transduction and the like nucleic acid construct containing the polynucleotide of the present invention.

Modification: The term "modification" here means any chemical modification of a polypeptide consisting of

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28; as well as genetic manipulations with DNA encoding this polypeptide. Modification (s) may (gut) be the replacement (s), deletion (s) and / or insertion (s) of amino acids (s), as well as the replacement (s) in the side chain (s) of amino acids; or using non-natural amino acids with similar characteristics in the amino acid sequence. In particular, the modification (s) may (gut) be the formation of an amide bond, such as the formation of an amide bond at the C-terminus.

Artificial variant: As used herein, the term "artificial variant" means an antimicrobial polypeptide obtained by an organism expressing a modified nucleotide sequence from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27. The modified nucleotide sequence is obtained by human intervention by modifying the nucleotide sequence disclosed in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27.

DETAILED DESCRIPTION OF THE INVENTION

ANTIMICROBIAL POLYPEPTIDES

In a first aspect, the invention relates to antimicrobial polypeptides that comprise the amino acid sequence represented by: Cx (3) -Cx (7.9) -C-C; C-C-x (8) -C-x-C; or Cx-Cx (8.11) -C.

In an embodiment of the invention, the polypeptides comprise an amino acid sequence represented by:

C-x (3) -C-x (7.9) -C-C and C-C-x (8) -C-x-C; or

C-C-x (8) -C-x-C and C-x-C-x (8.11) -C; or

C-x (3) -C-x (7.9) -C-C and C-x-C-x (8.11) -C; or

C-x (3) -C-x (7.9) -C-C and C-C-x (8) -C-x-C and C-x-C-x (8.11) -C.

Consensus C-x (3) -C-x (7.9) -C-C; C-C-x (8) -C-x-C and C-x-C-x (8,11) -C should be interpreted using the PROSITE (www.expasy.org/prosite/) consensus definition format:

- for amino acids the standard one-letter code IUPAC is used;

- the symbol "x" is used for the position where any amino acid is allowed;

- each element in the sample is separated from the neighboring one by means of “-”; and

- the repetition of an element in consensus is indicated by the following numerical value or numerical range in parentheses. For example: x (3) corresponds to x-x-x, x (2,4) corresponds to x-x or x-x-x or x-x-x-x.

For more information on applying the PROSITE principle, contact Sigrist et al. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 3: 265-274 (2002).

In a second aspect, which may be an embodiment of the first aspect, the invention relates to polypeptides comprising an amino acid sequence that has a degree of identity with amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28 (i.e., mature polypeptides) of at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75% more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even more preferably at least 97%, which possess antimicrobial action (below “homologous polypeptides”). In a preferred aspect, homologous polypeptides have an amino acid sequence that is characterized by ten amino acids, preferably five amino acids, more preferably four amino acids, even more preferably three amino acids, most preferably two amino acids, and even more preferably one amino acid from amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

The polypeptide of the present invention preferably contains the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or an allelic variant thereof; or a fragment thereof that has an antimicrobial effect. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28. In another preferred aspect, the polypeptide comprises amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28, or an allelic variant thereof; or a fragment thereof that has an antimicrobial effect. In another preferred aspect, the polypeptide comprises amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or an allelic variant thereof; or a fragment thereof that has an antimicrobial effect. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28. In another preferred aspect, the polypeptide consists of amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28 or an allelic variant thereof; or a fragment thereof that has an antimicrobial effect. In another preferred aspect, the polypeptide consists of amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

In a second aspect, the present invention relates to isolated polypeptides having antimicrobial activity, which are encoded by polynucleotides that hybridize under very mild conditions, preferably mild conditions, more preferably medium conditions, more preferably medium-hard conditions, even more preferably severe conditions and , most preferably, very stringent conditions with (i) nucleotides 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27;

(ii) a cDNA sequence contained in nucleotides 1 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 1 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 1 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 1 to 252 of SEQ ID NO: 21;

nucleotides 1 to 297 of SEQ ID NO: 23;

nucleotides 1 to 252 of SEQ ID NO: 25; or

nucleotides 1 to 321 of SEQ ID NO: 27;

(iii) a subsequence of (i) or (ii), or (iv) a complementary chain of (i), (ii) or (iii) (J. Sambrook, EF Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York). Subsequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 contains at least 100 conjugated nucleotides, or preferably 200 conjugated nucleotides . In addition, the subsequence can encode a polypeptide fragment that has an antimicrobial effect.

The nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 , SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 or their subsequences, as well as amino acid sequences from SEQ ID NO: 2 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, or fragments thereof, can be used to create a nucleic acid probe to detect and clone DNA encoding polypeptides having antimicrobial action, from strains of various genera or species according to methods well known in the art. In particular, such samples can be used for hybridization with a genomic or cDNA of an interesting genus or species, following the standard Southern blotting technique, to detect or isolate the corresponding gene in it. Such probes may be significantly shorter than the complete sequence, but should have at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 75 nucleotides in length. However, it is preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, a nucleic acid probe may have at least 200 nucleotides, preferably at least 250 nucleotides. Both DNA and RNA probes can be used. Probes are usually labeled to detect the corresponding gene (for example, ³² P, ³ H, ³⁵ S, biotin or avidin). Such probes are covered by the present invention.

Genomic DNA or cDNA libraries prepared from such organisms can thus be screened for DNA that hybridizes with the samples described above and which encodes a polypeptide having antimicrobial activity. Genomic or other DNA from such other organisms can be separated by agarose or polyacrylamide gel electrophoresis, or other separation methods. DNA from libraries or isolated DNA can be transferred and immobilized on nitrocellulose or other suitable carrier material. To detect a clone or DNA that is homologous to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 , SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, or a subsequence of the carrier material used in the southern blot.

For the purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 7 : 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, its complementary sequence, or its subsequence under very mild to very stringent hybridization conditions. The molecules with which the nucleic acid probe hybridizes can be detected using an x-ray film.

In a preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes polypeptides from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or subsequences. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is a region encoding a mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27.

For long probes with a length of at least 100 nucleotides, hybridization conditions from very mild to very severe are defined as prehybridization and hybridization at 42 ° C in 5X SSPE, 0.3% SDS, 200 μg / ml of destroyed and denatured salmon sperm DNA, and or 25% formamide for very mild or mild conditions, or 35% formamide for medium or medium-hard conditions, or 50% formamide for harsh or very harsh conditions, following standard Southern blotting techniques, optimally for 12 to 24 hours.

For long probes with a length of at least 100 nucleotides, the carrier material is finally washed three times, each for 15 minutes, using 2X SSC, 0.2% SDS, preferably at least 45 ° C (very mild washing), more preferably at least at 50 ° C (soft wash), more preferably at least 55 ° C (medium wash), more preferably at least 60 ° C (medium hard wash), even more preferably at least at 65 ° C (hard wash) and, most preferably, at least 70 ° C (very hard wash ka).

For short probes that have about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization and washing after hybridization at about 5 ° C to about 10 ° C below the calculated T _m (melting point) using calculation from Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48: 1390) at 0.9 M NaCI, 0.09 M Tris-HCI pH 7.6, 6 mM EDTA, 0 , 5% NP-40, 1X Denhart solution, 1 mM sodium pyrophosphate, 1 mM monobasic sodium phosphate, 0.1 mM ATP and 0.2 mg RNA from yeast per ml, following the standard Southern blotting procedure.

For short probes that have about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6X SSC plus 0.1% SDS for 15 minutes and twice for 15 minutes each, using 6X SSC at 5 ° C to 10 ° C below the calculated T _m .

In a third aspect, the present invention relates to artificial variants comprising a conservative substitution, deletion and / or insertion of one or more amino acids in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or in their mature polypeptides. Preferably, the amino acid substitutions are negligible, that is, conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, usually from one to about 30 amino acids; small extensions of the amino or carboxy terminus, such as a methionine base; a small linker peptide to about 20-25 bases; or a slight elongation that facilitates purification by altering the total charge or other function, such as a polyhistidine region, an antigenic epitope, or a binding domain.

Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions, which usually do not change specific activity, are known in the art and are described, for example, H. Neurath and RL Hill, 1979, In, The Proteins, Academic Press, New York. The most common substitutions are Ala / Ser, Val / lle, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / lle, Leu / Val, Ala / Glu and Asp / Gly.

In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxy-proline, 6- N- methyl-lysine, 2-aminobutyric acid, isovalin and alpha-methyl-serine) can be a substitute for the amino acid residues of the wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and non-natural amino acids can be used instead of amino acid residues. "Unnatural amino acids" were modified after protein synthesis and / or have a chemical structure in their side chains that is different from it in ordinary amino acids. Unnatural amino acids can be chemically synthesized and preferably commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline and 3,3-dimethylproline.

As an alternative, amino acid substitutions are substances with altered physico-chemical properties. For example, amino acid substitutions can improve the thermal stability of the polypeptide, change substrate specificity, change the optimum pH, and the like.

Essential amino acids in the parent polypeptide can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are inserted into each residue in the molecule and the resulting mutant molecules are tested for biological activity (i.e., antimicrobial activity) to detect amino acid residues that are critical to the activity of the molecule. See also Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of the structure, which is determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling in combination with an amino acid mutation of a possible contact site. See, for example, de Vos et al ., 1992, Science 255: 306-312; Smith et al., 1992, J. MoI. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The conclusion on the identification of essential amino acids can also be made from an identity analysis with polypeptides that are associated with the polypeptides of the invention.

Single or multiple amino acid substitutions can be made and tested using known mutagenase, recombination and / or rearrangement methods followed by appropriate screening procedures, such as those disclosed in Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include erroneously error-directed PCR, phage display (eg, Lowman et al., 1991, Biochem. 30: 10832-10837; US patent No. 5223409; WO 92/06204) and region-directed mutagenesis (Derbyshire et al., 1986; Gene 46: 145; Ner et al., 1988; DNA 7: 127).

Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned mutant polypeptides expressed in host cells. Mutated DNA molecules that encode active polypeptides can be isolated from host cells and quickly sequenced using standard methods in this field. These methods allow you to quickly determine the importance of individual amino acid residues in the polypeptide of interest and can be applied to polypeptides with unknown structure.

Total number of amino acid substitutions, deletions and / or insertions

in amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28 are 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably at most 5, more preferably 4, still more preferably 3, most preferably 2, and even more preferably 1.

In a preferred embodiment, the polypeptides of the invention are defensin polypeptides.

N-terminal extension

The N-terminal extension of the polypeptides of the invention may suitably be from 1 to 50 amino acids, preferably from 2 to 20 amino acids, especially from 3 to 15 amino acids. In one embodiment, the N-terminal peptide extension does not contain Arg (R). In another embodiment, the N-terminal extension comprises kex2 or kex2-like cleavage sites, as further defined below. In a preferred embodiment, the N-terminal extension is a peptide containing at least two Glu (E) and / or Asp (D) amino acid residues, such as an N-terminal extension, containing one of the following sequences: EAE, EE, DE and DD.

Kex2 sites

Kex2 sites (see, for example, Methods in Enzymology VoI 185, ed. D. Goeddel, Academic Press Inc. (1990), San Diego, CA, “Gene Expression Technology”) and kex2-like sites are dichotomous recognition sites (t ie, cleavage sites) found between the propeptide-coding region and the mature peptide region in some proteins.

In some cases, insertion of a kex2 or kex2-like site has been shown to improve the proper processing of endopeptidase at the propeptide cleavage site, leading to an increase in protein secretion.

In the context of the invention, the insertion of kex2 or kex2-like sites leads to the possibility of cleavage at a specific position in the N-terminal extension, leading to an extended antimicrobial peptide compared to the mature polypeptide shown in amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

Fusion Polypeptides

The polypeptides of the present invention also include fusion polypeptides or cleavable fusion polypeptides in which the other polypeptide is connected to the N-terminus or C-terminus of a polypeptide of the invention or a fragment thereof. A fusion polypeptide is obtained by fusing a nucleotide sequence (or part thereof) encoding another polypeptide with a nucleotide sequence (or part thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art and include ligation of coding sequences encoding the polypeptides in such a way that they are framed and that expression of the fusion polypeptide is controlled by the same promoter (s) and terminator.

Sources of antimicrobial polypeptides

The polypeptides according to the present invention can be obtained from microorganisms of any kind. For the purposes of the present invention, the term “derived from” as used herein, in connection with this source, should mean that the polypeptide encoded by the nucleotide sequence is produced by this source or strain into which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from this source is secreted outside the cell.

The polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be from gram-positive bacteria, such as a polypeptide from Bacillus , for example, a polypeptide from Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentusus bacillus megacomus Bacillus megacome Bacillus megacomus Bacillus megacomus subtilis or Bacillus thuringiensis ; or a polypeptide from Streptomyces , for example, a polypeptide from Streptomyces lividans or Streptomyces murinus ; or a polypeptide from gram-negative bacteria, for example, a polypeptide from E. coli or Pseudomonas sp. .

The polypeptide of the present invention may also be a fungal polypeptide and, more preferably, a yeast polypeptide, such as a polypeptide from Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia ; or, more preferably, a polypeptide from filamentous fungi, such as a polypeptide from Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastomycemicum, Neurospora, Paecilomyocomycema, Penecilomyocomycema, Penecilomycesomyceme, Pen Thielavia, Tolypocladium, or Trichoderma.

In a preferred aspect, the polypeptide is a polypeptide of Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis, which is effective.

In another preferred aspect, the polypeptide is a polypeptide of Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Fumarus fibrusarium fibrumarium fibrumarum Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum , Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa , Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride .

In another preferred aspect, the polypeptide is a polypeptide from Arenicola marina , for example, a polypeptide from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

It will be understood that for the above species, the invention encompasses both perfect and imperfect stages and other taxonomic equivalents, for example, anamorphs, regardless of the name of the species by which they are known. Those skilled in the art will easily recognize the identity of their respective equivalents.

Strains of these species are readily available in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS) and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center ( NRRL).

In addition, such polypeptides can be detected and obtained from other sources, including microorganisms isolated from nature (eg, soil, compost, water, etc.) using the above samples. Techniques for isolating microorganisms from natural habitats are well known in science. The polynucleotide can then be obtained by simple screening of a genomic or cDNA library of another microorganism. Once the polynucleotide sequence encoding the polypeptide is detected by the probe (s), the polynucleotide can be isolated or cloned using techniques that are well known to one skilled in the art (see, for example, Sambrook et al., 1989, supra ).

The polypeptides of the present invention also include fusion polypeptides or cleavable polypeptide fusions in which another polypeptide is fused to the N-terminus or C-terminus of the polypeptide or fragment thereof. A fusion polypeptide is obtained by fusing a nucleotide sequence (or part thereof) encoding another polypeptide with a nucleotide sequence (or fragment thereof) according to the present invention. Techniques for preparing polypeptide fusions are known in the art and include ligation of coding sequences encoding polypeptides so that they are framed and that expression of the fused polypeptide is controlled by the same promoter (s) and terminator.

Polynucleotides

The present invention also relates to isolated polynucleotides having a nucleotide sequence that encodes a polypeptide according to the present invention. In a preferred aspect, the nucleotide sequence is set to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27. In another preferred aspect, the nucleotide sequence is a region encoding the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27. The present invention also encompasses nucleotide sequences that encode a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28, or their mature peptides that differ from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 due to the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, which encode fragments from SEQ ID NO: 2 , SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28 having antimicrobial activity.

The present invention also relates to mutant polynucleotides containing at least one mutation in a sequence encoding a mature polypeptide from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, in which the mutant nucleotide sequence encodes a polypeptide consisting of

amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28.

Techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. Cloning of polynucleotides according to the present invention from such genomic DNA can be performed, for example, by using polymerase chain reaction (PCR) or antibody screening of expression libraries to detect DNA fragments with common structural properties. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application , Academic Press, New York. Other nucleic acid amplification techniques, such as ligase chain reaction (LCR), ligated activated transcription (LAT), and nucleotide sequence-based amplification (ONPA), may be used. Polynucleotides can be cloned from a strain of Aspergillus, or another, or a related organism, and therefore, for example, can be an allelic or species variant of a polypeptide-encoding region of a nucleotide sequence.

The present invention also relates to polynucleotides having nucleotide sequences that have a degree of identity of a sequence encoding a mature polypeptide from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27 (i.e., nucleotides 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9; nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13; nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19; nucleotides 118 to 252 of SEQ ID NO: 21; nucleotides from 151 to 297 of SEQ ID NO: 23; nucleotides from 121 to 252 of SEQ ID NO: 25; or nucleotides 145 to 321 of SEQ ID NO: 27) at least 60%, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 97% of the identities that encode the active polypeptide.

Modification of the nucleotide sequence encoding the polypeptide according to the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to polypeptides refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some design way from a polypeptide isolated from its natural source, for example, artificial variants that differ in a certain activity, thermal stability, optimum pH or the like. A variant sequence can be constructed based on the nucleic sequence represented as a polypeptide encoding region from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, for example, its subsequence and / or by introducing nucleotide substitutions that are not the source of another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to triplets of the host organism, started for the production of the enzyme, or by embedding nucleotide substitutions, which may be the source of an excellent amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutions can be made outside areas critical to the functioning of the molecule, and still produce an active polypeptide. Amino acid residues necessary for the activity of a polypeptide encoded by an isolated polynucleotide according to the invention and, therefore, preferably not objects for substitution, can be identified by methods known in science, such as site-directed mutagenesis or alanine scanning mutagenesis (see e.g. Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, mutations are introduced into each positively charged residue in the molecule and the resulting mutant molecules are tested for antimicrobial activity to detect amino acid residues that are critical to the activity of the molecule. The substrate-enzyme interaction sites can also be determined by analysis of a three-dimensional structure, which is determined by methods such as nuclear magnetic resonance, crystallography or photoaffinity labeling (see, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, Journal of Molecular Biology 224: 899-904; Wlodaver et al., 1992, FEBS Letters 309: 59-64).

The present invention also relates to isolated polynucleotides encoding a polypeptide according to the present invention, which hybridize under mild conditions, preferably moderate conditions, more preferably medium-severe conditions, even more preferably, severe conditions and, most preferably, very severe conditions with (i ) nucleotides from 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27;

(ii) a cDNA sequence contained in nucleotides 1 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 1 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 1 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 1 to 252 of SEQ ID NO: 21;

nucleotides 1 to 297 of SEQ ID NO: 23;

nucleotides 1 to 252 of SEQ ID NO: 25; or

nucleotides 1 to 321 of SEQ ID NO: 27; or

(iii) a complementary chain (i) or (ii); or their allelic variants or subsequences (Sambrook et al., 1989, supra ), which are defined here.

The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a DNA population under mild, medium, medium hard, hard or very severe conditions with (i) nucleotides from 151 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 118 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 112 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 118 to 252 of SEQ ID NO: 21;

nucleotides from 151 to 297 of SEQ ID NO: 23;

nucleotides 121 to 252 of SEQ ID NO: 25; or

nucleotides 145 to 321 of SEQ ID NO: 27;

(ii) a cDNA sequence contained in nucleotides 1 to 297 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9;

nucleotides 1 to 255 of SEQ ID NO: 11, SEQ ID NO: 13;

nucleotides 1 to 255 of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19;

nucleotides 1 to 252 of SEQ ID NO: 21;

nucleotides 1 to 297 of SEQ ID NO: 23;

nucleotides 1 to 252 of SEQ ID NO: 25; or

nucleotides 1 to 321 of SEQ ID NO: 27; or

(iii) a complementary chain (i) or (ii); and (b) isolating a hybridizable polynucleotide that encodes a polypeptide having an antimicrobial effect.

Nucleic acid constructs

The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide according to the present invention, operably attached to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

With the isolated polynucleotide encoding the polypeptide of the present invention, manipulations can be performed in various ways to allow expression of the polypeptide. Manipulation of the sequence of the polynucleotide before it is inserted into the vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

The control sequence may be the corresponding promoter sequence, a nucleotide sequence that is recognized by the host cell, for expression of a polynucleotide encoding the polypeptide of the present invention. The promoter sequence contains transcription control sequences that mediate the expression of the polypeptide. A promoter can be any nucleotide sequence that shows transcriptional activity in a selected host cell, including mutant, shortened, or hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, either homologous or heterologous to the host cell.

Examples of suitable promoters for directing transcription of nucleic acid constructs according to the present invention, especially in a bacterial host cell, are promoters derived from the lac operon of E. coli , the agarase gene ( dagA ) from Streptomyces coelicolor , the levansaccharase gene ( sacB ) from Bacillus subtilis , the gene alpha-amylase (amyL) from Bacillus licheniformis, a gene maltogenic amylase (amyM) from Bacillus stearothermophilus (amyM), alpha-amylase gene (amyQ) from Bacillus amyloliquefaciens, penicillinase gene (penP) of Bacillus licheniformis, xylA gene and the xylB of Bacillus subtilis , and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Additionally, promoters are described in "Useful proteins from recombinant bacteria" in Scientific American , 1980, 242: 74-94; and in Sambrook et al., 1989, supra .

Examples of suitable promoters for directing transcription of nucleic acid constructs according to the present invention in host cells from filamentous fungi are promoters derived from TAKA amylase genes from Aspergillus oryzae , aspartate protease from Rhizomucor miehei , neutral alpha-amylase from Aspergillus niger , amylase from Aspergillus niger, glucoamylase (glaA) of Aspergillus niger or Aspergillus awamori, lipase from Rhizomucor miehei, the alkaline protease from Aspergillus oryzae, triose-phosphate isomerase of Aspergillus oryzae, acetamidase of Aspergillus nidulans, amyloglucosidase from Fusarium venenatum (WO 00/56900), Daria from Fusarium venenatum (WO 00/56900), Quinn from Fusarium venenatum (WO 00/56900), trypsin-like protease from Fusarium oxysporum (WO 96/00787), beta glucose Trichoderma reesei, cellobiohydrolase I from Trichoderma reesei, endoglucanase I from Trichoderma reesei, endoglucanase II from Trichoderma reesei, endoglucanase III from Trichoderma reesei, endoglucanase IV from Trichoderma reesei, endoglucanase V from Trichoderma reesei, xylanase I from Trichoderma reesei, xylanase II from Trichoderma reesei , beta-xylosidase from Trichoderma reesei , as well as the NA2-tpi promoter (a hybrid of promoters from the neutral alpha-amylase genes from Aspergillus niger and triose-phosphate isomerase from Aspergillus or yzae ); and mutant, truncated, or hybrid promoters of this.

In the yeast hosts, the promoters used are derived from enolase (ENO-1) genes from Saccharomyces cerevisiae , galactokinase (GAL1) from Saccharomyces cerevisiae , alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2acocepharce , cereAce isomerase (TPI) from Saccharomyces cerevisiae ; metallothionine (CUP1) from Saccharomyces cerevisiae; and 3-phosphoglycerate kinase from Saccharomyces cerevisiae . Other passing promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to stop transcription. The termination sequence is functionally attached to the 3'-end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell may be used in the present invention.

Preferred terminators for host cells from filamentous fungi are those derived from TAKA genes of amylase from Aspergillus oryzae , glucoamylase from Aspergillus niger , anthranilate synthetase from Aspergillus nidulans , alpha-glucosidase from Aspergillus niger and trypsin-like protease oxidase from Figer .

Preferred terminators for yeast host cells are derived from enolase genes from Saccharomyces cerevisiae , cytochrome C (CYC1) from Saccharomyces cerevisiae , and glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae . Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra .

The control sequence may also be a leader sequence, an untranslated region of mRNA that is important for translation by the host cell. The leader sequence is functionally attached to the 5'-end of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in a selected host cell may be used in the present invention.

Preferred leaders for host cells from filamentous fungi are those derived from TAKA amylase genes from Aspergillus oryzae and triose phosphate isomerases from Aspergillus nidulans .

Suitable leaders for yeast host cells are enolase genes from Saccharomyces cerevisiae , 3-phosphoglycerate kinase from Saccharomyces cerevisiae , alpha-factor from Saccharomyces cerevisiae and alcohol dehydrogenase / glyceraldehyde-3-phosphate ishydrogenase-dehydrogenase-dehydrogenase-dehydrogenase-dehydrogenase .

The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-end of the nucleotide sequence, and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed RNA. Any polyadenylation sequence that is functional in a selected host cell may be used in the present invention.

Preferred polyadenylation sequences for the host cells from filamentous fungi are derived from the TAKA amylase genes from Aspergillus oryzae, glucoamylase from Aspergillus niger, anthranilate synthase of Aspergillus nidulans, a trypsin-like protease from Fusarium oxysporum, and alpha-glucosidase from Aspergillus niger.

Suitable polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide encoding region that encodes the amino acid sequence linked to the amino terminus of the polypeptide and directs the encoded polypeptide along the cellular secretory pathway. The 5 ′ end of the coding sequence of the nucleotide sequence may initially comprise a signal peptide coding region naturally linked in the translation reading frame to a segment of the coding region that encodes a secreted polypeptide. Alternatively, the 5 ′ end of the coding sequence may comprise a signal peptide coding region that is foreign to the coding region. A foreign signal peptide coding region may be required when the coding sequence does not inherently contain a signal peptide coding region. Alternatively, a foreign signal peptide coding region may simply replace the natural signal peptide coding region to enhance secretion of the polypeptide. However, any signal peptide coding region that directs an expressed polypeptide along the secretory pathway of a selected host cell can be used in the present invention.

Active signal peptide-coding regions for bacterial host cells are signal-peptide-coding regions derived from maltogenic amylase genes from Bacillus NCIB 11837, alpha-amylase from Bacillus stearothermophilus , subtilisin from Bacillus licheniformis , beta-lactamase from Bacillus licheniformis , nprS, nprM ) from Bacillus stearothermophilus , and prsA from Bacillus subtilis . Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

Operating signal peptide coding regions for the host cells of filamentous fungi are the signal peptide coding regions obtained from the genes for TAKA amylase of Aspergillus oryzae, neutral amylase from Aspergillus niger, glucoamylase from Aspergillus niger, aspartate protease from Rhizomucor miehei, the cellulase from Humicola insolens and lipase from Humicola lanuginose .

In a preferred aspect, the signal peptide coding region is nucleotides 1 to 57 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9, which encode amino acids from -50 up to -32 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10; nucleotides 1 to 63 of SEQ ID NO: 11 or SEQ ID NO: 13, which encode amino acids -39 to -19 of SEQ ID NO: 12 or SEQ ID NO: 14; nucleotides 1 to 63 of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, which encode amino acids -37 to -17 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: twenty; nucleotides 1 to 63 of SEQ ID NO: 21, which encode amino acids -39 to -19 of SEQ ID NO: 22; nucleotides 1 to 57 of SEQ ID NO: 23, which encode amino acids -50 to -32 of SEQ ID NO: 24; nucleotides 1 to 63 of SEQ ID NO: 25, which encode amino acids -40 to -20 of SEQ ID NO: 26; or nucleotides 1 to 66 of SEQ ID NO: 27, which encode amino acids -48 to -27 of SEQ ID NO: 28.

Suitable signal peptides for yeast host cells are derived from alpha factor genes from Saccharomyces cerevisiae and invertase from Saccharomyces cerevisiae . Other applicable regions encoding signal peptides are described by Romanos et al., 1992, supra .

The control sequence may also be a propeptide encoding region that encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a proenzyme or propolipeptide (or zymogen in some cases). A propolypeptide is usually inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of a propeptide from a propolipeptide. The propeptide coding region can be obtained from alkaline protease ( aprE ) genes from Bacillus subtilis , neutral protease ( nprT ) from Bacillus subtilis , alpha factor from Saccharomyces cerevisiae , aspartate protease from Rhizomucor miehei and laccase from Myceliophthora thermophila 95/336 Wophila (W).

In a preferred aspect, the propeptide encoding region is nucleotides 58 to 150 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, or SEQ ID NO: 9, which encode amino acids -31 to -1 from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10; nucleotides 64 to 117 of SEQ ID NO: 11 or SEQ ID NO: 13, which encode amino acids -18 to -1 of SEQ ID NO: 12 or SEQ ID NO: 14; nucleotides 64 to 111 of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19, which encode amino acids -16 to -1 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: twenty; nucleotides 64 to 117 of SEQ ID NO: 21, which encode amino acids -18 to -1 of SEQ ID NO: 22; nucleotides 58 to 150 of SEQ ID NO: 23, which encode amino acids -31 to -1 of SEQ ID NO: 24; nucleotides 64 to 120 of SEQ ID NO: 25, which encode amino acids -19 to -1 of SEQ ID NO: 26; or nucleotides 67 to 144 of SEQ ID NO: 27, which encode amino acids -26 to -1 of SEQ ID NO: 28.

Where regions of both the signal peptide and the propeptide are present at the amino terminus of the polypeptide, the propeptide region is located in front of the amino terminus of the polypeptide, and the region of the signal peptide is located in front of the amino terminus of the propeptide region.

It may also be desirable to add regulatory sequences that allow the expression of the polypeptide to be regulated relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of a gene to be turned on and off in response to chemical or physical stimuli, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac , tac, and trp operator systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the TAKA alpha-amylase promoter, the glucoamylase promoter from Aspergillus niger and the glucoamylase promoter from Aspergillus oryzae can be used as regulatory sequences. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, they include the dehydrofolate reductory gene, which amplifies in the presence of methotrexate, and the metallothionein genes, which amplify with heavy metals. In these cases, the polypeptide encoding the nucleotide sequence would be functionally attached to the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectors containing a polynucleotide according to the present invention, a promoter and stop signals of transcription and translation. The various nucleic acid and control sequences described above can be combined to produce a recombinant expression vector, which may include one or more convenient restriction sites, to allow insertion or replacement of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, the nucleotide sequence of the present invention can be expressed by embedding a nucleotide sequence or nucleic acid construct containing the sequence in an appropriate expression vector. When creating the expression vector, the coding sequence is located in the vector so that the coding sequence is functionally linked to the corresponding control sequences for expression.

The recombinant expression vector can be any vector (for example, a plasmid or virus), which can be conveniently subjected to recombinant DNA techniques and can express the nucleotide sequence. The choice of vector will usually depend on the compatibility of the vector with the host cell into which the vector is introduced. Vectors can be linear or closed circular plasmids.

A vector can be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal unit, the replication of which is independent of chromosomal replication, for example, a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector may contain any mechanisms for ensuring self-replication. Alternatively, the vector may be one that, when introduced into a host cell, integrates into the genome and replicates along with the chromosome (s) into which it integrates. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain complete DNA for insertion into the genome of the host cell, or transposon, can be used.

The vectors of the present invention preferably contain one or more selection markers that allow easy selection of transformed cells. A selection marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, properties of the prototroph auxotrophs and the like.

Examples of bacterial selection markers are dal genes from Bacillus subtilis or Bacillus licheniformis , or markers that confer resistance to antibiotics, such as resistance to ampicillin, kanamycin, chloramphenicol or tetracycline. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable marker for use in host cells of filamentous fungi include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyl transferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine -5'-phosphate decarboxylase), sC ( adenyl transferase sulfate) and trpC (anthranilate synthase), as well as their equivalents. AmdS and pyrG genes from Aspergillus nidulans or Aspergillus oryzae and the bar gene from Streptomyces hygroscopicus are preferred for use in Aspergillus cells.

The vectors of the present invention preferably comprise element (s) that allows the incorporation of the vector into the genome of the host cell or autonomous replication of the vector in the cell, regardless of the genome.

For insertion into the genome of the host cell, the vector may be based on the sequence of a polynucleotide encoding the polypeptide or any other element of the vector for integration into the genome via homologous or non-homologous recombination. Alternatively, the vector may contain additional nucleotide sequences to guide integration through homologous recombination in the genome of the host cell to the exact position (s) on the chromosome (s). To increase the likelihood of integration at the exact location, the integration elements should preferably contain a sufficient amount of nucleic acids, such as from 100 to 10,000 base pairs, preferably from 400 to 10,000 base pairs and, most preferably, from 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to enhance the likelihood of homologous recombination. An integration element can be any sequence that is homologous to the target sequence in the genome of the host cell. In addition, the integration elements may be non-coding or coding nucleotide sequences. Alternatively, the vector can be integrated into the genome of the host cell through non-homologous recombination.

For autonomous replication, the vector may further comprise a replication origin point enabling the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in the cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence enabling a plasmid or vector to replicate in vivo .

Examples of bacterial origin of replication are the origin of replication of plasmids pBR322, pUC19, pACYC177 and pACYC184 that allow replication in E. coli , and pUB110, pE194, pTA1060 and pAMβ1 that allow replication in Bacillus .

Examples of origin of replication for use in yeast host cells are 2 micron origin of replication, ARS1, ARS4, a combination of ARS1 and CEN3, and a combination of ARS4 and CEN6.

Examples of origin of replication for use in host cells from filamentous fungi are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al ., 1987, Nucleic Acids Research 15: 9163-9175; WO 00 / 24883). Isolation of the AMA1 gene and the construction of plasmids or vectors containing the gene can be performed according to the methods disclosed in WO 00/24883.

More than one copy of the polynucleotide according to the present invention can be inserted into the host cell to increase the production of the gene product. An increase in the number of copies of a polynucleotide can be obtained by inserting at least one additional copy of the sequence into the genome of the host cell or by incorporating an amplifiable selection marker gene with a polynucleotide, where cells containing amplified copies of the selection marker gene, and therefore additional copies of the polynucleotide, can be selected by culturing cells in the presence of an appropriate selection agent.

The techniques used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, for example, Sambrook et al., 1989, supra ).

Host cells

The present invention also relates to recombinant host cells containing a polynucleotide according to the present invention, which are mainly used in recombinant production of polypeptides. A vector containing a polynucleotide according to the present invention is introduced into the host cell in such a way that the vector is maintained as a chromosome insert or as a self-replicating extrachromosomal vector as previously described. The term “host cell” encompasses any progeny of the parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will be more dependent on the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, for example, a prokaryote, or a non-unicellular microorganism, for example, a eukaryote.

Suitable unicellular microorganisms are bacterial cells such as gram positive bacteria including, but not limited to, Bacillus cell, e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis , Bacillus megaterium , Bacillus stearothermophilus , Bacillus subtilis and Bacillus thuringiensis ; or Streptomyces cells, for example, Streptomyces lividans and Streptomyces murinus , or gram-negative bacteria, such as E. coli and Pseudomonas sp. In a preferred aspect, the bacterial host cell is a Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In another preferred aspect, the Bacillus cells are alkalophilic Bacillus cells.

The introduction of a vector into a bacterial host cell can, for example, be accomplished by transformation of protoplasts (see, for example, Chang and Cohen, 1979, Molecular General Genetics 168: 111-115) using competent cells (see, for example, Young and Spizizin , 1961, Journal of Bacteriology 81: 823-829 or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ) or conjugation ( see, for example, Koehler and Thone, 1987, Journal of Bacteriology 169: 5771-5278).

A host cell may also be eukaryotic, such as mammalian, insect, plant or fungal cells.

In a preferred aspect, the host cell is a fungal cell. “Mushrooms” used herein include the types of Ascomycetes , Basidiomycetes , Chitridiomycetes and Zygomycetes (defined by Hawksworth et al. , In, Ainsworth and Bisby's Dictionary of The Fungi , 8th edition, 1995, CAB International, University Press, Cambridge, UK), and also Oomycetes (cited in Hawksworth et al. , 1995, supra , page 171) and all mitospore forming fungi (Hawksworth et al. , 1995, supra ).

In a more preferred aspect, the fungal host cell is a yeast cell. The “yeast” used here includes ascospore yeast (endomycetic order), basidiospore yeast and yeast belonging to imperfect fungi (Blastomycetes). Since the classification of fungi may change in the future, for the purposes of the present invention, the yeast will be determined as described in Biology and Activities of Yeast (Skinner, FA, Passmore, SM, and Davenport, RR, eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell . In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentous fungal cell.

"Filamentous mushrooms" include all filamentous forms of the Eumitseta and Oomycetes division (as defined by Hawksworth et al., 1995, supra ). Filamentous fungi usually have a mycelium wall consisting of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. Vegetative growth is an extension of hyphae, and carbon catabolism is obligate aerobic. In contrast, vegetative growth of yeast, such as Saccharomyces cerevisiae, occurs by budding of unicellular thallus, and carbon catabolism can be enzymatic.

In an even more preferred aspect, the host cell of filamentous fungi is a cell of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliecophoma , Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes or Trichoderma .

In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell . In another most preferred aspect, the host cell of filamentous fungi is a cell of Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium frusarum ox sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides or Fusarium venenatum . In another most preferred aspect, the host cell of a filamentous fungal cell is a Bjerkandera adusta strains, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa , or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus , Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila , Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei or Trichoderma viride.

Fungal cells can be transformed by a process including protoplast formation, protoplast transformation and cell wall regeneration in a manner generally known per se . Suitable techniques for transforming host cells from Aspergillus and Trichoderma are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable techniques for transforming the Fusarium species are described by Malardier et al. 1989, Gene 78: 147-156 and WO 96/00787. Yeast can be transformed using the techniques described by Becker and Guarente, In Abelson, JN and Simon, MI, editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Production methods

The present invention also relates to methods for producing a polypeptide according to the present invention, comprising (a) culturing a cell which, in the form of a wild type, is capable of producing a polypeptide under conditions conducive to the production of the polypeptide; and (b) isolation of the polypeptide. Preferably, the cells are of the genus Arenicola and, even more preferably, are Arenicola marina .

The present invention also relates to methods for producing a polypeptide according to the present invention, comprising (a) culturing a cell under conditions conducive to the production of the polypeptide; and (b) isolation of the polypeptide.

The present invention also relates to methods for producing a polypeptide according to the present invention, comprising (a) culturing a cell under conditions conducive to the production of a polypeptide, where the host cell contains a mutant nucleotide sequence having at least one mutation in the region encoding the mature polypeptide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, where the mutated nucleotide sequence encodes a polypeptide that contains um from amino acids 1 to 49 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10;

amino acids 1 to 46 of SEQ ID NO: 12 or SEQ ID NO: 14;

amino acids 1 to 48 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20;

amino acids 1 to 45 of SEQ ID NO: 22;

amino acids 1 to 49 of SEQ ID NO: 24;

amino acids 1 to 44 of SEQ ID NO: 26; or

amino acids 1 to 59 of SEQ ID NO: 28; and (b) isolation of the polypeptide.

In the production methods of the present invention, cells are cultured in a growth medium suitable for producing a polypeptide using methods well known in the art. For example, cells can be cultured in flasks by shaking and fermenting on a small scale or on a large scale (including continuous, batch, fed or solid phase fermentation) in a laboratory or industrial fermenter, performed in a suitable medium and under conditions allowing expression and / or isolation of the polypeptide . The cultivation takes place in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using techniques known in the art. Suitable media are available from private suppliers and can be prepared using published compositions (e.g., catalogs of the American Type Culture Collection). If the polypeptide is secreted into the culture medium, the polypeptide can be directly isolated from the medium. If the polypeptide is not secreted, it can be isolated from cell lysates.

Polypeptides can be detected using methods known in the art that are selective for polypeptides. These detection methods may include the use of specific antibodies. For example, an antimicrobial detection method can be used to determine the activity of the polypeptide described herein.

The resulting polypeptide can be isolated using methods known in the art. For example, a polypeptide can be isolated from a culture medium by well-known techniques, including, but not limited to, centrifugation, filtration, extraction, convection drying, evaporation, or precipitation.

The polypeptides of the present invention can be purified using a variety of techniques known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing and gel filtration), electrophoretic techniques (e.g. preparative isoelectrofocusing) differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE or extraction (see, for example, Protein Purification , J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Plants

The present invention also relates to a transgenic plant, part of a plant or plant cell that is transformed with a nucleotide sequence encoding a polypeptide having an antimicrobial effect of the present invention in order to express and produce a polypeptide in isolated amounts. The polypeptide may be isolated from a plant or part of a plant. Alternatively, a plant or part of a plant containing a recombinant polypeptide can be used as such to improve the quality of food or feed, for example, improve nutritional value, palatability and rheological properties, or to destroy the anti-nutritional factor.

The transgenic plant may be dicotyledonous (dicotyledonous plant) or monocotyledonous (monocotyledonous plant). Examples of monocotyledonous plants are grasses such as bluegrass, forage grasses such as fescue, chaff, temperate grasses such as field grass, and cereals such as wheat, oats, rye, barley, rice, sorghum and maize (corn).

Examples of dicotyledonous plants are tobacco, legumes such as lupine, potatoes, sugar beets, peas, beans and soybeans and cruciferous plants (Cabbage family) such as cauliflower, rapeseed and a closely related model organism, Arabidopsis thaliana .

Examples of plant parts are the stem, callus, leaves, root, fruits, seeds and tubers, as well as the individual tissues constituting these parts, for example, the epidermis, mesophyll, parenchyma, vascular tissues, meristem. Individual plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and the cytoplasm, are also considered part of the plant. In addition, any plant cell, from any source of tissue, is considered part of the plant. Similarly, parts of plants, such as specific tissues and cells isolated to facilitate the use of the invention, are also considered parts of a plant, for example, embryos, endosperm, aleuron and seed coat.

Also included in the scope of the present invention is the progeny of such plants, plant parts, and plant cells.

A transgenic plant or plant cell expressing a polypeptide according to the present invention can be interpreted in accordance with methods known in the art. Briefly, a plant or plant cell is interpreted as including one or more expression constructs encoding a polypeptide of the present invention in the genome of a host plant and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

An expression construct is typically a nucleic acid construct that contains a polynucleotide encoding a polypeptide of the present invention operably linked to the appropriate regulatory sequences required for expression of the nucleotide sequence in a selected plant or part of a plant. In addition, the expression construct may contain a selection marker suitable for identifying host cells into which the expression construct has been inserted, and DNA sequences necessary for introducing the construct into the plant of interest (the latter depends on the method of DNA injection that will be used).

The choice of regulatory sequences, such as promoter and terminator sequences and, optionally, signal or transit sequences, is determined, for example, based on when, where and how it is desirable to express the polypeptide. For example, the expression of a gene encoding a polypeptide of the present invention may be constitutive or induced, or may be developmental specific to a stage (development) or tissue, and the gene product may target a specific tissue or part of a plant, such as leaves or seeds. Regulatory sequences are, for example, those described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter, the ubiquitin 1 promoter from maize and the actin 1 promoter from rice can be used (Franck et al ., 1980, Cell 21: 285-294, Christensen et al ., 1992, Plant Mo. Biol. 18 : 675-689; Zhang et al ., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters can be, for example, a promoter from storage tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303) or from tissues of metabolic sinks, such as meristems (Ito et al ., 1994, Plant MoI. Biol. 24: 863-878), a seed specific promoter such as glutelin, prolamin, globulin or albumin rice promoters (Wu et al ., 1998, Plant and Cell Physiology 39: 885-889), a promoter from Vicia faba legumin B4 and a promoter of an unknown protein gene from a seed from Vicia faba (Conrad et al. , 1998, Journal of Plant Physiology 152: 708-711), the promoter is vegetable oil protein (Chen et al ., 1998, Plant and Cell Physiology 39: 935-941), a promoter of the napA storage protein from Brassica napus or any other seed specific promoter known in the art, for example as described in WO 91 / 14772. In addition, the promoter may be a leaf-specific promoter, such as the rbcs promoter from rice or tomato (Kyozuka et al ., 1993, Plant Physiology 102: 991-1000), the promoter of the adenine methyltransferase gene from chlorella virus (Mitra and Higgins, 1994 Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al ., 1995, Molecular and General Genetics 248: 668-674), or an injury-induced promoter such as potato pin2 (Xu et al ., 1993, Plant Molecular Biology 22: 573-588). Similarly, the promoter can be induced by non-biological effects, such as temperature, dryness or changes in mineralization, or induced by endogenously used substances that activate the promoter, for example, ethanol, oestrogens, plant hormones, such as ethylene, abscisic acid and gibberelic acid , and heavy metals.

A promoter enhancer element can also be used to achieve higher expression of the polypeptide of the present invention in a plant. For example, a promoter enhancer element may be an intron placed between a promoter and a nucleotide sequence encoding a polypeptide of the present invention. For example, Xu et al ., 1993, supra , disclose the use of the first intron in the actin 1 gene of rice to enhance expression.

The selection marker gene and any other parts of the expression construct may be selected from those available in the art.

The nucleic acid construct is incorporated into the plant genome by well-known techniques known in the art, including Agrobacterium- mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation and electroporation (Gasser et al ., 1990, Science 244: 1293; Potrykus 1990, Bio / Technology 8: 535; Shimamoto et al. , 1989, Nature 338: 274).

To date, Agrobacterium tumefaciens- mediated gene transfer is the main way to obtain transgenic dicotyledonous plants (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and can also be used to transform monocotyledonous plants, although for these plants often use other methods of transformation. To date, the main way to produce transgenic monocotyledonous plants is to bombard particles (microscopic gold or tungsten particles coated with transforming DNA) of embryonic callus or developing embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al ., 1992, Bio / Technology 10: 667-674). An alternative method for transforming monocotyledonous plants is based on the transformation of protoplasts described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.

After transformation, transformants incorporating the expression construct are selected and regenerated into whole plants by methods well known in the art. Often, a transformation technique is designed to selectively remove selection genes either during regeneration or in subsequent generations by using, for example, co-transformation with two separate T-DNA constructs or site-specific excision of a selection gene by means of a specific recombinase.

The present invention also relates to methods for producing a polypeptide according to the present invention, comprising: (a) culturing a transgenic plant or plant cell containing a polynucleotide encoding a polypeptide having an antimicrobial effect according to the present invention under conditions conducive to obtaining a polypeptide; and (b) isolation of the polypeptide.

Songs

The present invention also relates to compositions, such as pharmaceutical compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide. The term "enriched" indicates that the antimicrobial activity of the composition is increased, for example, with an enrichment factor of 1.1.

The compositions may further comprise another pharmaceutically active agent, such as an additional biocide, such as another antimicrobial polypeptide exhibiting an antimicrobial effect, as defined above. The biocide may be an antibiotic known in the art. Classes of antibiotics include penicillins, for example, penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin and the like; penicillins in combination with beta-lactamase inhibitors; cephalosporins, for example, cefaclor, cefazolin, cefuroxime, moxalactam and the like; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; cinolones; chloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc. The biocide may also be an antifungal agent, including polyenes, for example, amphotericin B, nystatin; 5-flucosine; and azoles, for example miconazole, ketoconazole, itraconazole and fluconazole.

In an embodiment of the invention, the biocide is a non-enzyme chemical agent. In another embodiment, the biocide is a non-polypeptide chemical agent.

The composition may contain a suitable carrier material. The compositions may also contain a suitable delivery vehicle capable of delivering the antimicrobial polypeptides of the invention to the desired location where the compositions are used as a medicine.

The polypeptide compositions may be prepared by methods known in the art, and may be in the form of a liquid or dry composition. For example, the polypeptide composition may be in the form of granules or microgranules. The polypeptide for inclusion in the composition may be stabilized by methods known in the art.

The following are Examples of preferred use of the polypeptide compositions of the invention. Doses of the polypeptide composition of the invention and other conditions under which the composition is used can be determined based on methods known in the art.

Methods and application

The present invention is also directed to methods of using polypeptides having antimicrobial activity. Antimicrobial polypeptides are generally applicable anywhere contaminated with bacteria, fungi, yeast or algae. Typically, places are water systems, such as cooling water systems, laundry drains, oil systems, such as cutting fluids, lubricants, oil fields and the like, where microorganisms must be killed or where their growth must be controlled. However, the present invention can also be applied in all applications for which known antimicrobial compositions are applicable, such as wood protection, latex, adhesive, adhesive, paper, cardboard, textiles, leather, plastic, sealant and feed.

Other uses include the preservation of food, beverages, cosmetics, such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiprespirants, deodorants, mouthwashes, contact lens products, enzymatic pharmaceutical compositions or food ingredients.

Therefore, the antimicrobial polypeptides of the invention can be used as a disinfectant, for example, in the treatment of infections in the eyes and mouth, skin infections; in antiprespirants or deodorants; for cleaning and disinfecting contact lenses and teeth (oral care).

It is generally understood that the antimicrobial polypeptides of the present invention are useful for cleaning, disinfecting, or inhibiting the growth of microorganisms on any surface. Examples of surfaces for which contact with the antimicrobial polypeptides of the invention may be advantageous are surfaces of processing equipment used, for example, in the dairy industry, in chemical or pharmaceutical plants, in water disinfection systems, in oil refineries, in pulp mills, in water treatment plants and cooling towers. The antimicrobial polypeptides of the invention should be used in an amount that is effective for cleaning, disinfecting, or inhibiting the growth of microorganisms on the surface in question.

The antimicrobial polypeptides of the invention can additionally be used to clean surfaces and kitchen utensils in the food industry and at any place where food is prepared or served, such as hospitals, maternity hospitals and restaurants.

They can also be used as a preservative or disinfectant in water-based paints.

The invention also relates to the use of an antimicrobial polypeptide or composition of the invention as a medicine. Further, the antimicrobial polypeptide or composition of the invention can also be used to produce drugs for controlling and controlling microorganisms, such as fungi and bacteria, preferably gram-positive bacteria.

The composition and antimicrobial polypeptide of the invention can be used as a veterinary or medical therapeutic or prophylactic agent. Therefore, the composition and antimicrobial polypeptide of the invention can be used in the preparation of veterinary or medical therapeutic or prophylactic agents for the treatment of microbial infections, such as bacterial or fungal infections, preferably infections caused by gram-positive bacteria. In particular, microbial infections may be associated with pulmonary diseases, including but not limited to tuberculosis, pneumonia, and cystic fibrosis; and sexually transmitted diseases, including but not limited to gonorrhea and chlamydia.

The compositions of the invention comprise an effective amount of an antimicrobial polypeptide of the invention.

The term “effective amount” as used herein means an amount of the antimicrobial polypeptides of the invention that is sufficient to inhibit the growth of the microorganism in question.

The invention also relates to wound healing compositions or products, such as dressings, medical devices, such as, for example, catheters, and further to anti-dandruff products, such as shampoos.

The antimicrobial polypeptide compositions of the invention are administered to a person suffering from or predisposed to a microbial infection. The introduction may be local, localized or systemic depending on the particular microorganism, preferably, it will be localized. Typically, the dose of the antimicrobial polypeptides of the invention will be sufficient to reduce the population of microorganisms by at least 50%, usually at least 1 order and maybe 2 or more orders. The compounds of the present invention are administered in a dosage that reduces the population of microorganisms, while minimizing any side effects. It is envisaged that the composition will be obtained and used under the guidance of a therapist for in vivo use. The antimicrobial polypeptides of the invention are particularly useful for killing gram-negative bacteria, including Pseudomonas aeruginosa and Chlamydia trachomatis ; and gram-positive bacteria, including streptococci, such as Streptococcus pneumonia, S. uberis, S. hyointestinalis, S. pyogenes and S. agalactiae ; and staphylococci, such as Staphylococcus aureus, S. epidermidis, S. simulans, S. xylosus and S. carnosus .

The antimicrobial polypeptide compositions of the invention can be administered to a person suffering from or predisposed to a microbial pulmonary infection, such as pneumonia; or a microbial wound infection, such as a bacterial wound infection.

The antimicrobial polypeptide compositions of the invention can be administered to a person suffering from or predisposed to a skin infection, such as acne, atopic dermatitis or seborrheic dermatitis; preferably, the skin infection is a bacterial infection of the skin, for example, caused by Staphylococcus epidermidis, Staphylococcus aureus, Propionibacterium acnes, Pityrosporum ovale or Malassezia furfur .

The antimicrobial polypeptides of the invention are also useful for in vitro pharmaceutical formulations for killing microbes, especially where many conventional antibiotics are undesirable. For example, the antimicrobial polypeptides of the invention can be added to foods for animals and / or humans; or they can be included as an additive for in vitro cell cultures to prevent the growth of microbes in cell tissue culture.

The susceptibility of a particular microbe to lysis by the antimicrobial polypeptides of the invention can be determined by in vitro testing , described in detail in the experimental section. Typically, a microorganism culture is combined with an antimicrobial polypeptide at various concentrations for a period of time sufficient for the protein to act, usually between about one hour and one day. The surviving microbes are then counted and the level of lysis is determined.

Microbes of interest include, but are not limited to, gram-positive bacteria, for example: Citrobacter sp .; Enterobacter sp .; Escherichia sp., For example, E. coli; Klebsiella sp .; Morganella sp .; Proteus sp .; Providencia sp .; Salmonella sp., E.g. S. typhi, S. typhimurium; Serratia sp .; Shigella sp .; Pseudomonas sp., E.g. P. aeruginosa; Yersinia sp., E.g. Y. pestis, Y. pseudotuberculosis, Y. enterocolitica; Franciscella sp .; Pasturella sp .; Vibrio sp., E.g. V. cholerae, V. parahemolyticus; Campylobacter sp., E.g. C. jejuni; Haemophilus sp., E.g. H. influenzae, H. ducreyi; Bordetella sp., E.g. B. pertussis, B. bronchiseptica, B. parapertussis; Brucella sp., Neisseria sp., E.g. N. gonorrhoeae, N. meningitidis , etc. Other bacteria of interest include Legionella sp., For example, L pneumophila; Listeria sp., E.g. L. monocytogenes; Mycoplasma sp., E.g. M. hominis, M. pneumoniae; Mycobacterium sp., E.g. M. tuberculosis, M. leprae; Treponema sp., E.g. T. pallidum; Borrelia sp., E.g. B. burgdorferi; Leptospirae sp .; Rickettsia sp., E.g. R. rickettsii, R. typhi; Chlamydia sp., E.g. C. trachomatis, C. pneumoniae, C. psittaci; Helicobacter sp., E.g. H. pylori and the like.

Non-bacterial pathogens of interest include fungal and protozoal pathogens, for example, Plasmodia sp., For example P. falciparum, Trypanosoma sp., For example T. brucei; schistosomes; Entaemoeba sp., Cryptococcus sp., Candida sp., E.g. C. albicans , etc.

Various administration methods may be used. The polypeptide composition may be administered orally, or may be administered intravascularly, subcutaneously, intraperitoneally, in the form of an aerosol, through the eye, into the bladder, topically, and the like. For example, administration methods by inhalation are well known in the art. The dose of the therapeutic composition will vary widely depending on the particular antimicrobial polypeptide administered, the nature of the disease, frequency of administration, route of administration, withdrawal of the agent from the patient, and the like. The initial dose may be larger, with subsequent lower maintenance doses. The dose can be administered as infrequently as weekly, or once every two weeks, or divided into smaller doses and administered once or several times a day, twice a week, etc., to maintain an effective dosage level. In many cases, oral administration will require a higher dose than intravenous administration. Amide bonds, as well as amino and carboxy terminus, can be modified for greater oral stability. For example, a carboxy terminus may be amidated.

Compositions

The compounds of the invention may be included in a variety of compositions for therapeutic administration. More specifically, the compounds of the invention may be formulated in combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated in solid, soft, liquid or gaseous preparations, such as tablets, capsules, powders, granules, ointments, creams, foams , solutions, suppositories, injections, inhalers, gels, microspheres, lotions and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, and the like. introduction. The antimicrobial polypeptides of the invention can be systemic after administration or can be localized by the use of an implant or other prescription form that acts by keeping the active dosage at the site of implantation.

In one embodiment, the composition for topical use contains a chelating agent that reduces the effective concentration of divalent cations, especially calcium and magnesium. For example, agents such as citrate, EGTA or EDTA may be included, with citrate being preferred. The concentration of citrate is usually from about 1 to 10 mm.

The compounds of the present invention can be administered individually, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.). Compounds may be formulated into pharmaceutically acceptable salts in pharmaceutical dosage forms. The following methods and excipients are illustrative only and not in any way limiting.

For oral preparations, the compounds can be used individually or in combination with appropriate additives for the manufacture of tablets, powders, granules or capsules, for example, with well-known additives such as lactose, mannitol, corn or potato starch; with binders such as crystalline cellulose, cellulose derivatives, gum, corn starch or gelatins; with disintegrants, such as corn starch, potato starch or sodium carboxymethyl cellulose; with lubricants such as talc or magnesium stearate; and, if desired, with diluents, buffering agents, moisturizing agents, preservatives and flavorings.

The compounds can be formulated for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic saturated glycerides of saturated acids, esters of higher aliphatic acids or propylene glycol; and, if desired, with conventional additives such as solvents, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

The compounds can be used as an aerosol composition for administration by inhalation. The compounds of the present invention can be formulated into pressure-sensitive acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

The compounds can be used as lotions, for example, to prevent burn infections by being formulated with conventional additives such as solvents, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

In addition, the compounds can be made in suppositories by mixing with a variety of bases, such as emulsifying bases or water-based bases. The compounds of the present invention can be administered rectally through a suppository. A suppository may include carriers, such as cocoa butter, carboax, and polyethylene glycols, which dissolve at body temperature while remaining solid at room temperature.

Standard dosage forms for oral or rectal administration may be used, such as syrups, elixirs and suspensions, where each dosage unit, for example, a teaspoon, tablespoon, tablet or suppository, contains a predetermined amount of a composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may contain the compound of the present invention in the composition as a solution in sterile water, saline, or another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well known in the art. Implants are a prescription form, like microspheres, strips, etc. with biodegradable and non-biodegradable polymers. For example, polymers of lactic acid and / or glycolic acid form an erodible polymer that is well tolerated by the patient. An implant containing the antimicrobial polypeptides of the invention is placed in close proximity to the site of infection, so that the local concentration of the active agent is increased compared to the rest of the body.

The term “unit dosage form” as used herein refers to physically discrete units suitable as unit dosages for humans and animals, each unit containing a predetermined amount of the compounds of the present invention, calculated in an amount sufficient to produce the desired effect in combination with pharmaceutically acceptable diluent, carrier or delivery vehicle. The technical specifications for the unit dosage forms of the present invention depend on the particular compound used and the effect achieved, and the pharmacodynamics associated with the compound in the patient.

Pharmaceutically acceptable excipients, such as delivery vehicles, adjuvants, carriers or diluents, are readily available to all. Moreover, pharmaceutically acceptable excipients, such as pH adjusting agents and buffering agents, physiological salt concentration adjusting agents, stabilizers, wetting agents and the like, are readily available to all.

Typical dosages for systemic administration are in the range of 0.1 to 100 milligrams per kg of patient body weight per administration. The usual dosage may be one tablet taken two to six times a day, or one capsule or sustained release tablet taken once a day and containing a proportionally higher content of the active ingredient. The effect of delayed release can be obtained using capsule materials that dissolve at different pH values, using capsules that release slowly under the influence of osmotic pressure, or by any other known controlled release means.

One skilled in the art will appreciate that dose levels may vary depending on the particular compound, the severity of the symptoms, and the patient's susceptibility to side effects. Some of the specific compounds are stronger than others. Preferred dosages for this compound are readily determined by those skilled in the art by a variety of methods. A preferred method is to measure the physiological activity of the compound.

The use of liposomes as a delivery vehicle is one method of interest. Liposomes fuse with the cells at the target location and deliver the contents of the liposome inside the cell. For fusion, the liposomes are kept in contact with the cells for a sufficient time, using various means to maintain contact, such as isolation, binding agents and the like. In one aspect of the invention, liposomes are formulated as an aerosol for administration through the lungs. Liposomes can be prepared with purified proteins or peptides that mediate membrane fusion, such as Sendai virus or influenza virus and the like. Lipids can be any suitable combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipids will usually be neutral or acidic lipids, such as cholesterol, phosphatidylserine, phosphatidylglycerol and the like.

To obtain liposomes, the technique described by Kato et al. (1991) J. Biol. Chem. 266 : 3361. Briefly, lipids and a liposome lumen composition containing peptides are mixed in an appropriate aqueous medium, typically a physiological medium, where the total dry residue will be in the range of about 1-10 weight percent. After vigorous shaking for short periods from about 5-60 s. the tube is placed in a warm water bath with a temperature of about 25-40 ° C and the cycle is repeated about 5-10 times. The composition is then voiced for a suitable period of time, typically from about 1-10 seconds, and can then be shaken on a vortex. The volume is then increased by adding an aqueous medium, usually increasing the volume by about 1-2 times, followed by shaking and cooling. This method allows you to include in the lumen of liposomes molecules with high molecular weight.

Compositions with other active agents

For use in the methods under consideration, the antimicrobial polypeptides of the invention can be formulated with other pharmaceutically active agents, in particular with other antimicrobial agents. Other agents of interest include a wide range of antibiotics known in the art. Classes of antibiotics include penicillins, for example, penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin and the like; penicillins in combination with beta-lactamase inhibitors; cephalosporins, for example, cefaclor, cefazolin, cefuroxime, moxalactam and the like; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; cinolones; chloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc.

Antifungal agents are also suitable, including polyenes, for example, amphotericin B, nystatin; 5-flucosine; and azoles, for example miconazole, ketoconazole, itraconazole and fluconazole. Anti-TB drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in the antimicrobial polypeptides of the invention, for example, interferon gamma, tumor necrosis factor alpha, interleukin 12, and the like.

In vitro synthesis

The antimicrobial polypeptides of the invention can be prepared by in vitro synthesis using standard methods known in the art. Various commercial synthesis apparatuses are available, for example, automatic synthesizers from Applied Biosystems Inc., Beckman, and the like. When using synthesizers, naturally occurring amino acids can be replaced by unnatural amino acids, especially D-isomers (or D-forms), for example, D-alanine or D-isoleucine, diastereoisomers, side chains having an excellent length or other functions and like them. The particular sequence and method of preparation will be determined by convenience, economic considerations, required purity, and the like.

Chemical crosslinking can be provided for peptides or proteins containing convenient functionalities for binding, such as amino groups for the formation of amides or substituted amines, for example, reductive amination, thiol groups for the formation of thioethers or disulfide bonds, carboxyl groups for the formation of amides and the like.

If desired, various groups can be introduced into the peptide during synthesis or during expression, which allow crosslinking with other molecules or with the surface. So, cysteines can be used to create thioesters, histidines for binding to metal ion complexes, carboxyl groups for the formation of amides or esters, amino groups for the formation of amides and the like.

Polypeptides can be isolated and purified by well-known recombinant synthesis methods. You can obtain a lysate of expressing host cells and purify the lysate using HPLC, gel filtration, gel electrophoresis, affinity chromatography or other purification techniques. For the most part, the compositions used will contain at least 20% by weight of the desired product, preferably at least about 75% by weight, more preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99 , 5% by weight relative to the impurities associated with the method of obtaining the product and its purification. Typically, percentages will be based on total protein.

Pet food

The present invention is also directed to methods of using antimicrobial polypeptides in animal feed, as well as to feed compositions and feed additives containing the antimicrobial polypeptides of the invention.

The term animal includes all animals, including humans. Examples of animals are non-ruminant animals and ruminants such as cows, sheep and horses. In a specific embodiment, the animal is not a ruminant. Non-ruminant animals include animals with a single chamber stomach, such as pigs or boars (including, but not limited to, piglets, fattening pigs and sows); poultry such as turkeys and chickens (including, but not limited to, broiler chickens, laying hens); young calves; and fish (including, but not limited to, salmon).

The term food or food composition means any compound, preparation, mixture or composition suitable for or directed to the ingestion of animals.

In the use of the invention, antimicrobial polypeptides can be fed to an animal before, after, or at the same time as food. The latter is preferred.

In a specific embodiment, the antimicrobial polypeptide in the form in which it is added to the feed, or when included in the feed supplement, is strictly defined. Strictly defined means that the antimicrobial polypeptide preparation is at least 50% pure as determined by gel filtration chromatography (see Example 12 of WO 01/58275). In other particular embodiments, the antimicrobial polypeptide preparation has 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% purity determined by this method.

A strictly defined antimicrobial drug is beneficial. For example, it is much easier to dose the antimicrobial polypeptide that is practically free of interfering or contaminating other antimicrobial polypeptides correctly in the feed. The term dosage correctly refers in particular to the task of obtaining consistent and consistent results and the ability to optimize dosage based on the desired effect.

For use in animal feed, however, the antimicrobial polypeptide does not have to be of this purity; it may include, for example, other enzymes, in which case it may be called an antimicrobial polypeptide preparation.

The antimicrobial polypeptide preparation can be (a) added directly to the feed (or used directly in the processing of plant proteins) or (b) it can be used to produce one or more intermediate compositions, such as feed additives or pre-prepared mixtures, which are subsequently added feed (or used during processing). The degree of purity described above refers to the initial antimicrobial polypeptide preparation used either in (a) or (b).

Antimicrobial polypeptide preparations with a purity in this order of magnitude are in particular obtainable using recombinant production methods, while they are not easy to obtain and they are also subject to much higher variations from batch to batch when the antimicrobial polypeptide is prepared by conventional fermentation methods.

Such an antimicrobial polypeptide preparation can, of course, be mixed with other enzymes.

The term vegetable proteins, as used herein, refers to any compound, composition, preparation or mixture that includes at least one protein derived or derived from a plant, including modified proteins and protein derivatives. In specific embodiments, the vegetable protein content is at least 10, 20, 30, 40, 50, or 60% (w / w).

Plant proteins can be obtained from sources of plant proteins, such as legumes and grains, for example, materials from plants of the families Fabaceae ( Leguminosae ), Cruciferaceae , Chenopodiaceae and Poaceae , such as soybean flour, lupine flour and rapeseed flour.

In a specific embodiment, the vegetable protein source is material from one or more plants of the Fabaceae family , for example, soy, lupine, peas, or beans.

In another specific embodiment, the vegetable protein source is material from one or more plants from the Chenopodiaceae family, for example, beets, sugar beets, spinach or movie quinoa.

Other examples of vegetable protein sources are rapeseed and cabbage.

Soy is a preferred source of vegetable protein.

Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oats, maize (corn), rice and sorghum.

The antimicrobial polypeptide can be added to the feed in any form, being either a relatively pure antimicrobial polypeptide or in admixture with other components intended to be added to animal feed, i.e. in the form of additives in animal feed, such as the so-called pre-prepared mixture for animal feed.

In a further aspect, the present invention relates to compositions for use in animal feed, such as animal feed and additives in animal feed, for example, pre-prepared mixtures.

In addition to the antimicrobial polypeptide of the invention, the animal feed additives of the invention contain at least one fat-soluble vitamin and / or at least one water-soluble vitamin, and / or at least one trace element, and / or at least one macroelement.

Further optional feed additives are colorants, aromatics, stabilizers and / or at least one enzyme selected from phytases EC 3.1.3.8. or 3.1.3.26; EU xylanase 3.2.1.8 .; EU galactanase 3.2.1.89; and / or EC beta-glucanases 3.2.1.4.

In a specific embodiment, these other enzymes are strictly defined (as defined above for antimicrobial polypeptide preparations).

Examples of other antimicrobial polypeptides (AMPs) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Tanatin, Defensin, Ovispirin, such as Novispirin (Robert Lehrer, 2000), and variants or fragments thereof that retain antimicrobial activity.

Examples of other antifungal polypeptides (AFPs) are peptides from Aspergillus giganteus and Aspergillus niger , as well as variants or fragments thereof that retain the antifungal activity described in WO 94/01459 and WO 02/090384.

Typically, fat and water soluble vitamins, as well as trace elements, form a part, a so-called pre-prepared mixture, intended to be added to the feed, while macro elements are usually separately added to the feed. Any of these types of compositions, when enriched with the antimicrobial polypeptide of the invention, is an additive to the animal feed of the invention.

In a specific embodiment, the animal feed additive of the invention is intended to be included (or prescribed as it should be) in animal or animal nutrition at a level of from 0.01 to 10%; more specifically, from 0.05 to 5.0%; or from 0.2 to 1% (% means the value in grams of the additive per 100 g of feed). This is, in particular, for pre-prepared mixtures.

The following is a non-exclusive list of examples of these compounds:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E and vitamin K, for example vitamin K3.

Examples of water soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, and pantothenate, for example Ca-D pantothenate.

Examples of trace elements are manganese, zinc, iron, copper, iodine, selenium and cobalt.

Examples of macronutrients are calcium, phosphorus and sodium.

The need for these compounds (illustrated in poultry and piglets / pigs) is shown in Table A of WO 01/58275. Need means that these compounds must be provided in food at the indicated concentrations.

Alternatively, the animal feed additive of the invention contains at least one of the individual components defined in Table A of WO 01/58275. At least one means either one, or more than one, or two, or three, or four, etc. to all thirteen or to all fifteen individual components. More specifically, this at least one individual component is included in the additive of the invention in such an amount as to provide a concentration in the feed within the limits indicated in column four, or column five, or column six of Table A.

The present invention also relates to animal feed compositions. Animal feed compositions or nutrition have a relatively high protein content. The nutrition of poultry or pigs may vary as indicated in Table B of WO 01/58275, columns 2-3. Fish nutrition may vary as indicated in column 4 of this Table B. Furthermore, such fish nutrition usually has a total fat content of 200-310 g / kg.

The animal feed composition of the invention has a total protein content of 50-800 g / kg and, in addition, contains at least one antimicrobial polypeptide as claimed herein.

In addition or alternatively (to the total protein content indicated above), the animal feed composition of the invention has a metabolizable energy content of 10-30 MJ / kg; and / or a calcium content of 0.1-200 g / kg; and / or the content of available phosphorus is 0.1-200 g / kg; and / or a methionine content of 0.1-100 g / kg; and / or methionine plus cysteine 0.1-150 g / kg; and / or a lysine content of 0.5-50 g / kg.

In specific embodiments, the content of metabolizable energy, total protein, calcium, phosphorus, methionine, methionine plus cysteine and / or lysine is in any one of rows 2, 3, 4 or 5 in Table B of WO 01/58275 (P. 2 -5).

Total protein is calculated as nitrogen (N) times 6.25, i.e. Total protein (g / kg) = N (g / kg) x 6.25. The nitrogen content is determined using the Kjeldahl method (A.O.A., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington DC).

Metabolizable energy can be calculated based on NRC Nutrient requirements in swine, ninth revised edition 1988, subcommittee on swine nutrition, committee on animal nutrition, board of agriculture, national research council. National Academy Press, Washington, D. C, p. 2-6 and European Table of Energy Values for Poultry Feed-stuffs, Spelderholt center for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen & looijen bv, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids in animal nutrition is calculated based on tables such as Veevoedertabel 1997, gegevens over chemische samenstelling, verteerbaarheid en voederwaarde van voedermiddelen, Central Veevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a specific embodiment, the animal feed composition of the invention comprises at least one vegetable protein or protein source as defined above.

In still further particular embodiments, the animal feed composition of the invention comprises 0-80% maize; and / or 0-80% sorghum; and / or 0-70% wheat; and / or 0-70% of barley; and / or 0-30% oats; and / or 0-40% soy flour; and / or 0-10% of fishmeal; and / or 0-20% whey. Animal diets can, for example, be made as a mash (not granular) or granular food. Usually, milled fodder products are mixed and a sufficient amount of necessary vitamins and minerals is added according to the technical conditions for this species. Enzymes can be added as solid or liquid enzyme compositions. For example, a solid enzyme composition is usually added before or during the mixing step; and a liquid enzyme composition is usually added after the granulation step. The enzyme may also be included in a feed additive or a pre-prepared mixture.

The final concentration of the enzyme in the diet is in the range from 0.01-200 mg of protein enzyme per kg of diet, for example, in the range of 5-30 mg of protein enzyme per kg of animal diet.

The antimicrobial polypeptide may be introduced in one or more of the following amounts (dose ranges): 0.01-200; or 0.01-100; or 0.05-100; or 0.05-50; or 0.10-10 - all of these limits, which are in mg of the antimicrobial polypeptide protein per kilogram of feed (ppm).

To determine the mg of the antimicrobial polypeptide per kg of feed, the antimicrobial polypeptide is purified from the lump composition, and the specific activity of the purified antimicrobial polypeptide is determined using an appropriate analysis method (see antimicrobial activity, substrates and analysis methods). The antimicrobial activity of the feed composition as such is also determined using the same analysis method, and based on these two definitions, the dose in mg of the antimicrobial polypeptide protein per kg of feed is calculated.

The same principles apply to the determination of mg antimicrobial polypeptide protein in feed additives. Of course, if a sample of the antimicrobial polypeptide used to prepare the feed additive or feed is available, the specific activity is determined from that sample (there is no need to purify the antimicrobial polypeptide from the feed composition or additive).

Signal Peptide or Propeptide

The present invention also relates to nucleic acid constructs comprising a gene encoding a protein operably linked to one or both of the first nucleotide sequence consisting of nucleotides 1 to 57 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 encoding a signal peptide consisting of amino acids -50 to -32 of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10; nucleotides 1 to 63 of SEQ ID NO: 11 or SEQ ID NO: 13 encoding a signal peptide consisting of amino acids -39 to -19 of SEQ ID NO: 12 or SEQ ID NO: 14; nucleotides 1 to 63 of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 encoding a signal peptide consisting of amino acids -37 to -17 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20; nucleotides 1 to 63 of SEQ ID NO: 21 encoding a signal peptide consisting of amino acids -39 to -19 of SEQ ID NO: 22; nucleotides 1 to 57 of SEQ ID NO: 23 encoding a signal peptide consisting of amino acids -50 to -32 of SEQ ID NO: 24; nucleotides 1 to 63 of SEQ ID NO: 25 encoding a signal peptide consisting of amino acids -40 to -20 of SEQ ID NO: 26; or nucleotides 1 to 66 of SEQ ID NO: 27 encoding a signal peptide consisting of amino acids -48 to -27 of SEQ ID NO: 28;

and a second nucleotide sequence consisting of nucleotides 58 to 150 of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or SEQ ID NO: 9 encoding a propeptide consisting of amino acids from -31 to -1 from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 10; nucleotides 64 to 117 of SEQ ID NO: 11 or SEQ ID NO: 13 encoding a propeptide consisting of amino acids -18 to -1 of SEQ ID NO: 12 or SEQ ID NO: 14; nucleotides 64 to 111 of SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 encoding a propeptide consisting of amino acids -16 to -1 of SEQ ID NO: 16, SEQ ID NO: 18 or SEQ ID NO: 20; nucleotides 64 to 117 of SEQ ID NO: 21 encoding a propeptide consisting of amino acids -18 to -1 of SEQ ID NO: 22; nucleotides 58 to 150 of SEQ ID NO: 23 encoding a propeptide consisting of amino acids -31 to -1 of SEQ ID NO: 24; nucleotides 64 to 120 of SEQ ID NO: 25 encoding a propeptide consisting of amino acids -19 to -1 of SEQ ID NO: 26; or nucleotides 67 to 144 of SEQ ID NO: 27 encoding a propeptide consisting of amino acids -26 to -1 of SEQ ID NO: 28; where the gene is foreign to the first and second nucleotide sequences.

The present invention also relates to recombinant expression vectors and recombinant host cells containing such nucleic acid constructs.

The present invention also relates to methods for producing a protein, comprising (a) culturing such a recombinant host cell under conditions suitable for producing a protein; and (b) protein isolation.

The first and second nucleotide sequences can be functionally attached to foreign genes separately with other control sequences or in combination with other control sequences. Such other control sequences are described supra . As described above, where the regions of both the signal peptide and the propeptide are at the amino terminus of the protein, the propeptide region is located in front of the amino acid end of the protein and the region of the signal peptide is located in front of the amino acid end of the propeptide region.

The protein may be native or heterologous to the host cell. The term “protein” is not intended to refer to the specific length of the encoded product and thus encompasses peptides, oligopeptides and proteins. The term “protein” also encompasses two or more polypeptides combined to form an encoded product. Proteins also include hybrid polypeptides that contain a combination of partial or complete polypeptide sequences derived from at least two different proteins, where one or more may be heterologous or native to the host cell. Proteins further include naturally occurring allelic or artificially created variations of the aforementioned proteins and fusion proteins.

Preferably, the protein is a hormone or variant thereof, an enzyme, a receptor or part thereof, an antibody or part thereof, or a reporter. In a more preferred aspect, the protein is an oxidoreductase, transferase, hydrolase, lyase, isomerase or ligase. In an even more preferred aspect, the protein is aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, kutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase glucose-beta-beta , laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylan Zoi.

The gene can be obtained from any prokaryotic, eukaryotic or other source.

The present invention is further described by means of the following examples, which should not be construed as limiting the scope of the invention.

EXAMPLES

The chemicals used as buffers and substrates were a commercial product of at least reagent purity (corresponding to our PSA).

EXAMPLE 1

Obtaining cDNA library from Arenicola marina

A cDNA library was obtained from the intestines of A. marina . Enriched polyA mRNA was purified, cDNA was synthesized, and a library was created using standard molecular biology techniques. A detailed protocol of the entire process can be found in the examples of international patent application WO 01/12794. The vector used for cloning was pMHAs7i. Plasmid pMHas7i is a derivative of pMhas5 (see WO 03/044049) in which SfiI cloning sites compatible with SfiI-adapted cDNA were created using the SMART protocol. Briefly, the EcoRI-NotI cDNA fragment of human adolase from lambdaTripIX2 (Clontech) was cloned into Eco-RI pMHas5 cloning sites. The resulting plasmid, pMHas7i contains human adolase cDNA flanked by the corresponding SfiI sites necessary for cloning the SMART adapted cDNA.

Obtaining pMHas7i for cloning cDNA: The vector was treated with SfiI restriction endonuclease and gel purified from the adolase insert by agarose gel electrophoresis. A strip containing the restriction endonuclease treated plasmid was purified by GFX treatment (GE Healthcare).

EXAMPLE 2

Discovery of the peptide family of marinazines by a signal capture method from a cDNA library from A. marina

The plasmid cDNA pool was obtained from 20,000 total transformants of the initial cDNA ligation in the pMHas5 vector and carried out the signal capture method as described in WO 01/77315, resulting in 381 sequence contigs.

An individual database search was performed for all 381 contigs and the results were analyzed. One contig (ZY151351) had some amino acid homology with known antimicrobial polypeptides (defensin-like polypeptides). The deduced polypeptide was named Marinazine 1A (SEQ ID NO: 6).

After re-examining the 381 contigs by searching for homology with the sequence of the marinazine 1A polypeptide, several other “marinazines” were found (SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 , SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28).

EXAMPLE 3

Creation of an Aspergillus Expression Vector for Marinazines

cDNA encoding the predicted mature portion of marinazine 1 B (amino acids 1 to 49 of SEQ ID NO: 8), marinazine 2A (amino acids 1 to 46 of SEQ ID NO: 12), marinazine 3B (amino acids 1 to 48 of SEQ ID NO: 18), Marinezine 4 (amino acids 1 to 45 of SEQ ID NO: 22), Marinezine 5 (amino acids 1 to 49 of SEQ ID NO: 24), Marinezine 6 (amino acids 1 to 44 of SEQ ID NO: 26) and Marinazine 7 (amino acids 1 through 59 of SEQ ID NO: 28), were amplified from the cDNA library from the intestines of A. marina and connected to a cDNA fragment encoding the pre-pro region of plectasin, as follows: 10 ng cDNA was used as matri PCR reactions using the oligonucleotides shown in Table 1.

Table 1 Marinezin1B-F 5 'GGATGCGAACCAACTTCAGAAACGTAGCTGGCTATGTAACTGGTTGGGC 3' Marinezin1B-R 5 'CCCAAGCTTCACATGGTGTATGGTTGATCTCTCC 3' Marinezin 2A-F 5 'GGATGCGAACCAACTTCAGAAACGTGGTTGGTGCTGGCAGTGGACATGT 3' Marinezin 2B-R 5 'CCCAAGCTTGCCCTTCTAGCGTTCAGCTCTT 3' Marinezin3B-F 5 'GGATGCGAACCAACTTCAGAAACGTCATTGGTGTTTCGAGTGGTCATGT 3' Marinezin3B-R 5 'CCCAAGCTTTCAGTGGAGAACCGTTACAGCA 3' Marinezin4-F 5 'GGATGCGAACCAACTTCAGAAACGTATCCCCTGTTGGACTCCGACATGT 3' Marinezin4-R 5 'CCCAAGCTTAGCCCAGTATGCTCTGCACGT 3' Marinezin5-F 5 'GGATGCGAACCAACTTCAGAAACGTGGGGGCCGGCCCTGTCATAGGCAT 3' Marinezin5-R 5 'CCCAAGCTTAGTTGTTCGCTCCATTAGCACCT 3' Marinezin6-F 5 'ACCAACTTCAGAAACGTGGCAGGTCGTGTAATTTCTGGTT 3' Marinezin6-R 5 'CCCAAGCTTTGGGCGATTCTATCTGCCTCTTA 3' Marinezin7-F 5 'ACCAACTTCAGAAACGTGGTGGGATGTGTGGTGACGA 3' Marinezin7-R 5 'CCCAAGCTTGCACATCGTTTCCACCGCAA 3'

5 pmol of each primer F (direct) and R (reverse) (e.g., Marinazin1B-F and Marinazin1B-R) were used in 25 μl of reaction volume with the Expand High Fidelity PCR System (Roche). After denaturation at 94 ° C for 2 minutes, 35 cycles of PCR were carried out according to the following program: 94 ° C for 15 s and 60 ° C for 60 s.

Pre-pro area of plectasin, including an 58 bp intron (see Examples from WO 03/044049), was amplified from a plasmid matrix using the oligonucleotides shown in Table 2.

table 2 Plecbhi-f 5 'CGCGGATCCCACCATGCAATTTACCACCATCCTCTC 3' Plec-mar1b-r 5 'GCCCAACCAGTTACATAGCCAGCTACGTTTCTGAAGTTGGTTCGCATCC 3' Plec-mar2a-r 5 'ACATGTCCACTGCCAGCACCAACCACGTTTCTGAAGTTGGTTCGCATCC 3' Plec-mar3b-r 5'ACATGACCACTCGAAACACCAATGACGTTTCTGAAGTTGGTTCGCATCC 3 ' Plec-mar4-r 5'TCCAACAGGGGATACGTTTCTGAAGTTGGTTCGCATCC 3 ' Plec-mar5-r 5 'ATGCCTATGACAGGGCCGGCCCCCACGTTTCTGAAGTTGGTTCGCATCC 3' Plec-mar6-r 5 'AAATTACACGACCTGCCACGTTTCTGAAGTTGGTTCGCATCC 3' Plec-mar7-r 5'CACACATCCCACCACGTTTCTGAAGTTGGTTCGCATCC 3 '

5 pmol of each primer F and R (e.g., PlecBHI-F and Marinazin1B-R) were used in 25 μl of reaction volume with the Expand High Fidelity PCR System (Roche). After denaturation at 94 ° C for 2 minutes, 35 cycles of PCR were carried out according to the following program: 94 ° C for 15 s and 60 ° C for 60 s.

PCR reactions were separated on a 2% agarose gel. Bands of the expected size for both marinazine cDNA and the pre-pro site of plectasin were isolated using the GFX DNA purification kit (Amersham) according to the manufacturer's protocol. 1/50 of the purified material was used as a template for PCR for the compound using a direct primer from plectasin and a reverse primer from marinazine (for example, for Marinazin 1B: Product Marinazina 1B-F and Marinazina 1B-R combined with PlecBHI-F and Plec-Mar1B -R as a template using PlecBHI-F and Marinazin1B-R as primers). 5 pmol of each primer F and R (e.g., Marinazin1B-F and Marinazin1B-R) was used in 25 μl of reaction volume with the Expand High Fidelity PCR System (Roche). After denaturation at 94 ° C for 2 minutes, 25 PCR cycles were performed according to the following program: 94 ° C for 15 s and 60 ° C for 60 s. PCR reactions were separated on a 2% agarose gel. The expected size bands encoding the constructs of the plectasin / marinazine fusions were isolated using the GFX DNA purification kit (Amersham) according to the manufacturer's protocol. Fragments were digested with BamHI and HindIII, which cleaved protruding ends introduced by PCR primers. Cleaved fragments were isolated and cloned into an expression plasmid pDAu109 (see Examples from WO 2005/042735).

EXAMPLE 4

The expression of marinazines in Aspergillus

Plasmids expressing marinazines based on pDAu109 were transformed into Aspergillus orizae BECh2 strain (see WO 00/393229). Ten to 20 transformants of each strain were re-isolated twice under selective and non-inducing conditions on plates with minimal Cova medium with sucrose and acetamide. To test the expression of marinazines, transformants were grown for 3 days at 26 degrees C in test tubes with 10 ml of Dap2C-1 (see "Media" in Examples from WO 2004/032648). Expression was checked using Maldi-TOF and SDS-PAGE from culture supernatants on 16% NuPage SDS gels (Invitrogen) as recommended by the manufacturer.

EXAMPLE 5

Determination of antimicrobial activity by radial diffusion

The supernatants described above in Example 4 were analyzed using the radial diffusion method following the previously published protocol for determining the antimicrobial activity of Lehrer et al., (1991) Ultra sensitive assays for endogenous antimicrobial polypeptides J Immunol Methods 137: 167-173). Target bacteria (10 ⁶ colony forming units (CFU)) were added to 10 ml of the lower agarose layer (1% low melting electroosmotic agarose, 0.03% trypticase soy medium, 10 mM sodium phosphate, pH 7.4, 37 degrees Celsius). The suspension solidified on INTEGRID Petri Dish Petri dishes (Becton Dickinson Labware, NJ). A 3 mm Gel Puncher was used to make holes in lower agarose (Amersham Pharmacia Biotech, Sweden). Samples were added to the hole and incubated at 37 degrees Celsius for 3 hours. The top layer was poured onto the surface and the plate was incubated overnight (LB medium, 7.5% agar). An antimicrobial effect was seen as bacteria-free zones around the wells. Living cells were stained with 10 ml of 0.2 mM MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium thiazole blue bromide). All standard protocols have been described elsewhere (Sambrook, Fritsch, and Maniatis, 1989). Electronic photographs were taken. Inhibition of bacterial growth is seen as transparent zones, which were measured with an accuracy of 0.1 nm and subtracted the diameter of the hole. The results are shown as zones of inhibition of the original samples (Table 3) and concentrated samples (Table 4) against a number of test strains:

A: Staphylococcus simulans (ATCC11631)

B: Staphylococcus carnosus (ATCC51365)

C: Streptococcus hyointestinalis (ATCC49169)

D: Bacillus subtilis (ATCC23857)

E: Enterococcus saccharolyticus (ATCC43076)

F: Escherichia coli (ATCC10536)

G: Acinetobacter anitratus (ATCC17903)

H: Erwinia chrysanthemi (ATCC11663)

J: Pseudomonas boreopolis (ATCC15452)

The samples were concentrated by precipitating 45 ml of the culture supernatant with 2.5 ml of 100% TCA and resuspending the precipitated precipitate in 450 μl of resuspension buffer (100 mM Tris, pH 8, 100 mM NaCl).

Table 3 The results of the radial diffusion method for the initial culture supernatants of recombinant A. oryzae clones expressing marinazines. 10 units in the radial diffusion method correspond to 1 mm of the diameter of the transparent zone around the 3 mm hole for the sample Test strain AT FROM D E Marinezin 1B 21 10 10 9 Marinezin 2A 26 12 16 fourteen Marinezin 4 60 54 32 43 Marinezin 6 - 45 39 40 The control 0 0 0 0

Table 4 The results of the radial diffusion method for TCA precipitated culture supernatants from recombinant A. oryzae clones expressing marinazines (100-fold concentrated). 10 units in the radial diffusion method correspond to 1 mm of the diameter of the transparent zone around the 3 mm hole for the sample. Test strain A B C D E F G N J Marinezin 1B 22 25 22 28 thirty 25 26 25 28 Marinezin 2A 34 35 35 38 41 25 39 40 39 Marinezin 4 fourteen 81 59 32 68 0 6 0 0 Marinezin 5 22 13 24 25 23 19 26 26 22 The control 0 0 0 0 0 0 0 0 0

EXAMPLE 6

Using HMM files from the PFAM database to identify defensin

Sequence analysis using hidden Markov model profiles (HMM profiles) can be performed either online on the Internet or locally on a computer using the well-known open-access NMMER software package. The current version is NMMER 2.3.2, dated October 2003.

HMM profiles can be obtained from the well-known PFAM database. The current version is PFAM 16.0, dated November 2004. Both NMMER and PFAM are available on all computer platforms from, for example, Washington University in St. Louis (USA), School of Medicine (http://pfam.wustl.edu and http : //hmmer.wustl.edu).

If the requested amino acid sequence or fragment thereof belongs to one of the following five PFAM families, then the amino acid sequence is the defensin of the invention:

Beta Defensin or Beta Defensin, access number: PF00711;

Defensin propep or Defensin propeptide, access number: PF00879;

Defensin_1 or "Defensin of mammals", access number: PF00323;

Defensin_2 or "Arthropod Defensin", access number: PF01097;

Gamma-thionine or the "Gamma-thionine Family", access number: PF00304.

An amino acid sequence belongs to the PFAM family of the invention if it forms an E-value that is greater than 0.1 and a count that is greater than or equal to zero when the PFAM database is used online or when the hmmpfam program (from the HMMER software package) is used locally.

When sequence analysis is performed locally using the hmmpfam program, it is necessary to obtain (download) HMM profiles from the PFAM database. Two profiles exist for each family: “xxx_ls.hmm” for general searches and “xxx_fs.hmm” for local searches (“xxx” is the name of the family). This amounts to only 10 profiles for the five families mentioned above.

These ten profiles can be used individually or combined (added) in one profile (using a text editor - profiles are ASCII files), which can be called, for example, defensin.hmm. The requested amino acid sequence can then be evaluated using the following command line:

hmmpfam -E 0.1 defensin.hmm sequence_file

- where “sequence_file” is a file with the requested amino acid sequence in any of the formats recognized by the HMMER software package.

If the score is greater than or equal to zero (0.0), and the E-value is greater than 0.1, then the requested amino acid sequence is the defensin of the invention.

The PFAM database is further described in Bateman et al. (2004) "The Pfam Protein Families Database", Nucleic Acids Research, Vol. 32 (Database Issue) p. D138-D141.

Claims (23)

1. The defensin polypeptide having antimicrobial activity, which contains the amino acid sequence represented
Cx (3) -Cx (7.9) -C-C and C-Cx (8) -C-x-C and Cx-Cx (8.11) -C. 2. The polypeptide according to claim 1, which is:
(a) a polypeptide containing an amino acid sequence that has at least 60% identity with:
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26,
or amino acids 1 to 59 of SEQ ID NO: 28; or
(b) a polypeptide that is encoded by a nucleotide sequence that hybridizes under at least average conditions with (i):
nucleotides from 151 to 297 of SEQ ID NO: 1,
nucleotides from 151 to 297 of SEQ ID NO: 3,
nucleotides from 151 to 297 of SEQ ID NO: 5,
nucleotides from 151 to 297 of SEQ ID NO: 7,
nucleotides from 151 to 297 of SEQ ID NO: 9,
nucleotides from 118 to 255 of SEQ ID NO: 11,
nucleotides from 118 to 255 of SEQ ID NO: 13,
nucleotides 112 to 255 of SEQ ID NO: 15,
nucleotides 112 to 255 of SEQ ID NO: 17,
nucleotides 112 to 255 of SEQ ID NO: 19,
nucleotides from 118 to 252 of SEQ ID NO: 21,
nucleotides from 151 to 297 of SEQ ID NO: 23,
nucleotides 121 to 252 of SEQ ID NO: 25 or
nucleotides 145 to 321 of SEQ ID NO: 27; or (ii) a complementary chain (i). 3. The polypeptide according to claim 2, containing an amino acid sequence that has at least 70% identity with:
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26 or
amino acids 1 to 59 of SEQ ID NO: 28. 4. A polypeptide containing an amino acid sequence that has at least 80% identity with:
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26 or
amino acids 1 to 59 of SEQ ID NO: 28. 5. The polypeptide according to claim 2, containing an amino acid sequence that has at least 90% identity with:
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26 or
amino acids 1 to 59 of SEQ ID NO: 28. 6. The polypeptide according to claim 2, which is encoded by a nucleotide sequence that hybridizes under medium-severe conditions with (i):
nucleotides from 151 to 297 of SEQ ID NO: 1,
nucleotides from 151 to 297 of SEQ ID NO: 3,
nucleotides from 151 to 297 of SEQ ID NO: 5,
nucleotides from 151 to 297 of SEQ ID NO: 7,
nucleotides from 151 to 297 of SEQ ID NO: 9,
nucleotides from 118 to 255 of SEQ ID NO: 11,
nucleotides from 118 to 255 of SEQ ID NO: 13,
nucleotides 112 to 255 of SEQ ID NO: 15,
nucleotides 112 to 255 of SEQ ID NO: 17,
nucleotides 112 to 255 of SEQ ID NO: 19,
nucleotides from 118 to 252 of SEQ ID NO: 21,
nucleotides from 151 to 297 of SEQ ID NO: 23,
nucleotides from 121 to 252 of SEQ ID NO: 25,
or nucleotides from 145 to 321 of SEQ ID NO: 27 or
(ii) a complementary chain (i). 7. The polypeptide according to claim 2, which is encoded by a nucleotide sequence that hybridizes under stringent conditions with (i):
nucleotides from 151 to 297 of SEQ ID NO: 1,
nucleotides from 151 to 297 of SEQ ID NO: 3,
nucleotides from 151 to 297 of SEQ ID NO: 5,
nucleotides from 151 to 297 of SEQ ID NO: 7,
nucleotides from 151 to 297 of SEQ ID NO: 9,
nucleotides from 118 to 255 of SEQ ID NO: 11,
nucleotides from 118 to 255 of SEQ ID NO: 13,
nucleotides 112 to 255 of SEQ ID NO: 15,
nucleotides 112 to 255 of SEQ ID NO: 17,
nucleotides 112 to 255 of SEQ ID NO: 19,
nucleotides from 118 to 252 of SEQ ID NO: 21,
nucleotides from 151 to 297 of SEQ ID NO: 23,
nucleotides from 121 to 252 of SEQ ID NO: 25,
or nucleotides from 145 to 321 of SEQ ID NO: 27 or
(ii) a complementary chain (i). 8. The polypeptide according to any one of claims 1 to 7, which consists of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28. 9. The polypeptide according to claim 2, which consists of
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26 or
amino acids 1 to 59 of SEQ ID NO: 28. 10. Polynucleotide containing a nucleotide sequence that encodes a polypeptide according to any one of claims 1 to 9. 11. The polynucleotide of claim 10, having at least one mutation in a coding mature polypeptide sequence from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27, in which the mutant nucleotide sequence encodes a polypeptide consisting of
amino acids 1 to 49 of SEQ ID NO: 2,
amino acids 1 to 49 of SEQ ID NO: 4,
amino acids 1 to 49 of SEQ ID NO: 6,
amino acids 1 to 49 of SEQ ID NO: 8,
amino acids 1 to 49 of SEQ ID NO: 10,
amino acids 1 to 46 of SEQ ID NO: 12,
amino acids 1 to 46 of SEQ ID NO: 14,
amino acids 1 to 48 of SEQ ID NO: 16,
amino acids 1 to 48 of SEQ ID NO: 18,
amino acids 1 to 48 of SEQ ID NO: 20,
amino acids 1 to 45 of SEQ ID NO: 22,
amino acids 1 to 49 of SEQ ID NO: 24,
amino acids 1 to 44 of SEQ ID NO: 26 or
amino acids 1 to 59 of SEQ ID NO: 28. 12. The nucleic acid construct containing the polynucleotide of claim 10, operably linked to one or more coding sequences that direct the production of the polypeptide in the expression host. 13. Recombinant expression vector containing the nucleic acid construct of claim 12. 14. Recombinant host cell containing the nucleic acid construct of claim 12. 15. A method for producing a polypeptide according to any one of claims 1 to 9, comprising (a) culturing a cell which, in the form of a wild type, is capable of producing a polypeptide under conditions conducive to the production of the polypeptide; and (b) isolation of the polypeptide. 16. A method for producing a polypeptide according to any one of claims 1 to 9, comprising (a) culturing a host cell containing a nucleic acid construct containing a nucleotide sequence encoding a polypeptide under conditions conducive to the production of the polypeptide; and (b) isolation of the polypeptide. 17. An isolated polynucleotide obtained by (a) hybridizing a DNA population under medium conditions, preferably medium hard or stringent conditions with (i)
nucleotides from 151 to 297 of SEQ ID NO: 1,
nucleotides from 151 to 297 of SEQ ID NO: 3,
nucleotides from 151 to 297 of SEQ ID NO: 5,
nucleotides from 151 to 297 of SEQ ID NO: 7,
nucleotides from 151 to 297 of SEQ ID NO: 9,
nucleotides from 118 to 255 of SEQ ID NO: 11,
nucleotides from 118 to 255 of SEQ ID NO: 13,
nucleotides 112 to 255 of SEQ ID NO: 15,
nucleotides 112 to 255 of SEQ ID NO: 17,
nucleotides 112 to 255 of SEQ ID NO: 19,
nucleotides from 118 to 252 of SEQ ID NO: 21,
nucleotides from 151 to 297 of SEQ ID NO: 23,
nucleotides 121 to 252 of SEQ ID NO: 25 or
nucleotides 145 to 321 of SEQ ID NO: 27; or (ii) a complementary chain (i); and (b) isolating a hybridizable polynucleotide that encodes a polypeptide having an antimicrobial effect. 18. A method for producing a polypeptide according to any one of claims 1 to 9, comprising (a) cultivating a transgenic plant or plant cell containing a polynucleotide encoding a polypeptide having an antimicrobial effect according to the present invention under conditions conducive to the production of the polypeptide; and (b) isolation of the polypeptide. 19. A transgenic plant, part of a plant or plant cell that has been transformed with a polynucleotide encoding a polypeptide according to any one of claims 1 to 9. 20. A method of lysing or inhibiting the growth of microbial cells, comprising contacting the microbial cells with an antimicrobial polypeptide as defined in any one of claims 1 to 9. 21. An antimicrobial polypeptide, as defined in any one of claims 1 to 9, for use as a medicine or antimicrobial veterinary or medical therapeutic or prophylactic agent. 22. The use of an antimicrobial polypeptide, as defined in any one of claims 1 to 9, in the preparation of a veterinary or medical therapeutic or prophylactic agent for the treatment of microbial infection or for prophylactic use. 23. The use of at least one antimicrobial polypeptide, as defined in any one of claims 1 to 9, in animal feed.