Patent Publication Number: US-2010120690-A1

Title: Polypeptides Having Antimicrobial Activity and Polynucleotides Encoding Same

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional of U.S. application Ser. No. 11/446,896 filed Jun. 5, 2006, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 2005 00823 and PA 2005 01435 filed Jun. 6, 2005 and Oct. 13, 2005, respectively, and U.S. provisional application Nos. 60/726,992 and 60/688,589 filed Oct. 14, 2005 and June 8, respectively, the contents of which are fully incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to isolated polypeptides having antimicrobial activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides. 
     BACKGROUND OF THE INVENTION 
     It is an object of the present invention to provide polypeptides having improved antimicrobial activity. The polypeptides may exhibit reduced hemolytic activity and/or reduced cytotoxicity. The polypeptides may also exhibit reduced sensitivity towards cations, such as Ca 2+ , Mg 2+ , Na + . The polypeptides may also exhibit a different antimicrobial spectrum compared to SEQ ID NO: 1. 
     SUMMARY OF THE INVENTION 
     The present invention provides a polypeptide having antimicrobial activity which comprises, preferably consists of, an amino acid sequence which has at least 80% identity with amino acids 1 to 40 of the amino acid sequence: 
                    G-X 1 -G- C -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 - C -H-X 12 -               X 13 - C -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -G-G-X 21 - C -X 22 -               X 23 -X 24 -X 25 -X 26 -X 27 - C -X 28 - C -X 29 ;            
wherein
 
X 1 =F, L, W or I; preferably X 1 =F;
 
X 2 =N, R, Q, V, G, S, A, K, L, M, D, H or Y; preferably X 2 =N, R, Q, V, G, S, A, K or Y;
 
X 3 =G, R, A or K; preferably X 3 =G;
 
X 4 =P, A, L, V, K or R; preferably X 4 =P, K or R;
 
     X 5 =W or R; 
     X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or Y; preferably X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, V or Y;
 
X 7 =E, G, A, L, C, Q or S; preferably X 7 =E, G or S;
 
X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M, A, C or R; preferably X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or R;
 
X 9 =D or P; preferably X 9 =D;
 
X 10 =M, R, S, V, A, F, G, L, T, Y, W, E or K; preferably X 10 =M, R, S, V, G, Y, L, F, T, W or K;
 
X 11 =Q, R, L, F, G, H, S, A, C, I, K, M, P, T, V, W or Y; preferably X 11 =Q, R, L, F, G, H, S, K or Y;
 
     X 12 =N, R, I, Y, V, K, T, Q, S, F, A, W, E or H; 
     X 13 =H, A, F, Q, T, V or L; preferably X 13 =H or L;
 
X 14 =K, Q or R; preferably X 14 =K or R;
 
     X 15 =S, A, V, N or F; 
     X 16 =I, L, M, T, W or V; preferably X 16 =I, L or V; 
     X 17 =K, T or R; 
     X 18 =G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably X 18 =G, H, R, K or N;
 
X 19 =Y, H, K, L, M, N, Q, S, V or R; preferably X 19 =Y or R;
 
X 20 =K, F, H, T, C or R; preferably X 20 =K or R;
 
X 21 =Y, F, R, A, H, L, M, S or W; preferably X 21 =Y, F, R or W;
 
X 22 =A, K, N, Q, T, E, H, I, R, S, V, G or Y; preferably X 22 =A, K, N, Q, T, S or Y;
 
X 23 =K, R or T; preferably X 23 =K or R;
 
X 24 =G, K, Q, E, N, S, T, A or R; preferably X 24 =G, K, Q, A or R;
 
X 25 =G, K, H, W or R; preferably X 25 =G, K or R;
 
X 26 =F, A, H, I, M, V, W, R or L; preferably X 26 =F or L;
 
X 27 =V, L, M, I, K, Q, R or T; preferably X 27 =V, L, M or T;
 
X 28 =K, H, N or R; preferably X 28 =K or R;
 
X 29 =Y, I, YRCG or YR; preferably X 29 =Y or YR;
 
and which has less than 100% identity with amino acids 1 to 40 of SEQ ID NO: 1.
 
     The present invention also relates to nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising the polynucleotides. 
     The present invention also relates to methods for producing such polypeptides having antimicrobial activity comprising (a) cultivating a recombinant host cell comprising a nucleic acid construct comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     The present invention also relates to methods of using the polypeptides of the invention. 
     DEFINITIONS 
     Antimicrobial activity: The term “antimicrobial activity” is defined herein as an activity which is capable of killing or inhibiting growth of microbial cells. In the context of the present invention the term “antimicrobial” is intended to mean that there is a bactericidal and/or a bacteriostatic and/or fungicidal and/or fungistatic effect and/or a virucidal effect, wherein the term “bactericidal” is to be understood as capable of killing bacterial cells. The term “bacteriostatic” is to be understood as capable of inhibiting bacterial growth, i.e., inhibiting growing bacterial cells. The term “fungicidal” is to be understood as capable of killing fungal cells. The term “fungistatic” is to be understood as capable of inhibiting fungal growth, i.e., inhibiting growing fungal cells. The term “virucidal” is to be understood as capable of inactivating virus. The term “microbial cells” denotes bacterial or fungal cells (including yeasts). 
     In the context of the present invention the term “inhibiting growth of microbial cells” is intended to mean that the cells are in the non-growing state, i.e., that they are not able to propagate. 
     For purposes of the present invention, antimicrobial activity may be determined according to the procedure described by Lehrer et al., 1991,  Journal of Immunological Methods  137(2): 167-174. Alternatively, antimicrobial activity may be determined according to the NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards). 
     Polypeptides having antimicrobial activity may be capable of reducing the number of living cells of  Escherichia coli  (DSM 1576) to 1/100 after 24 hours (preferably after 12 hours, more preferably after 8 hours, more preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and in particular after 30 minutes) incubation at 20° C. in an aqueous solution of 25% (w/w); preferably in an aqueous solution of 10% (w/w); more preferably in an aqueous solution of 5% (w/w); even more preferably in an aqueous solution of 1% (w/w); most preferably in an aqueous solution of 0.5% (w/w); and in particular in an aqueous solution of 0.1% (w/w) of the polypeptides having antimicrobial activity. 
     Polypeptides having antimicrobial activity may also be capable of inhibiting the outgrowth of  Escherichia coli  (DSM 1576) for 24 hours at 25° C. in a microbial growth substrate, when added in a concentration of 1000 ppm; preferably when added in a concentration of 500 ppm; more preferably when added in a concentration of 250 ppm; even more preferably when added in a concentration of 100 ppm; most preferably when added in a concentration of 50 ppm; and in particular when added in a concentration of 25 ppm. 
     Polypeptides having antimicrobial activity may be capable of reducing the number of living cells of  Bacillus subtilis  (ATCC 6633) to 1/100 after 24 hours (preferably after 12 hours, more preferably after 8 hours, more preferably after 4 hours, more preferably after 2 hours, most preferably after 1 hour, and in particular after 30 minutes) incubation at 20° C. in an aqueous solution of 25% (w/w); preferably in an aqueous solution of 10% (w/w); more preferably in an aqueous solution of 5% (w/w); even more preferably in an aqueous solution of 1% (w/w); most preferably in an aqueous solution of 0.5% (w/w); and in particular in an aqueous solution of 0.1% (w/w) of the polypeptides having antimicrobial activity. 
     Polypeptides having antimicrobial activity may also be capable of inhibiting the outgrowth of  Bacillus subtilis  (ATCC 6633) for 24 hours at 25° C. in a microbial growth substrate, when added in a concentration of 1000 ppm; preferably when added in a concentration of 500 ppm; more preferably when added in a concentration of 250 ppm; even more preferably when added in a concentration of 100 ppm; most preferably when added in a concentration of 50 ppm; and in particular when added in a concentration of 25 ppm. 
     The polypeptides of the present invention have 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 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the antimicrobial activity of the polypeptide consisting of the amino acid sequence shown as amino acids 1 to 40 of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. 
     Defensin: The term “defensin” as used herein refers to polypeptides recognized by a person skilled in the art as belonging to the defensin class of antimicrobial peptides. To determine if a polypeptide is a defensin according to the invention, the amino acid sequence is preferably compared with the hidden markov model profiles (HMM profiles) of the PFAM database by using the freely available HMMER software package (see Example 7). 
     The PFAM defensin families include Defensin — 1 or “Mammalian defensin” (accession no. PF00323), Defensin — 2 or “Arthropod defensin” (accession no. PF01097), Defensin_beta or “Beta Defensin” (accession no. PF00711), Defensin_propep or “Defensin propeptide” (accession no. PF00879) and Gamma-thionin or “Gamma-thionins family” (accession no. PF00304). 
     The defensins may belong to the alpha-defensin class, the beta-defensin class, the theta-defensin class, the insect or arthropod defensin classes, or the plant defensin class. 
     In an embodiment, the amino acid sequence of a defensin according to the invention comprises 4, 5, 6, 7, or 8 cysteine residues, preferably 4, 5, or 6 cysteine residues, more preferably 4 or 6 cysteine residues, and most preferably 6 cysteine residues. 
     The defensins may also be synthetic defensins sharing the characteristic features of any of the defensin classes. 
     Examples of such defensins include, but are not limited to, α-Defensin HNP-1 (human neutrophil peptide) HNP-2 and HNP-3; β-Defensin-12, Drosomycin, Heliomicin, γ1-purothionin, Insect defensin A, and the defensins disclosed in PCT applications WO 99/53053, WO 02/06324, WO 02/085934, PCT/DK2005/000725, PCT/DK2005/000735 and PCT/DK2006/000155. 
     Isolated polypeptide: The term “isolated polypeptide” as used herein refers to a polypeptide which is at least 20% pure, preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, most preferably at least 90% pure, and even most preferably at least 95% pure, as determined by SDS-PAGE. 
     Substantially pure polypeptide: The term “substantially pure polypeptide” denotes herein a polypeptide preparation which 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 most preferably at most 0.5% by weight of other polypeptide material with which it is natively associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. 
     The polypeptides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polypeptides are in “essentially pure form”, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively associated. This can be accomplished, for example, by preparing the polypeptide by means of well-known recombinant methods or by classical purification methods. 
     Herein, the term “substantially pure polypeptide” is synonymous with the terms “isolated polypeptide” and “polypeptide in isolated form.” 
     Variant: The term “variant” is defined herein as an antimicrobial polypeptide comprising one or more alterations, such as substitutions, insertions, deletions, and/or truncations of one or more specific amino acid residues at one or more specific positions in the polypeptide. 
     Numbering of Variants: In the present invention, a specific numbering of amino acid residue positions in the antimicrobial polypeptide variants is employed. For example, by aligning the amino acid sequences of known antimicrobial polypeptides, it is possible to designate an amino acid position number to any amino acid residue in any antimicrobial polypeptide. 
     Using the numbering system originating from the amino acid sequence of the antimicrobial polypeptide disclosed in SEQ ID NO: 1, aligned with the amino acid sequence of a number of other antimicrobial polypeptides, it is possible to indicate the position of an amino acid residue in an antimicrobial polypeptide in regions of structural homology. 
     Multiple alignments of protein sequences may be made, for example, using “ClustalW” (Thompson, Higgins, and Gibson, 1994, CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice,  Nucleic Acids Research  22: 4673-4680). Multiple alignments of DNA sequences may be done using the protein alignment as a template, replacing the amino acids with the corresponding codon from the DNA sequence. 
     Pairwise sequence comparison algorithms in common use are adequate to detect similarities between protein sequences that have not diverged beyond the point of approximately 20-30% sequence identity (Doolittle, 1992,  Protein Sci.  1: 191-200; Brenner et al., 1998,  Proc. Natl. Acad. Sci. USA  95, 6073-6078). However, truly homologous proteins with the same fold and similar biological function have often diverged to the point where traditional sequence-based comparison fails to detect their relationship (Lindahl and Elofsson, 2000,  J. Mol. Biol.  295: 613-615). Greater sensitivity in sequence-based searching can be attained using search programs that utilize probabilistic representations of protein families (profiles) to search databases. For example, the PSI-BLAST program generates profiles through an iterative database search process and is capable of detecting remote homologs (Atschul et al., 1997,  Nucleic Acids Res.  25: 3389-3402). Even greater sensitivity can be achieved if the family or superfamily for the protein of interest has one or more representatives in the protein structure databases. Programs such as GenTHREADER (Jones, 1999,  J. Mol. Biol.  287: 797-815; McGuffin and Jones, 2003,  Bioinformatics  19: 874-881) utilize information from a variety of sources (PSI-BLAST, secondary structure prediction, structural alignment profiles, and solvation potentials) as input to a neural network that predicts the structural fold for a query sequence. Similarly, the method of Gough et al., 2000,  J. Mol. Biol.  313: 903-919, can be used to align a sequence of unknown structure with the superfamily models present in the SCOP database. These alignments can in turn be used to generate homology models for the protein of interest, and such models can be assessed for accuracy using a variety of tools developed for that purpose. 
     For proteins of known structure, several tools and resources are available for retrieving and generating structural alignments. For example the SCOP superfamilies of proteins have been structurally aligned, and those alignments are accessible and downloadable. These alignments can be used to predict the structurally and functionally corresponding amino acid residues in proteins within the same structural superfamily. This information, along with information derived from homology modeling and profile searches, can be used to predict which residues to mutate when moving mutations of interest from one protein to a close or remote homolog. 
     In describing the various antimicrobial polypeptide variants of the present invention, the nomenclature described below is adapted for ease of reference. In all cases, the accepted IUPAC single letter or triple letter amino acid abbreviation is employed. 
     For an amino acid substitution, the following nomenclature is used: Original amino acid, position, substituted amino acid(s). Accordingly, the substitution of threonine with alanine at position 226 is designated as “T226A”; and substitution of tyrosine with tyrosine and arginine at position 40 (effectively adding arginine after tyrosine) is designated as “Y40YR”. Multiple mutations are separated by addition marks (“+”), e.g., “G205R+S411F”, representing mutations at positions 205 and 411 substituting glycine (G) with arginine (R), and serine (S) with phenylalanine (F), respectively. 
     Parent antimicrobial polypeptide: The term “parent” antimicrobial polypeptide as used herein means an antimicrobial polypeptide to which modifications, e.g., substitution(s), insertion(s), deletion(s), and/or truncation(s), are made to produce the antimicrobial polypeptide variants of the present invention. This term also refers to the polypeptide with which a variant is compared and aligned. The parent may be a naturally occurring (wild type) polypeptide, or it may even be a variant thereof, prepared by any suitable means. For instance, the parent protein may be a variant of a naturally occurring polypeptide which has been modified or altered in the amino acid sequence. A parent may also be an allelic variant which is a polypeptide encoded by any of two or more alternative forms of a gene occupying the same chromosomal locus. 
     Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”. 
     For purposes of the present invention, the degree of identity between two amino acid sequences is determined by using the program FASTA included in version 2.0× of the FASTA program package (see Pearson and Lipman, 1988, “Improved Tools for Biological Sequence Analysis”,  PNAS  85: 2444-2448; and Pearson, 1990, “Rapid and Sensitive Sequence Comparison with FASTP and FASTA”,  Methods in Enzymology  183: 63-98). The scoring matrix used was BLOSUM50, gap penalty was −12, and gap extension penalty was −2. 
     The degree of identity between two nucleotide sequences is determined using the same algorithm and software package as described above. The scoring matrix used was the identity matrix, gap penalty was −16, and gap extension penalty was −4. 
     Alternatively, an alignment of two amino acid sequences is determined by using the Needle program from the EMBOSS package (http://emboss.org) version 2.8.0. The Needle program implements the global alignment algorithm described in Needleman and Wunsch, 1970,  J. Mol. Biol.  48: 443-453. The substitution matrix used is BLOSUM62, gap opening penalty is 10, and gap extension penalty is 0.5. The degree of identity between an amino acid sequence of the present invention (such as amino acids 1 to 40 of SEQ ID NO: 1) and a different amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length (number of amino acid residues) of the sequence of the present invention; or alternatively the output of Needle labeled “longest identity” is used as the percent identity and is calculated as follows: (Identical Residues×100)/(Length of Alignment Number of Gaps in Alignment). The result is expressed in percent identity. 
     Polypeptide Fragment: The term “polypeptide fragment” is defined herein as a polypeptide having one or more amino acids deleted from the amino and/or carboxyl terminus of SEQ ID NO: 2 or a homologous sequence thereof, wherein the fragment has antimicrobial activity. 
     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 or a homologous sequence thereof, wherein the subsequence encodes a polypeptide fragment having antimicrobial activity. 
     Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. 
     Substantially pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide 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 most preferably at most 0.5% by weight of other polynucleotide material with which it is natively associated. 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 is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form. In particular, it is preferred that the polynucleotides disclosed herein are in “essentially pure form”, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively associated. Herein, the term “substantially pure polynucleotide” is synonymous with the terms “isolated polynucleotide” and “polynucleotide in isolated form.” The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof. 
     cDNA: The term “cDNA” is defined herein as a DNA molecule which can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA which is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences. 
     Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise 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 a coding sequence of the present invention. 
     Control sequence: The term “control sequences” is defined herein to include all components, which are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide. 
     Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide. 
     Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG. The coding sequence may a DNA, cDNA, or recombinant nucleotide sequence. 
     Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. 
     Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the invention, and which is operably linked to additional nucleotides that provide for its expression. 
     Host cell: The term “host cell”, as used herein, includes any cell type which is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct comprising a polynucleotide of the present invention. 
     Modification: The term “modification” means herein any chemical modification of the polypeptide consisting of amino acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274 as well as genetic manipulation of the DNA encoding that polypeptide. The modification(s) can be substitution(s), deletion(s) and/or insertions(s) of the amino acid(s) as well as replacement(s) of amino acid side chain(s); or use of unnatural amino acids with similar characteristics in the amino acid sequence. In particular the modification(s) can be amidations, such as amidation of the C-terminus. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Polypeptides Having Antimicrobial Activity 
     In a first aspect, the present invention provides a polypeptide having antimicrobial activity which comprises, preferably consists of, an amino acid sequence which has at least 70% identity (preferably at least 80% identity, more preferably at least 85% identity, even more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97% identity, and in particular 100% identity) with amino acids 1 to 40 of the amino acid sequence (I): 
                    G-X 1 -G- C -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -X 9 -X 10 -X 11 - C -H-X 12 -               X 13 - C -X 14 -X 15 -X 16 -X 17 -X 18 -X 19 -X 20 -G-G-X 21 - C -X 22 -               X 23 -X 24 -X 25 -X 26 -X 27 - C -X 28 - C -X 29 ;            
wherein
 
X 1 =F, L, W or I; preferably X 1 =F;
 
X 2 =N, R, Q, V, G, S, A, K, L, M, D, H or Y; preferably X 2 =N, R, Q, V, G, S, A, K or Y; more preferably X 2 =N, R, S or G;
 
X 3 =G, R, A or K; preferably X 3 =G;
 
X 4 =P, A, L, V, K or R; preferably X 4 =P, K or R;
 
     X 5 =W or R; 
     X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, C, I, R, V or Y; preferably X 6 =D, A, G, K, L, T, N, F, H, M, P, Q, S, V or Y; more preferably X 6 =D, S, A, G or N;
 
X 7 =E, G, A, L, C, Q or S; preferably X 7 =E, G or S;
 
X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M, A, C or R; preferably X 8 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or R; more preferably X 8 =D, G or N;
 
X 9 =D or P; preferably X 9 =D;
 
X 10 =M, R, S, V, A, F, G, L, T, Y, W, E or K; preferably X 10 =M, R, S, V, G, Y, L, F, T, W or K; more preferably X 10 =M, L, G or V;
 
X 11 =Q, R, L, F, G, H, S, A, C, I, K, M, P, T, V, W or Y; preferably X 11 =Q, R, L, F, G, H, S, K or Y; more preferably X 11 =Q, K, R or F;
 
X 12 =N, R, I, Y, V, K, T, Q, S, F, A, W, E or H; more preferably X 12 =N, R, V or Q;
 
X 13 =H, A, F, Q, T, V or L; preferably X 13 =H or L;
 
X 14 =K, Q or R; preferably X 14 =K or R;
 
     X 15 =S, A, V, N or F; 
     X 16 =I, L, M, T, W or V; preferably X 16 =I, L or V; 
     X 17 =K, T or R; 
     X 18 =G, H, K, A, P, F, I, Q, R, S, T, Y or N; preferably X 18 =G, H, R, K or N; more preferably 
     X 18 =G or R; 
     X 19 =Y, H, K, L, M, N, Q, S, V or R; preferably X 19 =Y or R;
 
X 20 =K, F, H, T, C or R; preferably X 20 =K or R;
 
X 21 =Y, F, R, A, H, L, M, S or W; preferably X 21 =Y, F, R or W;
 
X 22 =A, K, N, Q, T, E, H, I, R, S, V, G or Y; preferably X 22 =A, K, N, Q, T, S or Y; more preferably X 22 =A, S or T;
 
X 23 =K, R or T; preferably X 23 =K or R;
 
X 24 =G, K, Q, E, N, S, T, A or R; preferably X 24 =G, K, Q, A or R; more preferably X 24 =G or A;
 
X 25 =G, K, H, W or R; preferably X 25 =G, K or R;
 
X 26 =F, A, H, I, M, V, W, R or L; preferably X 26 =F or L;
 
X 27 =V, L, M, I, K, Q, R or T; preferably X 27 =V, L, M or T; more preferably X 27 =V or L;
 
X 28 =K, H, N or R; preferably X 28 =K or R;
 
X 29 =Y, I, YRCG or YR; preferably X 29 =Y or YR;
 
and which has less than 100% identity with amino acids 1 to 40 of SEQ ID NO: 1.
 
     In an embodiment, the polypeptide of the invention is a polypeptide having antimicrobial activity which comprises, preferably consists of, an amino acid sequence which has at least 70% identity (preferably at least 80% identity, more preferably at least 85% identity, even more preferably at least 90% identity, even more preferably at least 95% identity, most preferably 97% identity, and in particular 100% identity) with amino acids 1 to 40 of the amino acid sequence (II): 
                    G-F-G- C -X 1 -G-X 2 -X 3 -X 4 -X 5 -X 6 -D-X 7 -X 8 - C -H-X 9 -X 10 - C -               X 11 -S-X 12 -X 13 -X 14 -X 15 -X 16 -G-G-X 17 - C -X 18 -K-X 19 -X 20 -               X 21 -X 22 - C -K- C -X 23 ;            
wherein
 
X 1 =N, R, Q, V, G, S, A, K or Y; preferably X i =N, R, S or G;
 
     X 2 =P, K or R; 
     X 3 =W or R; 
     X 4 =D, A, G, K, L, T, N, F, H, M, P, Q, S, V or Y; preferably X 4 =D, S, A, G or N; 
     X 5 =E, G or S; 
     X 6 =D, F, G, N, V, Y, H, K, L, P, S, T, W, I, M or R; preferably X 6 =D, G or N;
 
X 7 =M, R, S, V, G, Y, L, F, T, W or K; preferably X 7 =M, L, G or V;
 
X 8 =Q, R, L, F, G, H, S, K or Y; preferably X 8 =Q, K, R or F;
 
X 9 =N, R, I, Y, V, K, T, S, Q or H; preferably X 9 =N, R, V or Q;
 
     X 10 =H or L; 
     X 11 =K or R; 
     X 12 =I, L or V; 
     X 13 =K or R; 
     X 14 =G, H, R, K or N; preferably X 14 =G or R; 
     X 15 =Y or R; 
     X 16 =K or R; 
     X 17 =Y, F, R or W; 
     X 18 =A, K, N, Q, T, S or Y; preferably X 18 =A, S or T;
 
X 19 =G, K, Q, A or R; preferably X 19 =G or A;
 
     X 20 =G, K or R; 
     X 21 =F or L; 
     X 22 =V, L, M or T; preferably X 22 =V or L; 
     X 23 =Y or YR; 
     and which has less than 100% identity with amino acids 1 to 40 of SEQ ID NO: 1. 
     In another embodiment, the amino acid sequence (I) and/or (II) has 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences compared to the amino acid sequence of SEQ ID NO: 1. Preferably 1, 2, 3, 4, 5 or 6; more preferably 1, 2, 3, 4 or 5; even more preferably 1, 2, 3 or 4; even more preferably 1, 2 or 3; and most preferably 1 or 2 amino acids are different compared to the amino acid sequence of SEQ ID NO: 1. 
     In another embodiment, the amino acid sequence (I) and/or (II) has at least 60% identity with amino acids 1 to 40 of SEQ ID NO: 1, preferably at least 65% identity, at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity with amino acids 1 to 40 of SEQ ID NO: 1. 
     In another embodiment, the amino acid sequence (I) and/or (II) has 0, 1, 2, 3, 4 or 5 insertions, preferably 0, 1, 2 or 3 insertions, more preferably 0, 1 or 2 insertions; and 0, 1, 2, 3, 4 or 5 deletions, preferably 0, 1, 2 or 3 deletions, more preferably 0, 1 or 2 deletions, as compared to SEQ ID NO: 1 or anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. 
     In another embodiment, the polypeptide of the invention comprises, preferably consists of, an amino acid sequence which has at least 60% identity (preferably 70% identity, more preferably 80% identity, even more preferably 85% identity, even more preferably 90% identity, even more preferably 95% identity, and most preferably 100% identity) with amino acids 1 to 40 of anyone of SEQ ID NO: 3 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274, preferably anyone of SEQ ID NO: 3 to SEQ ID NO: 117 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID 
     NO: 252 to SEQ ID NO: 274. 
     The term “anyone of SEQ ID NO: 3 to SEQ ID NO: 117” is intended to mean SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, or SEQ ID NO: 117. 
     The term “anyone of SEQ ID NO: 118 to SEQ ID NO: 225” is intended to mean anyone of SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, or SEQ ID NO: 225. 
     The term “anyone of SEQ ID NO: 226 to SEQ ID NO: 251” is intended to mean anyone of SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, or SEQ ID NO: 251. 
     The term “anyone of SEQ ID NO: 252 to SEQ ID NO: 274” is intended to mean anyone of SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, or SEQ ID NO: 274. 
     The term “anyone of SEQ ID NO: 3 to SEQ ID NO: 225” is intended to mean anyone of SEQ ID NO: 3 to SEQ ID NO: 117 or anyone of SEQ ID NO: 118 to SEQ ID NO: 225. 
     The term “anyone of SEQ ID NO: 2 to SEQ ID NO: 225” is intended to mean SEQ ID NO: 2 or anyone of SEQ ID NO: 3 to SEQ ID NO: 225. 
     The amino acids making up the polypeptides of the invention may independently be selected from D or L forms. Preferably the polypeptide of the invention is a defensin polypeptide; more preferably an alpha defensin, a beta defensin, or an insect (arthropod) defensin. 
     The polypeptides of the invention may exhibit higher or at least equal, preferably higher, antimicrobial activity compared to the polypeptide of SEQ ID NO: 1, determined as the Minimum Inhibitory Concentration (MIC), against  Staphylococcus carnosus  ATCC51365,  Staphylococcus aureus  ATCC29213 or  Staphylococcus aureus  ATCC25923 according to the NCCLS/CLSI guidelines, Protocol M7-A6, Vol. 20, No. 2: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. 
     In an embodiment, the present invention relates to artificial variants comprising a conservative substitution, deletion, and/or insertion of one or more amino acids of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 10 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, 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 do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979,  In, The Proteins , Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly. 
     In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. “Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline. 
     Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like. 
     Essential amino acids in the parent polypeptide can be identified according to procedures 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 introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (i.e., antimicrobial activity) to identify 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 biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992,  Science  255: 306-312; Smith et al., 1992,  J. Mol. Biol.  224: 899-904; Wlodaver et al., 1992,  FEBS Lett.  309:59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides which are related to a polypeptide according to the invention. 
     Single or multiple amino acid substitutions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by 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 error-prone PCR, phage display (e.g., Lowman et al., 1991,  Biochem.  30:10832-10837; U.S. Pat. No. 5,223,409; 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 activity of cloned, mutagenized polypeptides expressed by host cells. Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure. 
     The total number of amino acid substitutions, deletions and/or insertions of amino acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274 is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably at most 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1. 
     In another aspect, the present invention provides a variant of a parent antimicrobial polypeptide, which parent antimicrobial polypeptide has at least 60% identity with the amino acid sequence of SEQ ID NO: 1, wherein the variant has antimicrobial activity and comprises a substitution in one or more positions, and wherein the substitutions are selected from: 
     F2L, F2W or F2I; 
     N5R, N5Q, N5V, N5G, N5S, N5A, N5K, N5L, N5M, N5D, N5H or N5Y; 
     G6R, G6A or G6K; 
     P7A, P7L, P7V, P7K or P7R; 
     W8R; 
     D9A, D9G, D9K, D9L, D9T, D9N, D9F, D9H, D9M, D9P, D9Q, D9S, D9C, D91, D9R, D9V or D9Y; 
     E10G, E10A, E10L, E10C, E10Q or E10S; 
     D11F, D11G, D11N, D11V, D11Y, D11H, D11K, D11L, D11P, D115, D11T, D11W, D11I, D11M, D11A, D11C or D11R; 
     D12P; 
     M13R, M13S, M13V, M13A, M13F, M13G, M13L, M13T, M13Y, M13W, M13E or M13K; 
     Q14R, Q14L, Q14F, Q14G, Q14H, Q14S, Q14A, Q14C, Q14I, Q14K, Q14M, Q14P, Q14T, Q14V, Q14W or Q14Y; 
     N17R, N17I, N17Y, N17V, N17K, N17T, N17S, N17Q, N17F, N17A, N17W, N17E or N17H; 
     H18A, H18F, H18Q, H18T, H18V or H18L; 
     K20Q or K20R; 
     S21A, S21V, S21N or S21F; 
     I22L, I22M, I22T, I22W or I22V; 
     K23R or K23T; 
     G24H, G24K, G24A, G24P, G24F, G241, G24Q, G24R, G24S, G24T, G24Y or G24N; 
     Y25H, Y25K, Y25L, Y25M, Y25N, Y25Q, Y25S, Y25V or Y25R; 
     K26F, K26H, K26T, K26C or K26R; 
     Y29F, Y29R, Y29A, Y29H, Y29L, Y29M, Y29S or Y29W; 
     A31K, A31N, A31Q, A31T, A31E, A31H, A31I, A31R, A31S, A31V, A31G or A31Y; 
     K32R or K32T; 
     G33K, G33Q, G33E, G33N, G33S, G33T, G33A or G33R; 
     G34K, G34H, G34W or G34R; 
     F35A, F35H, F35I, F35M, F35V, F35W, F35R or F35L; 
     V36L, V36M, V361, V36K, V36Q, V36R or V36T; 
     K38H, K38N or K38R; and 
     Y40I, Y40YRCG or Y40YR. 
     Preferably the parent antimicrobial polypeptide has at least 70% identity, more preferably at least 80% identity, even more preferably at least 90% identity, and most preferably 95% identity with the amino acid sequence of SEQ ID NO: 1. In particular the parent antimicrobial polypeptide may be identical to the amino acid sequence of SEQ ID NO: 1. 
     In an embodiment the parent antimicrobial polypeptide is a defensin polypeptide; more preferably an alpha defensin, a beta defensin, or an insect (arthropod) defensin. Preferably the parent amino acid sequence exhibit antimicrobial activity. 
     In another embodiment, the parent antimicrobial polypeptide has 1, 2, 3, 4, 5, 6, 7 or 8 amino acid differences compared to the amino acid sequence of SEQ ID NO: 1. Preferably 1, 2, 3, 4, 5 or 6; more preferably 1, 2, 3, 4 or 5; even more preferably 1, 2, 3 or 4; even more preferably 1, 2 or 3; and most preferably 1 or 2 amino acids are different compared to the amino acid sequence of SEQ ID NO: 1. 
     N-Terminal Extension 
     An N-terminal extension of the polypeptides of the invention may suitably consist of from 1 to 50 amino acids, preferably 2-20 amino acids, especially 3-15 amino acids. In one embodiment N-terminal peptide extension does not contain an Arg (R). In another embodiment the N-terminal extension comprises a kex2 or kex2-like cleavage site as will be defined further below. In a preferred embodiment the N-terminal extension is a peptide, comprising at least two Glu (E) and/or Asp (D) amino acid residues, such as an N-terminal extension comprising one of the following sequences: EAE, EE, DE and DD. 
     Kex2 Sites 
     Kex2 sites (see, e.g.,  Methods in Enzymology  Vol. 185, ed. D. Goeddel, Academic Press Inc. (1990), San Diego, Calif., “Gene Expression Technology”) and kex2-like sites are di-basic recognition sites (i.e., cleavage sites) found between the pro-peptide encoding region and the mature region of some proteins. 
     Insertion of a kex2 site or a kex2-like site have in certain cases been shown to improve correct endopeptidase processing at the pro-peptide cleavage site resulting in increased protein secretion levels. 
     In the context of the invention insertion of a kex2 or kex2-like site result in the possibility to obtain cleavage at a certain position in the N-terminal extension resulting in an antimicrobial polypeptide being extended in comparison to amino acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. 
     Fused Polypeptides 
     The polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the invention or a fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. 
     Sources of Polypeptides Having Antimicrobial Activity 
     A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly. 
     A polypeptide of the present invention may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a  Bacillus  polypeptide, e.g., a  Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis , or  Bacillus thuringiensis  polypeptide; or a  Streptomyces  polypeptide, e.g., a  Streptomyces lividans  or  Streptomyces murinus  polypeptide; or a gram negative bacterial polypeptide, e.g., an  E. coli  or a  Pseudomonas  sp. polypeptide. 
     A polypeptide of the present invention may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a  Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , or  Yarrowia  polypeptide; or more preferably a filamentous fungal polypeptide such as an  Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium , or  Trichoderma  polypeptide. 
     In a preferred aspect, the polypeptide is a  Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis , or  Saccharomyces oviformis  polypeptide having antimicrobial activity. 
     In another preferred aspect, the polypeptide is an  Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Fusarium bacteridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, 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  polypeptide. 
     It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. 
     Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL). 
     Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. 
     Polynucleotides 
     The present invention also relates to polynucleotides having a nucleotide sequence which encodes for a polypeptide of the invention. In particular, the present invention relates to polynucleotides consisting of a nucleotide sequence which encodes for a polypeptide of the invention. Due to the degeneracy of the genetic code, the skilled person will easily recognize that several nucleotide sequences encoding each of the polypeptides of the invention may be prepared. It is well known in the art which nucleotides make up codons encoding the amino acids of the polypeptides of the invention. 
     The present invention also relates to polynucleotides which encode fragments of the amino acid sequence shown as anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274 that have antimicrobial activity. A subsequence of the polynucleotides is a nucleotide sequence wherein one or more nucleotides from the 5′ and/or 3′ end have been deleted. 
     The nucleotide sequence may be obtained by standard cloning procedures used in genetic engineering to relocate the nucleotide sequence from one location to a different site where it will be reproduced. The cloning procedures may involve excision and isolation of a desired fragment comprising the nucleotide sequence encoding the polypeptide, insertion of the fragment into a vector molecule, and incorporation of the recombinant vector into a host cell where multiple copies or clones of the nucleotide sequence will be replicated. The nucleotide sequence may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof. 
     Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of a polypeptide, which comprises an amino acid sequence that has at least one substitution, deletion and/or insertion as compared to amino acids 1 to 40 of anyone of SEQ ID NO: 2 to SEQ ID NO: 225 or anyone of SEQ ID NO: 226 to SEQ ID NO: 251 or anyone of SEQ ID NO: 252 to SEQ ID NO: 274. These artificial variants may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. 
     It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, 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 at every positively charged residue in the molecule, and the resultant mutant molecules are tested for antimicrobial activity to identify amino acid residues that are critical to the activity of the molecule. Sites of interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labelling (see, e.g., 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). 
     Moreover, a nucleotide sequence encoding a polypeptide of the present invention may be modified by introduction of nucleotide substitutions which do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the antimicrobial polypeptide. 
     Nucleic Acid Constructs 
     The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more control sequences which direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. 
     An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide&#39;s sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art. 
     The control sequence may be an appropriate promoter sequence, a nucleotide sequence which is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences which mediate the expression of the polypeptide. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. 
     Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the  E. coli  lac operon,  Streptomyces coelicolor  agarase gene (dagA),  Bacillus subtilis  levansucrase gene (sacB),  Bacillus licheniformis  alpha-amylase gene (amyL),  Bacillus stearothermophilus  maltogenic amylase gene (amyM),  Bacillus amyloliquefaciens  alpha-amylase gene (amyQ),  Bacillus licheniformis  penicillinase gene (penP),  Bacillus subtilis  xylA and xylB genes, 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). Further 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 the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Rhizomucor miehei  aspartic proteinase,  Aspergillus niger  neutral alpha-amylase,  Aspergillus niger  acid stable alpha-amylase,  Aspergillus niger  or  Aspergillus awamori  glucoamylase (glaA),  Rhizomucor miehei  lipase,  Aspergillus oryzae  alkaline protease,  Aspergillus oryzae  triose phosphate isomerase,  Aspergillus nidulans  acetamidase,  Fusarium venenatum  amyloglucosidase (WO 00/56900),  Fusarium venenatum  Daria (WO 00/56900),  Fusarium venenatum  Quinn (WO 00/56900),  Fusarium oxysporum  trypsin-like protease (WO 96/00787),  Trichoderma reesei  beta-glucosidase,  Trichoderma reesei  cellobiohydrolase I,  Trichoderma reesei  endoglucanase I,  Trichoderma reesei  endoglucanase II,  Trichoderma reesei  endoglucanase III,  Trichoderma reesei  endoglucanase IV,  Trichoderma reesei  endoglucanase V,  Trichoderma reesei  xylanase I,  Trichoderma reesei  xylanase II,  Trichoderma reesei  beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for  Aspergillus niger  neutral alpha-amylase and  Aspergillus oryzae  triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof. 
     In a yeast host, useful promoters are obtained from the genes for  Saccharomyces cerevisiae  enolase (ENO-1),  Saccharomyces cerevisiae  galactokinase (GAL1),  Saccharomyces cerevisiae  alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),  Saccharomyces cerevisiae  triose phosphate isomerase (TPI),  Saccharomyces cerevisiae  metallothionine (CUP1), and  Saccharomyces cerevisiae  3-phosphoglycerate kinase. Other useful 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 a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. 
     Preferred terminators for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger  glucoamylase,  Aspergillus nidulans  anthranilate synthase,  Aspergillus niger  alpha-glucosidase, and  Fusarium oxysporum  trypsin-like protease. 
     Preferred terminators for yeast host cells are obtained from the genes for  Saccharomyces cerevisiae  enolase,  Saccharomyces cerevisiae  cytochrome C (CYC1), and  Saccharomyces cerevisiae  glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra. 
     The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention. 
     Preferred leaders for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase and  Aspergillus nidulans  triose phosphate isomerase. 
     Suitable leaders for yeast host cells are obtained from the genes for  Saccharomyces cerevisiae  enolase (ENO-1),  Saccharomyces cerevisiae  3-phosphoglycerate kinase,  Saccharomyces cerevisiae  alpha-factor, and  Saccharomyces cerevisiae  alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP). 
     The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3′ terminus of the nucleotide sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the present invention. 
     Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger  glucoamylase,  Aspergillus nidulans  anthranilate synthase,  Fusarium oxysporum  trypsin-like protease, and  Aspergillus niger  alpha-glucosidase. 
     Useful 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 coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell&#39;s secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region which encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding region which is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used in the present invention. Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions obtained from the genes for  Bacillus  NCIB 11837 maltogenic amylase,  Bacillus stearothermophilus  alpha-amylase,  Bacillus licheniformis  subtilisin,  Bacillus licheniformis  beta-lactamase,  Bacillus stearothermophilus  neutral proteases (nprT, nprS, nprM), and  Bacillus subtilis  prsA. Further signal peptides are described by Simonen and Palva, 1993,  Microbiological Reviews  57: 109-137. 
     Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for  Aspergillus oryzae  TAKA amylase,  Aspergillus niger  neutral amylase,  Aspergillus niger  glucoamylase,  Rhizomucor miehei  aspartic proteinase,  Humicola insolens  cellulase, and  Humicola lanuginosa  lipase. 
     Useful signal peptides for yeast host cells are obtained from the genes for  Saccharomyces cerevisiae  alpha-factor and  Saccharomyces cerevisiae  invertase. Other useful signal peptide coding regions are described by Romanos et al., 1992, supra. 
     The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for  Bacillus subtilis  alkaline protease (aprE),  Bacillus subtilis  neutral protease (nprT),  Saccharomyces cerevisiae  alpha-factor,  Rhizomucor miehei  aspartic proteinase, and  Myceliophthora thermophila  laccase (WO 95/33836). 
     Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region. 
     It may also be desirable to add regulatory sequences which allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, 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 GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter,  Aspergillus niger  glucoamylase promoter, and  Aspergillus oryzae  glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene which is amplified in the presence of methotrexate, and the metallothionein genes which are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence. 
     Expression Vectors 
     The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a nucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression. 
     The recombinant expression vector may be any vector (e.g., a plasmid or virus) which can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. 
     The vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. 
     The vectors of the present invention preferably contain one or more selectable markers which permit easy selection of transformed cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. 
     Examples of bacterial selectable markers are the dal genes from  Bacillus subtilis  or  Bacillus licheniformis , or markers which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an  Aspergillus  cell are the amdS and pyrG genes of  Aspergillus nidulans  or  Aspergillus oryzae  and the bar gene of  Streptomyces hygroscopicus.    
     The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell&#39;s genome or autonomous replication of the vector in the cell independent of the genome. 
     For integration into the host cell genome, the vector may rely on the polynucleotide&#39;s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. 
     For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication which functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo. 
     Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in  E. coli , and pUB110, pE194, pTA1060, and pAM31 permitting replication in  Bacillus.    
     Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. 
     Examples of origins of replication useful in a filamentous fungal cell 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 construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883. 
     More than one copy of a polynucleotide of the present invention may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. 
     The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra). 
     Host Cells 
     The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. 
     The host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. 
     Useful unicellular microorganisms are bacterial cells such as gram positive bacteria including, but not limited to, a  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 a  Streptomyces  cell, e.g.,  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  cell is an alkalophilic  Bacillus.    
     The introduction of a vector into a bacterial host cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979,  Molecular General Genetics  168: 111-115), using competent cells (see, e.g., 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, e.g., Koehler and Thorne, 1987,  Journal of Bacteriology  169: 5771-5278). 
     The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell. 
     In a preferred aspect, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al.,  In, Ainsworth and Bisby&#39;s Dictionary of The Fungi,  8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra). 
     In a more preferred aspect, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in  Biology and Activities of Yeast  (Skinner, Passmore, and Davenport, 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 fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as  Saccharomyces cerevisiae  is by budding of a unicellular thallus and carbon catabolism may be fermentative. 
     In an even more preferred aspect, the filamentous fungal host cell is an  Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes , or  Trichoderma  cell. 
     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 filamentous fungal host cell is a  Fusarium bacteridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides , or  Fusarium venenatum  cell. In another most preferred aspect, the filamentous fungal host cell is a  Bjerkandera adusta, 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  strain cell. 
     Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of  Aspergillus  and  Trichoderma  host cells are described in EP 238 023 and Yelton et al., 1984,  Proceedings of the National Academy of Sciences USA  81: 1470-1474. Suitable methods for transforming  Fusarium  species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., 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. 
     Methods of Production 
     The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     The present invention also relates to methods for producing a polypeptide of the present invention, comprising (a) cultivating a host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates. 
     The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies. For example, an antimicrobial activity assay may be used to determine the activity of the polypeptide as described herein. 
     The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. 
     The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989). 
     Plants 
     The present invention also relates to a transgenic plant, plant part, or plant cell which has been transformed with a nucleotide sequence encoding a polypeptide having antimicrobial activity of the present invention so as to express and produce the polypeptide in recoverable quantities. The polypeptide may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor. 
     The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass,  Poa ), forage grass such as  Festuca, Lolium , temperate grass, such as  Agrostis , and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn). 
     Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism  Arabidopsis thaliana.    
     Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats. 
     Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells. 
     The transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more expression constructs encoding a polypeptide of the present invention into the plant host genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell. 
     The expression construct is conveniently a nucleic acid construct which comprises a polynucleotide encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used). 
     The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences, is determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed. For instance, the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988,  Plant Physiology  86: 506. 
     For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may 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 may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards &amp; Coruzzi, 1990,  Ann. Rev. Genet.  24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994,  Plant Mol. Biol.  24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998,  Plant and Cell Physiology  39: 885-889), a  Vicia faba  promoter from the legumin B4 and the unknown seed protein gene from  Vicia faba  (Conrad et al., 1998,  Journal of Plant Physiology  152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998,  Plant and Cell Physiology  39: 935-941), the storage protein napA promoter from  Brassica napus , or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, 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 chlorella virus adenine methyltransferase gene promoter (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 a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993,  Plant Molecular Biology  22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals. 
     A promoter enhancer element may also be used to achieve higher expression of a polypeptide of the present invention in the plant. For instance, the promoter enhancer element may be an intron which is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression. 
     The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art. 
     The nucleic acid construct is incorporated into the plant genome according to conventional 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). 
     Presently,  Agrobacterium tumefaciens -mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992,  Plant Molecular Biology  19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli 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 transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993,  Plant Molecular Biology  21: 415-428. 
     Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase. 
     The present invention also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding a polypeptide having antimicrobial activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. 
     Compositions 
     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 has been increased, e.g., with an enrichment factor of 1.1. 
     The compositions may further comprise another pharmaceutically active agent, such as an additional biocidal or biostatic agent, such as another antimicrobial polypeptide exhibiting antimicrobial activity as defined above. The biocidal agent may be an antibiotic, as known in the art. Classes of antibiotics include penicillins, e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with beta-lactamase inhibitors, cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc. The biocidal agent may also be an anti-mycotic agent, including polyenes, e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol and fluconazol. 
     In an embodiment the biocidal agent is a non-enzymatic chemical agent. In another embodiment the biocidal agent is a non-polypeptide chemical agent. 
     The compositions may comprise a suitable carrier material. The compositions may also comprise a suitable delivery vehicle capable of delivering the antimicrobial polypeptides of the invention to the desired locus when the compositions are used as a medicament. 
     The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art. Examples are given below of preferred uses of the polypeptide compositions of the invention. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art. 
     Methods and Uses 
     The present invention is also directed to methods for using the polypeptides having antimicrobial activity. The antimicrobial polypeptides are typically useful at any locus subject to contamination by bacteria, fungi, yeast or algae. Typically, loci are in aqueous systems such as cooling water systems, laundry rinse water, oil systems such as cutting oils, lubricants, oil fields and the like, where microorganisms need to be killed or where their growth needs to be controlled. However, the present invention may also be used in all applications for which known antimicrobial compositions are useful, such as protection of wood, latex, adhesive, glue, paper, cardboard, textile, leather, plastics, caulking, and feed. 
     Other uses include preservation of foods, beverages, cosmetics such as lotions, creams, gels, ointments, soaps, shampoos, conditioners, antiperspirants, deodorants, mouth wash, contact lens products, enzyme formulations, or food ingredients. 
     Thus, the antimicrobial polypeptides of the invention may by useful as a disinfectant, e.g., in the treatment of infections in the eye or the mouth, skin infections; in antiperspirants or deodorants; for cleaning and disinfection of contact lenses and teeth (oral care). 
     In general it is contemplated that the antimicrobial polypeptides of the present invention are useful for cleaning, disinfecting or inhibiting microbial growth on any surface. Examples of surfaces, which may advantageously be contacted with the antimicrobial polypeptides of the invention are surfaces of process equipment used, e.g., dairies, chemical or pharmaceutical process plants, water sanitation systems, oil processing plants, paper pulp processing plants, water treatment plants, and cooling towers. The antimicrobial polypeptides of the invention should be used in an amount, which is effective for cleaning, disinfecting or inhibiting microbial growth on the surface in question. 
     The antimicrobial polypeptides of the invention may additionally be used for cleaning surfaces and cooking utensils in food processing plants and in any area in which food is prepared or served such as hospitals, nursing homes and restaurants. 
     It may also be used as a preservation agent or a disinfection agent in water based paints. 
     The invention also relates to the use of an antimicrobial polypeptide or composition of the invention as a medicament. Further, an antimicrobial polypeptide or composition of the invention may also be used for the manufacture of a medicament for controlling or combating microorganisms, such as fungal organisms or bacteria, preferably gram positive bacteria. 
     The composition and antimicrobial polypeptide of the invention may be used as an antimicrobial veterinarian or human therapeutic or prophylactic agent. Thus, the composition and antimicrobial polypeptide of the invention may be used in the preparation of veterinarian or human therapeutic agents or prophylactic agents for the treatment of microbial infections, such as bacterial or fungal infections, preferably gram positive bacterial infections. In particular the microbial infections may be associated with lung diseases including, but not limited to, tuberculosis, pneumonia and cystic fibrosis; and sexual transmitted diseases including, but not limited to, gonorrhea and chlamydia. 
     The composition of the invention comprises an effective amount of the antimicrobial polypeptide of the invention. 
     The term “effective amount” when used herein is intended to mean an amount of the antimicrobial polypeptides of the invention, which is sufficient to inhibit growth of the microorganisms in question. 
     The invention also relates to wound healing compositions or products such as bandages, medical devices such as, e.g., catheters and further to anti-dandruff hair products, such as shampoos. 
     Formulations of the antimicrobial polypeptides of the invention are administered to a host suffering from or predisposed to a microbial infection. Administration may be topical, localized or systemic, depending on the specific microorganism, preferably it will be localized. Generally the dose of the antimicrobial polypeptides of the invention will be sufficient to decrease the microbial population by at least about 50%, usually by at least 1 log, and may be by 2 or more logs of killing. The compounds of the present invention are administered at a dosage that reduces the microbial population while minimizing any side-effects. It is contemplated that the composition will be obtained and used under the guidance of a physician 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.    
     Formulations of the antimicrobial polypeptides of the invention may be administered to a host suffering from or predisposed to a microbial lung infection, such as pneumonia; or to a microbial wound infection, such as a bacterial wound infection. 
     Formulations of the antimicrobial polypeptides of the invention may also be administered to a host suffering from or predisposed to a skin infection, such as acne, atopic dermatitis or seborrheic dermatitis; preferably the skin infection is a bacterial skin infection, e.g., 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 formulations to kill microbes, particularly where one does not wish to introduce quantities of conventional antibiotics. For example, the antimicrobial polypeptides of the invention may be added to animal and/or human food preparations; or they may be included as an additive for in vitro cultures of cells, to prevent the overgrowth of microbes in tissue culture. 
     The susceptibility of a particular microbe to killing with the antimicrobial polypeptides of the invention may be determined by in vitro testing, as detailed in the experimental section. Typically a culture of the microbe is combined with the antimicrobial polypeptide at varying concentrations for a period of time sufficient to allow the protein to act, usually between about one hour and one day. The viable microbes are then counted, and the level of killing determined. 
     Microbes of interest include, but are not limited to, Gram-negative bacteria, for example:  Citrobacter  sp.;  Enterobacter  sp.;  Escherichia  sp., e.g.,  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., e.g.,  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 , etc. 
     Non-bacterial pathogens of interest include fungal and protozoan pathogens, e.g.,  Plasmodia  sp., e.g.,  P. falciparum, Trypanosoma  sp., e.g.,  T. brucei ; shistosomes;  Entaemoeba  sp.,  Cryptococcus  sp.,  Candida  sp., e.g.,  C. albicans ; etc. 
     Various methods for administration may be employed. The polypeptide formulation may be given orally, or may be injected intravascularly, subcutaneously, peritoneally, by aerosol, opthalmically, intra-bladder, topically, etc. For example, methods of administration by inhalation are well-known in the art. The dosage of the therapeutic formulation will vary widely, depending on the specific antimicrobial polypeptide to be administered, the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. The initial dose may be larger, followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered once or several times daily, semi-weekly, etc. to maintain an effective dosage level. In many cases, oral administration will require a higher dose than if administered intravenously. The amide bonds, as well as the amino and carboxy termini, may be modified for greater stability on oral administration. For example, the carboxy terminus may be amidated. 
     Formulations 
     The compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, creams, foams, solutions, suppositories, injections, inhalants, 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, intratracheal, etc., administration. The antimicrobial polypeptides of the invention may be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation. 
     In one embodiment, a formulation for topical use comprises a chelating agent that decreases the effective concentration of divalent cations, particularly calcium and magnesium. For example, agents such as citrate, EGTA or EDTA may be included, where citrate is preferred. The concentration of citrate will usually be from about 1 to 10 mM. 
     The compounds of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., perforin, anti-inflammatory agents, antibiotics, etc.). In pharmaceutical dosage forms, the compounds may be administered in the form of their pharmaceutically acceptable salts. The following methods and excipients are merely exemplary and are in no way limiting. 
     For oral preparations, the compounds can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. 
     The compounds can be formulated into preparations for injections by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. 
     The compounds can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. 
     The compounds can be used as lotions, for example to prevent infection of burns, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. 
     Furthermore, the compounds can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature. 
     Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compounds of the present invention. Similarly, unit dosage forms for injection or intravenous administration may comprise the compound of the present invention in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier. 
     Implants for sustained release formulations are well-known in the art. Implants are formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or glycolic acid form an erodible polymer that is well-tolerated by the host. The implant containing the antimicrobial polypeptides of the invention is placed in proximity to the site of infection, so that the local concentration of active agent is increased relative to the rest of the body. 
     The term “unit dosage form”, as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with the compound in the host. 
     The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public. 
     Typical dosages for systemic administration range from 0.1 pg to 100 milligrams per kg weight of subject per administration. A typical dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release. 
     Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific compounds are more potent than others. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means. A preferred means is to measure the physiological potency of a given compound. 
     The use of liposomes as a delivery vehicle is one method of interest. The liposomes fuse with the cells of the target site and deliver the contents of the lumen intracellularly. The liposomes are maintained in contact with the cells for sufficient time for fusion, using various means to maintain contact, such as isolation, binding agents, and the like. In one aspect of the invention, liposomes are designed to be aerosolized for pulmonary administration. Liposomes may be prepared with purified proteins or peptides that mediate fusion of membranes, such as Sendai virus or influenza virus, etc. The lipids may be any useful combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid will be normally neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like. 
     For preparing the liposomes, the procedure described by Kato et al., 1991,  J. Biol. Chem.  266: 3361 may be used. Briefly, the lipids and lumen composition containing peptides are combined in an appropriate aqueous medium, conveniently a saline medium where the total solids will be in the range of about 1-10 weight percent. After intense agitation for short periods of time, from about 5-60 sec., the tube is placed in a warm water bath, from about 25-40° C. and this cycle repeated from about 5-10 times. The composition is then sonicated for a convenient period of time, generally from about 1-10 sec. and may be further agitated by vortexing. The volume is then expanded by adding aqueous medium, generally increasing the volume by about from 1-2 fold, followed by shaking and cooling. This method allows for the incorporation into the lumen of high molecular weight molecules. 
     Formulations with Other Active Agents 
     For use in the subject methods, the antimicrobial polypeptides of the invention may be formulated with other pharmaceutically active agents, particularly other antimicrobial agents. Other agents of interest include a wide variety of antibiotics, as known in the art. Classes of antibiotics include penicillins, e.g., penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc.; penicillins in combination with beta-lactamase inhibitors, cephalosporins, e.g., cefaclor, cefazolin, cefuroxime, moxalactam, etc.; carbapenems; monobactams; aminoglycosides; tetracyclines; macrolides; lincomycins; polymyxins; sulfonamides; quinolones; cloramphenical; metronidazole; spectinomycin; trimethoprim; vancomycin; etc. 
     Anti-mycotic agents are also useful, including polyenes, e.g., amphotericin B, nystatin; 5-flucosyn; and azoles, e.g., miconazol, ketoconazol, itraconazol and fluconazol. Antituberculotic drugs include isoniazid, ethambutol, streptomycin and rifampin. Cytokines may also be included in a formulation of the antimicrobial polypeptides of the invention, e.g., interferon gamma, tumor necrosis factor alpha, interleukin 12, etc. 
     In Vitro Synthesis 
     The antimicrobial peptides of the invention may be prepared by in vitro synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids, particularly D-isomers (or D-forms), e.g., D-alanine and D-isoleucine, diastereoisomers, side chains having different lengths or functionalities, and the like. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. 
     Chemical linking may be provided to various peptides or proteins comprising convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g., reductive amination, thiol groups for thioether or disulfide formation, carboxyl groups for amide formation, and the like. 
     If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. 
     The polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein 
     Animal Feed 
     The present invention is also directed to methods for using the polypeptides having antimicrobial activity in animal feed, as well as to feed compositions and feed additives comprising the antimicrobial polypeptides of the invention. 
     The term animal includes all animals, including human beings. Examples of animals are non-ruminants, and ruminants, such as cows, sheep and horses. In a particular embodiment, the animal is a non-ruminant animal. Non-ruminant animals include mono-gastric animals, e.g., pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys and chicken (including but not limited to broiler chicks, layers); young calves; and fish (including but not limited to salmon). 
     The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. 
     In the use according to the invention the antimicrobial polypeptide can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred. 
     In a particular embodiment, the antimicrobial polypeptide, in the form in which it is added to the feed, or when being included in a feed additive, is well defined. Well-defined means that the antimicrobial polypeptide preparation is at least 50% pure as determined by Size-exclusion chromatography (see Example 12 of WO 01/58275). In other particular embodiments the antimicrobial polypeptide preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or at least 95% pure as determined by this method. 
     A well-defined antimicrobial polypeptide preparation is advantageous. For instance, it is much easier to dose correctly to the feed an antimicrobial polypeptide that is essentially free from interfering or contaminating other antimicrobial polypeptides. The term dose correctly refers in particular to the objective of obtaining consistent and constant results, and the capability of optimising dosage based upon the desired effect. 
     For the use in animal feed, however, the antimicrobial polypeptide need not be that pure; it may, e.g., include other enzymes, in which case it could be termed an antimicrobial polypeptide preparation. 
     The antimicrobial polypeptide preparation can be (a) added directly to the feed (or used directly in a treatment process of vegetable proteins), or (b) it can be used in the production of one or more intermediate compositions such as feed additives or premixes that is subsequently added to the feed (or used in a treatment process). The degree of purity described above refers to the purity of the original antimicrobial polypeptide preparation, whether used according to (a) or (b) above. 
     Antimicrobial polypeptide preparations with purities of this order of magnitude are in particular obtainable using recombinant methods of production, whereas they are not so easily obtained and also subject to a much higher batch-to-batch variation when the antimicrobial polypeptide is produced by traditional fermentation methods. 
     Such antimicrobial polypeptide preparation may 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 from or originating from a vegetable, including modified proteins and protein-derivatives. In particular embodiments, the protein content of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60% (w/w). 
     Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example materials from plants of the families Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, and Poaceae, such as soy bean meal, lupin meal and rapeseed meal. 
     In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae, e.g., soybean, lupine, pea, or bean. 
     In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae, e.g., beet, sugar beet, spinach or quinoa. 
     Other examples of vegetable protein sources are rapeseed, and cabbage. 
     Soybean is a preferred vegetable protein source. 
     Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum. 
     The antimicrobial polypeptide can be added to the feed in any form, be it as a relatively pure antimicrobial polypeptide, or in admixture with other components intended for addition to animal feed, i.e., in the form of animal feed additives, such as the so-called pre-mixes for animal feed. 
     In a further aspect the present invention relates to compositions for use in animal feed, such as animal feed, and animal feed additives, e.g., premixes. 
     Apart from 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 mineral, and/or at least one macro mineral. 
     Further, optional, feed-additive ingredients are colouring agents, aroma compounds, stabilisers, and/or at least one other enzyme selected from amongst phytases EC 3.1.3.8 or 3.1.3.26; xylanases EC 3.2.1.8; galactanases EC 3.2.1.89; and/or beta-glucanases EC 3.2.1.4. 
     In a particular embodiment these other enzymes are well defined (as defined above for antimicrobial polypeptide preparations). 
     Examples of other antimicrobial peptides (AMPs) are CAP18, Leucocin A, Tritrpticin, Protegrin-1, Thanatin, Defensin, Ovispirin such as Novispirin (Robert Lehrer, 2000), and variants, or fragments thereof which retain antimicrobial activity. 
     Examples of other antifungal polypeptides (AFPs) are the  Aspergillus giganteus , and  Aspergillus niger  peptides, as well as variants and fragments thereof which retain antifungal activity, as disclosed in WO 94/01459 and WO 02/090384. 
     Usually fat and water soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Either of these composition types, when enriched with an antimicrobial polypeptide of the invention, is an animal feed additive of the invention. 
     In a particular embodiment, the animal feed additive of the invention is intended for being included (or prescribed as having to be included) in animal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05 to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This is so in particular for premixes. 
     The following are non-exclusive lists of examples of these components: 
     Examples of fat soluble vitamins are vitamin A, vitamin D3, vitamin E, and vitamin K, e.g., vitamin K3. 
     Examples of water soluble vitamins are vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g., Ca-D-panthothenate. 
     Examples of trace minerals are manganese, zinc, iron, copper, iodine, selenium, and cobalt. 
     Examples of macro minerals are calcium, phosphorus and sodium. 
     The nutritional requirements of these components (exemplified with poultry and piglets/pigs) are listed in Table A of WO 01/58275. Nutritional requirement means that these components should be provided in the diet in the concentrations indicated. 
     In the alternative, the animal feed additive of the invention comprises at least one of the individual components specified in Table A of WO 01/58275. At least one means either of, one or more of, one, or two, or three, or four and so forth up to all thirteen, or up 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 an in-feed-concentration within the range 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 diets have a relatively high content of protein. Poultry and pig diets can be characterised as indicated in Table B of WO 01/58275, columns 2-3. Fish diets can be characterised as indicated in column 4 of this Table B. Furthermore such fish diets usually have a crude fat content of 200-310 g/kg. 
     An animal feed composition according to the invention has a crude protein content of 50-800 g/kg, and furthermore comprises at least one antimicrobial polypeptide as claimed herein. 
     Furthermore, or in the alternative (to the crude protein content indicated above), the animal feed composition of the invention has a content of metabolizable energy of 10-30 MJ/kg; and/or a content of calcium of 0.1-200 g/kg; and/or a content of available phosphorus of 0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or a content of methionine plus cysteine of 0.1-150 g/kg; and/or a content of lysine of 0.5-50 g/kg. 
     In particular embodiments, the content of metabolisable energy, crude protein, calcium, phosphorus, methionine, methionine plus cysteine, and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO 01/58275 (R. 2-5). 
     Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25, i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content is determined by the Kjeldahl method (A.O.A.C., 1984, Official Methods of Analysis 14th ed., Association of Official Analytical Chemists, Washington D.C.). 
     Metabolizable energy can be calculated on the basis of the NRC publication 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., pp. 2-6, and the European Table of Energy Values for Poultry Feed-stuffs, Spelderholt centre for poultry research and extension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen &amp; looijen bv, Wageningen. ISBN 90-71463-12-5. 
     The dietary content of calcium, available phosphorus and amino acids in complete animal diets is calculated on the basis of feed 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 particular embodiment, the animal feed composition of the invention contains at least one vegetable protein or protein source as defined above. 
     In still further particular embodiments, the animal feed composition of the invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey. Animal diets can, e.g., be manufactured as mash feed (non pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. Enzymes can be added as solid or liquid enzyme formulations. For example, a solid enzyme formulation is typically added before or during the mixing step; and a liquid enzyme preparation is typically added after the pelleting step. The enzyme may also be incorporated in a feed additive or premix. 
     The final enzyme concentration in the diet is within the range of 0.01-200 mg enzyme protein per kg diet, for example in the range of 5-30 mg enzyme protein per kg animal diet. 
     The antimicrobial polypeptide may be administered in one or more of the following amounts (dosage ranges): 0.01-200; or 0.01-100; or 0.05-100; or 0.05-50; or 0.10-10—all these ranges being in mg antimicrobial polypeptide protein per kg feed (ppm). 
     For determining mg antimicrobial polypeptide protein per kg feed, the antimicrobial polypeptide is purified from the feed composition, and the specific activity of the purified antimicrobial polypeptide is determined using a relevant assay (see under antimicrobial activity, substrates, and assays). The antimicrobial activity of the feed composition as such is also determined using the same assay, and on the basis of these two determinations, the dosage in mg antimicrobial polypeptide protein per kg feed is calculated. 
     The same principles apply for determining mg antimicrobial polypeptide protein in feed additives. Of course, if a sample is available of the antimicrobial polypeptide used for preparing the feed additive or the feed, the specific activity is determined from this sample (no need to purify the antimicrobial polypeptide from the feed composition or the additive). 
     The present invention is further described by the following examples which should not be construed as limiting the scope of the invention. 
     EXAMPLES 
     Chemicals used as buffers and substrates were commercial products of at least reagent grade. 
     Example 1 
     Evaluation of Minimal Effective Concentration 
     Three antimicrobial polypeptides, which are variants of SEQ ID NO: 1, were tested for antimicrobial activity. A Minimal Effective Concentration assay (MEC, expressed as micrograms/mL) against different microorganisms was performed using the three plectasin mutants, Y40YR, N17R and Y25R following the protocol described in the book of Methods in Molecular Biology, Vol. 78, Antibacterial peptide protocol William M. Shafer, Human Press. 
     The plectasin mutant “Y40YR” was constructed by adding an arginine residue at the end of the plectasin amino acid sequence. 
     The results showed improved activity of all three antimicrobial peptides compared to wildtype plectasin (SEQ ID NO: 1) against the bacteria  Bacillus subtilis, Micrococcus luteus  and  Staphylococcus epidermidis . 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 MEC values; all values are micrograms/mL 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 MEC against 
                 MEC against 
                 MEC against 
               
               
                 Mutation 
                 SEQ ID NO: 
                 
                   B. subtilis 
                 
                 
                   M. luteus 
                 
                 
                   S. epidermis 
                 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 wildtype 
                 1 
                 0.09 
                 1.89 
                 0.98 
               
               
                 Y40YR 
                 99 
                 0.05 
                 0.18 
                 0.31 
               
               
                 N17R 
                 95 
                 0.07 
                 0.32 
                 0.59 
               
               
                 Y25R 
                 117 
                 0.04 
                 1.40 
                 ND 
               
               
                   
               
            
           
         
       
     
     Example 2 
     Evaluation of Antimicrobial Activity 
     A range of antimicrobial polypeptides, which are variants of SEQ ID NO: 1 (Plectasin), were tested for antimicrobial activity by expressing them in  S. cerevisae  and screening the supernatant of the yeast transformants for antimicrobial activity against  Staphylococcus carnosus  ATCC51365. 
     Growth media and solutions were prepared as described in Sambrook, Fritsch and Maniatis (1989),  Molecular cloning , Cold Spring Harbour, Laboratory Press, New York. 
     Radial Diffusion Assay was carried out as described in Methods in Molecular Biology, Vol. 78, Antibacterial peptide protocol, William M. Shafer, Human Press. 
     200-300 yeast transformant colonies were plated on 14 cm round plates containing 25 mL SC growth medium supplemented with 1.5% galactose, 0.5% glucose and 1.5% agarose. Plates were incubated for three hours at room temperature, overlaid with 25 mL of the same growth medium and allowed to grow for three days at 30° C. Then the plates were overlaid with 25 mL of LB growth medium containing 1.5% agarose and 10 5  cells of the indicator strain  Staphylococcus carnosus  and incubated at 30° C. overnight to allow growth of the bacterial cells. Next day, the plates were stained with 1.5 mM MTT to facilitate visualization of the clearing zones. 
     Yeast colonies creating clearing zones were transferred to microtiter plates containing 200 micro-L of SC growth medium supplemented with 2% glucose and ampicillin (100 mg/L). Such plates designated “master plates” were incubated for 2 days at 30° C. with shaking at 450 rpm to allow yeast growth. 
     10 micro-L SC growth medium from each well of the master plates were transferred to new microtiter plates containing 200 micro-L SC growth medium with 1.5% galactose and 0.5% glucose. These plates were called daughter plates and were incubated for 3 days at 30° C. under 450 rpm shaking to allow yeast growth and peptide synthesis. 
     Finally a Radial Diffusion Assay (RDA) was performed following the protocol described in Methods in Molecular Biology to analyse and quantify the antimicrobial activity of the yeast supernatants against  S. carnosus.    
     Briefly, 30 mL of minimal underlay medium containing 1% agarose and 5×10 5  cfu/mL of  S. carnosus  was poured in an omnitray plate (Nunc, 242811). A Nunc TSP plate (#445497) was inserted immediately on the plate to allow a 96 well pattern formation. Once the media had solidified, the TSP plate was removed and 10 micro-L of yeast supernatant samples were applied on the holes. Plates were incubated 3 hours at 37° C. and overlaid with 15 mL of LB agar growth medium. Finally, plates were incubated overnight at 37° C. and coloured with 1.5 mM MTT to visualize the clearing zones. 
     Inspection and measurements of the clearing zones was performed on the plates. The corresponding yeast clones resulting in clearing zones of similar or increased size than the clones encoding for wild type plectasin were picked from the master plates and transferred to agar plates containing SC growth medium with 2% glucose and ampicillin (100 mg/L). Such plates were incubated for further 2 days at 30° C. to allow yeast growth. Subsequently, colony PCR was performed followed by sequence analysis to identify amino acid changes in the plectasin sequence. 
     The mutations and corresponding antimicrobial activities, relative to the activity of plectasin, are shown in Table 2. An activity of 2 corresponds to the activity of Plectasin. An activity of 3 is better than Plectasin, and 1 is worse than Plectasin. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Antimicrobial activity data from the yeast screening assay 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ ID 
                   
                   
               
               
                 Sequence 
                 Mutation(s) 
                 NO: 
                 Activity 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 wildtype 
                 1 
                 2 
                   
               
               
                   
               
               
                 GFGCRGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5R 
                 3 
                 3 
               
               
                   
               
               
                 GFGCQGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5Q 
                 4 
                 3 
               
               
                   
               
               
                 GFGCVGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5V 
                 5 
                 3 
               
               
                   
               
               
                 GFGCGGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5G 
                 6 
                 3 
               
               
                   
               
               
                 GFGCSGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5S 
                 7 
                 3 
               
               
                   
               
               
                 GFGCAGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5A 
                 8 
                 3 
               
               
                   
               
               
                 GFGCNGKWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 P7K 
                 9 
                 3 
               
               
                   
               
               
                 GFGCNGRWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 P7R 
                 10 
                 3 
               
               
                   
               
               
                 GFGCNGPRDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 W8R 
                 11 
                 3 
               
               
                   
               
               
                 GFGCNGPWAEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9A 
                 12 
                 3 
               
               
                   
               
               
                 GFGCNGPWGEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9G 
                 13 
                 3 
               
               
                   
               
               
                 GFGCNGPWKEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9K 
                 14 
                 3 
               
               
                   
               
               
                 GFGCNGPWLEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9L 
                 15 
                 3 
               
               
                   
               
               
                 GFGCNGPWTEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9T 
                 16 
                 3 
               
               
                   
               
               
                 GFGCNGPWYEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9Y 
                 17 
                 3 
               
               
                   
               
               
                 GFGCNGPWFEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9F 
                 18 
                 3 
               
               
                   
               
               
                 GFGCNGPWHEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9H 
                 19 
                 3 
               
               
                   
               
               
                 GFGCNGPWMEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9M 
                 20 
                 3 
               
               
                   
               
               
                 GFGCNGPWNEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9N 
                 21 
                 3 
               
               
                   
               
               
                 GFGCNGPWPEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9P 
                 22 
                 3 
               
               
                   
               
               
                 GFGCNGPWQEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9Q 
                 23 
                 3 
               
               
                   
               
               
                 GFGCNGPWSEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9S 
                 24 
                 3 
               
               
                   
               
               
                 GFGCNGPWVEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9V 
                 25 
                 3 
               
               
                   
               
               
                 GFGCNGPWDGDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10G 
                 26 
                 3 
               
               
                   
               
               
                 GFGCNGPWDSDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10S 
                 27 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEFDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11F 
                 28 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11G 
                 29 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEHDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11H 
                 30 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEKDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11K 
                 31 
                 3 
               
               
                   
               
               
                 GFGCNGPWDELDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11L 
                 32 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEPDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11P 
                 33 
                 3 
               
               
                   
               
               
                 GFGCNGPWDESDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11S 
                 34 
                 3 
               
               
                   
               
               
                 GFGCNGPWDETDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11T 
                 35 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEVDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11V 
                 36 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEWDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11W 
                 37 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEIDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11I 
                 38 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEMDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11M 
                 39 
                 3 
               
               
                   
               
               
                 GFGCNGPWDENDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11N 
                 40 
                 3 
               
               
                   
               
               
                 GFGCNGPWDERDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11R 
                 41 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEYDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11Y 
                 42 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDRQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13R 
                 43 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDSQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13S 
                 44 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDVQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13V 
                 45 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMFCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14F 
                 46 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMGCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14G 
                 47 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMHCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14H 
                 48 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMSCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14S 
                 49 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMYCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14Y 
                 50 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNLCKSIKGYKGGYCAKGGFVCKCY 
                 H18L 
                 51 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSLKGYKGGYCAKGGFVCKCY 
                 I22L 
                 52 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSVKGYKGGYCAKGGFVCKCY 
                 I22V 
                 53 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKHYKGGYCAKGGFVCKCY 
                 G24H 
                 54 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKKYKGGYCAKGGFVCKCY 
                 G24K 
                 55 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKNYKGGYCAKGGFVCKCY 
                 G24N 
                 56 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGFCAKGGFVCKCY 
                 Y29F 
                 57 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGRCAKGGFVCKCY 
                 Y29R 
                 58 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGWCAKGGFVCKCY 
                 Y29W 
                 59 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCKKGGFVCKCY 
                 A31K 
                 60 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCNKGGFVCKCY 
                 A31N 
                 61 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCQKGGFVCKCY 
                 A31Q 
                 62 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCTKGGFVCKCY 
                 A31T 
                 63 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCYKGGFVCKCY 
                 A31Y 
                 64 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKKGFVCKCY 
                 G33K 
                 65 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKQGFVCKCY 
                 G33Q 
                 66 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKRGFVCKCY 
                 G33R 
                 67 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGKFVCKCY 
                 G34K 
                 68 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGRFVCKCY 
                 G34R 
                 69 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGLVCKCY 
                 F35L 
                 70 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFLCKCY 
                 V36L 
                 71 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFMCKCY 
                 V36M 
                 72 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFTCKCY 
                 V36T 
                 73 
                 3 
               
               
                   
               
               
                 GFGCKGPWDEDDMQCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 N5K + K26R 
                 74 
                 3 
               
               
                   
               
               
                 GFGCKGPWDEGDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5K + D11G 
                 75 
                 3 
               
               
                   
               
               
                 GFGCSGPWDEDDMRCHNHCKAIRGYKGGYCAKGGFVCKCY 
                 N5S + Q14R + S21A + 
                 76 
                 3 
               
               
                   
                 K23R 
               
               
                   
               
               
                 GFGCSGPWDEDDMRCHSHCKSIRGYKGGYCAKGGFVCKCY 
                 N5S + Q14R + N17S + 
                 77 
                 3 
               
               
                   
                 K23R 
               
               
                   
               
               
                 GFGCNGPRDEDDRQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 W8R + M13R 
                 78 
                 3 
               
               
                   
               
               
                 GFGCNGPWGEDDMRCHNHCKSIRGYKGGYCAKGGFVCKCY 
                 D9G + Q14R + K23R 
                 79 
                 3 
               
               
                   
               
               
                 GFGCNGPWDGDDMRCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10G + Q14R 
                 80 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHNHCKSIKGYKGGYCARGGFVCKCY 
                 D11G + K32R 
                 81 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHSHCKSIKGYKGGYCAKGGFVCKCY 
                 D11G + N17S 
                 82 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHNHCKSVKGYKGGYCAKGGFVCKCY 
                 D11G + I22V 
                 83 
                 3 
               
               
                   
               
               
                 GFGCNGPWDENDMQCHNHCKSIKGYKGGYCAKGGFICKCY 
                 D11N + V36I 
                 84 
                 3 
               
               
                   
               
               
                 GFGCNGPWDERDIQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11R + M13I 
                 85 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMVCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 Q14V + K26R 
                 86 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIKGYRGGYCAKGGFVCRCY 
                 Q14R + K26R + K38R 
                 87 
                 3 
               
               
                   
               
               
                 GLGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 F2L 
                 118 
                 2 
               
               
                   
               
               
                 GWGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 F2W 
                 119 
                 2 
               
               
                   
               
               
                 GIGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 F2I 
                 120 
                 2 
               
               
                   
               
               
                 GFGCLGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5L 
                 121 
                 2 
               
               
                   
               
               
                 GFGCMGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5M 
                 122 
                 2 
               
               
                   
               
               
                 GFGCNRPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 G6R 
                 123 
                 2 
               
               
                   
               
               
                 GFGCNAPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 G6A 
                 124 
                 2 
               
               
                   
               
               
                 GFGCNKPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 G6K 
                 125 
                 2 
               
               
                   
               
               
                 GFGCNGAWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 P7A 
                 126 
                 2 
               
               
                   
               
               
                 GFGCNGLWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 P7L 
                 127 
                 2 
               
               
                   
               
               
                 GFGCNGVWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 P7V 
                 128 
                 2 
               
               
                   
               
               
                 GFGCNGPWCEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9C 
                 129 
                 2 
               
               
                   
               
               
                 GFGCNGPWIEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9I 
                 130 
                 2 
               
               
                   
               
               
                 GFGCNGPWREDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9R 
                 131 
                 2 
               
               
                   
               
               
                 GFGCNGPWWEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D9W 
                 132 
                 2 
               
               
                   
               
               
                 GFGCNGPWDADDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10A 
                 133 
                 2 
               
               
                   
               
               
                 GFGCNGPWDLDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10L 
                 134 
                 2 
               
               
                   
               
               
                 GFGCNGPWDCDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10C 
                 135 
                 2 
               
               
                   
               
               
                 GFGCNGPWDQDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 E10Q 
                 136 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEADMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11A 
                 137 
                 2 
               
               
                   
               
               
                 GFGCNGPWDECDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11C 
                 138 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDPMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D12P 
                 139 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDAQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13A 
                 140 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDFQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13F 
                 141 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDGQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13G 
                 142 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDLQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13L 
                 143 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDTQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13T 
                 144 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDYQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13Y 
                 145 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMACHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14A 
                 146 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMCCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14C 
                 147 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMICHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14I 
                 148 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMKCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14K 
                 149 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMMCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14M 
                 150 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMPCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14P 
                 151 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMTCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14T 
                 152 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMVCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14V 
                 153 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMWCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14W 
                 154 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNACKSIKGYKGGYCAKGGFVCKCY 
                 H18A 
                 155 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNFCKSIKGYKGGYCAKGGFVCKCY 
                 H18F 
                 156 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNQCKSIKGYKGGYCAKGGFVCKCY 
                 H18Q 
                 157 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNTCKSIKGYKGGYCAKGGFVCKCY 
                 H18T 
                 158 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNVCKSIKGYKGGYCAKGGFVCKCY 
                 H18V 
                 159 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCQSIKGYKGGYCAKGGFVCKCY 
                 K20Q 
                 160 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSMKGYKGGYCAKGGFVCKCY 
                 I22M 
                 161 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSTKGYKGGYCAKGGFVCKCY 
                 I22T 
                 162 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSWKGYKGGYCAKGGFVCKCY 
                 I22W 
                 163 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKAYKGGYCAKGGFVCKCY 
                 G24A 
                 164 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKPYKGGYCAKGGFVCKCY 
                 G24P 
                 165 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKFYKGGYCAKGGFVCKCY 
                 G24F 
                 166 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKIYKGGYCAKGGFVCKCY 
                 G24I 
                 167 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKQYKGGYCAKGGFVCKCY 
                 G24Q 
                 168 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKRYKGGYCAKGGFVCKCY 
                 G24R 
                 169 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKSYKGGYCAKGGFVCKCY 
                 G24S 
                 170 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKTYKGGYCAKGGFVCKCY 
                 G24T 
                 171 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKYYKGGYCAKGGFVCKCY 
                 G24Y 
                 172 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGHKGGYCAKGGFVCKCY 
                 Y25H 
                 173 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGKKGGYCAKGGFVCKCY 
                 Y25K 
                 174 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGLKGGYCAKGGFVCKCY 
                 Y25L 
                 175 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGMKGGYCAKGGFVCKCY 
                 Y25M 
                 176 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGNKGGYCAKGGFVCKCY 
                 Y25N 
                 177 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGQKGGYCAKGGFVCKCY 
                 Y25Q 
                 178 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGSKGGYCAKGGFVCKCY 
                 Y25S 
                 179 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGVKGGYCAKGGFVCKCY 
                 Y25V 
                 180 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYFGGYCAKGGFVCKCY 
                 K26F 
                 181 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYHGGYCAKGGFVCKCY 
                 K26H 
                 182 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYTGGYCAKGGFVCKCY 
                 K26T 
                 183 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGACAKGGFVCKCY 
                 Y29A 
                 184 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGHCAKGGFVCKCY 
                 Y29H 
                 185 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGLCAKGGFVCKCY 
                 Y29L 
                 186 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGMCAKGGFVCKCY 
                 Y29M 
                 187 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGSCAKGGFVCKCY 
                 Y29S 
                 188 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCEKGGFVCKCY 
                 A31E 
                 189 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCHKGGFVCKCY 
                 A31H 
                 190 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCIKGGFVCKCY 
                 A31I 
                 191 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCRKGGFVCKCY 
                 A31R 
                 192 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCSKGGFVCKCY 
                 A31S 
                 193 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCVKGGFVCKCY 
                 A31V 
                 194 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCARGGFVCKCY 
                 K32R 
                 195 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCATGGFVCKCY 
                 K32T 
                 196 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKEGFVCKCY 
                 G33E 
                 197 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKNGFVCKCY 
                 G33N 
                 198 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKSGFVCKCY 
                 G33S 
                 199 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKTGFVCKCY 
                 G33T 
                 200 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGHFVCKCY 
                 G34H 
                 201 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGWFVCKCY 
                 G34W 
                 202 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGAVCKCY 
                 F35A 
                 203 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGHVCKCY 
                 F35H 
                 204 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGIVCKCY 
                 F35I 
                 205 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGMVCKCY 
                 F35M 
                 206 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGVVCKCY 
                 F35V 
                 207 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGWVCKCY 
                 F35W 
                 208 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFICKCY 
                 V36I 
                 209 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFKCKCY 
                 V36K 
                 210 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFQCKCY 
                 V36Q 
                 211 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFRCKCY 
                 V36R 
                 212 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCHCY 
                 K38H 
                 213 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCNCY 
                 K38N 
                 214 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCRCY 
                 K38R 
                 215 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCI 
                 Y40I 
                 216 
                 2 
               
               
                   
               
               
                 GFGCNGPWLEDDMQCHNHCKSIKGYNGGYCAKGGFVCKCY 
                 D9L + K26N 
                 217 
                 2 
               
               
                   
               
               
                 GFGCNGPWDELDIQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 D11L + M13I 
                 218 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDRQCHNHCKSIKGYKGGFCAKGGFVCKCY 
                 M13R + Y29F 
                 219 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIRGYRGGYCAKGGFVCKCY 
                 Q14R + K23R + K26R 
                 220 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCRSIKGYKGGYCAKGGFVCKCY 
                 Q14R + K20R 
                 221 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMSCHNHCKSIKGYKGGYCAKGGFVCRCY 
                 Q14S + K38R 
                 222 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHSHCKSIRGYKGGYCAKGGFVCKCY 
                 N17S + K23R 
                 223 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGFCARGGFVCKCY 
                 Y29F + K32R 
                 224 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCARGKFVCKCY 
                 K32R + G34K 
                 225 
                 2 
               
               
                   
               
            
           
         
       
     
     Example 3 
     Identification of Antimicrobial Peptides with Improved Antimicrobial Activity 
     A range of antimicrobial polypeptides, which are variants of SEQ ID NO: 1, were tested in the TAPS assay. TAPS may be used to identify new or improved genes encoding peptides that can kill or inhibit the growth of target cells (see PCT application WO 2004/033715). The TAPS assay, an acronym for Trans Acting Peptide System, is based on having a sensitive host producing a peptide followed by screening for its activity in trans against an indicator strain. The advantage of this system is based on that the antimicrobial peptide is expressed in the Gram-negative bacterium  E. coli  and its antimicrobial activity can be monitored on different microbes including Gram-negatives, positives or fungi. Additionally, TAPS offers the possibility to produce correctly folded AMPs containing disulfide bonds in the host cells, thereby retaining their antimicrobial activities. 
     The TAPS approach requires first, that expression of the peptide is under control of an inducible promoter with tight regulation, because the host cells are sensitive to the peptide when producing it. Secondly, the produced peptide has to be released to the media so that it can interact with the target organism. 
     The TAPS screening can be carried out either in solid or liquid media. On solid media, a plasmid library is initially introduced into  E. coli  host cells. It is important that the transformants are cultivated on the surface of a cellulose acetate filter placed on LB growth medium without inducer (arabinose) to avoid expression of the antimicrobial peptide and hence growth inhibition. In the next step, the filter containing the colonies is transferred to LB growth medium containing inducer (0.1% arabinose) to permit peptide synthesis. Subsequently, the target strain, for example  S. carnosus , is overlaid onto the plate and allowed to grow for 12-16 hours at 37° C. Finally, visual inspection of the host cells capable of reducing the proliferation of the target cells is performed and the nucleotide sequence encoding for the antimicrobial peptide is recovered from the host cells. DNA sequence analysis of the variants is obtained to elucidate the nature of the peptide. 
     As mentioned above, the TAPS screening can also be performed using liquid medium. This procedure requires the use of robotics to analyze large number of clones. In this system, the host  E. coli  origami cells are transformed with the plasmid library and plated out on LB medium+0.2% glucose+ampicillin (200 mg/L). Independent colonies are then inoculated into 96 or 384-well plates containing 200 micro-L of TB medium+ampicillin (200 mg/L) and cultured overnight at 37° C. These cultures are then replicated robotically and grown to exponential phase until inducer (0.1% arabinose) is added to trigger peptide synthesis. The next step consists in hydrolyzing the cells such that the peptide is released to the media by hot acid hydrolysis. This treatment consists on adding 1 M sodium Phosphate buffer pH 2.3 to obtain a final pH approximately of 2.3 and incubating the cultures overnight at 80° C. Next day, a 25 micro-L aliquot of the hydrolyzed cultures is used to perform an activity test against the desired target organisms. The activity test performed was a Radial Diffusion Assay (RDA) where an aliquot of the hydrolyzed cultures was added to the agarose media inoculated with the target strain,  S. carnosus . RDAs obtained from the screening plates containing clearing zones corresponding to clones exhibiting antimicrobial activity were easily identified. Measurements of the diameter of the clearing zones were performed to quantify the potency of the antimicrobial activity of the peptides. 
     The antimicrobial activity (against  Staphylococcus carnosus ) of the tested plectasin variants, which was measured using the TAPS assay, is shown in table 3. The antimicrobial activity corresponds to the clearing zone size and has been classified as 4&gt;3&gt;2&gt;1; whereas 4 is better than 1, and wildtype activity corresponds to 1. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Antimicrobial activity data from the TAPS assay 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 SEQ ID 
                   
                   
               
               
                 Sequence 
                 Mutation(s) 
                 NO: 
                 Activity 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                   
                 control 
                   
                 0 
                   
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 wildtype 
                 1 
                 1 
               
               
                   
               
               
                 GFGCKGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5K 
                 88 
                 2 
               
               
                   
               
               
                 GFGCYGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5Y 
                 89 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 M13K 
                 90 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMLCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14L 
                 91 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 Q14R 
                 92 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHHHCKSIKGYKGGYCAKGGFVCKCY 
                 N17H 
                 93 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHIHCKSIKGYKGGYCAKGGFVCKCY 
                 N17I 
                 94 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHRHCKSIKGYKGGYCAKGGFVCKCY 
                 N17R 
                 95 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHYHCKSIKGYKGGYCAKGGFVCKCY 
                 N17Y 
                 96 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCRSIKGYKGGYCAKGGFVCKCY 
                 K20R 
                 97 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 K26R 
                 98 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGFVCKCYR 
                 Y40YR 
                 99 
                 2 
               
               
                   
               
               
                 GFGCYGPWDEDDMLCHNHCKSIKGYKGGYCAKGGFVCKCY 
                 N5Y + Q14L 
                 100 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 D11G + K26R 
                 101 
                 2 
               
               
                   
               
               
                 GFGCNGPWDEGDKQCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 D11G + M13K + K26R 
                 102 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHRHCKSIKGYKGGYCAKGGFVCKCYR 
                 M13K + N17R + Y40YR 
                 103 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHRHCKSIKGYKGGYCAKGGFVCKCYR 
                 N17R + Y40YR 
                 104 
                 3 
               
               
                   
               
               
                 GFGCNGPWDENDMQCHNHCKFIKGYKGGYCAKGGFVCKCY 
                 D11N + S21F 
                 105 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHRHCKSIKGYKGGYCAKGGFVCKCY 
                 M13K + N17R 
                 106 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCKCYR 
                 M13K + Y40YR 
                 107 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 M13K + K26R 
                 108 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDKQCHNHCKSIKGYKGGYCAKGGFVCRCY 
                 M13K + K38R 
                 109 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEGDMQCHNHCKSIKGYRGGYCAKGGFVCKCYR 
                 D11G + K26R + Y40YR 
                 110 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEGDKQCHNHCKSIKGYRGGYCAKGGFVCKCYR 
                 D11G + M13K + K26R + 
                 111 
                 4 
               
               
                   
                 Y40YR 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFVCKCYR 
                 Q14R + Y40YR 
                 112 
                 3 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIKGYKGGYCAKGGFICKCY 
                 Q14R + V36I 
                 113 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDMRCHNHCKSIKGYRGGYCAKGGFVCKCY 
                 Q14R + K26R 
                 114 
                 4 
               
               
                   
               
               
                 GFGCNGPWDEDDMQCHNHCKFIKGYKGGYCTKGGFVCKCY 
                 S21F + A31T 
                 115 
                 2 
               
               
                   
               
            
           
         
       
     
     Example 4 
     Evaluation of Antimicrobial Activity 
     Plectasin wild-type (SEQ ID NO: 1) and five variants of Plectasin, which showed improved activity in the TAPS method and/or in the yeast system, were expressed and purified. The Minimal Inhibitory Concentration (MIC, micrograms/mL) was determined to test for their antimicrobial activity following the NCCLS guidelines (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards). 
     The results showed that all plectasin variants had improved activity, compared to wildtype plectasin, against  Staphylococcus aureus, Micrococcus luteus  and  Bacillus subtilis . 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 MIC values; all values are micrograms/mL 
               
            
           
           
               
               
               
               
               
            
               
                   
                 SEQ 
                 MIC against 
                 MIC against 
                 MIC against 
               
               
                 Mutation(s) 
                 ID NO: 
                 
                   S. aureus 
                 
                 
                   M. luteus 
                 
                 
                   B. subtilis 
                 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Wildtype 
                 1 
                 8 
                 32 
                 1 
               
               
                 M13K + Y40YR 
                 107 
                 2 
                 2 
                 0.25 
               
               
                 D11G + K26R 
                 101 
                 2 
                 8 
                 0.50 
               
               
                 Q14K + K26R 
                 116 
                 2 
                 1 
                 0.13 
               
               
                 D11G + K26R + 
                 110 
                 0.50 
                 0.25 
                 0.06 
               
               
                 Y40YR 
               
               
                 D11G + M13K + 
                 111 
                 0.50 
                 0.50 
                 &lt;0.03 
               
               
                 K26R + Y40YR 
               
               
                   
               
            
           
         
       
     
     Example 5 
     Evaluation of Antimicrobial Activity 
     Plectasin wild-type (SEQ ID NO: 1) and several variants of Plectasin were expressed and purified. The Minimal Inhibitory Concentration (MIC) was determined to test for their antimicrobial activity following the NCCLS guidelines from CLSI (Clinical and Laboratory Standards Institute; formerly known as National Committee for Clinical and Laboratory Standards). 
     The peptides were tested against the following strains:
     A:  Staphylococcus aureus , ATCC 29213;   B:  Staphylococcus aureus , ATCC 25923;   C:  Staphylococcus aureus , ATCC 29737;   D:  Staphylococcus aureus , MRSA ST5 (2001), multi-resistant clinical human isolate from Statens Serum Institut, Denmark;   E:  Staphylococcus aureus , MRSA ST80 (2003), multi-resistant clinical human isolate from Statens Serum Institut, Denmark.   

     All MIC values represent an average of two independent experiments; and all results in Table 5 are expressed relative to wildtype Plectasin: 
     0: &gt;100% of Plectasin wildtype MIC;
 
1: 80-100% of Plectasin wildtype MIC;
 
2: 50-80% of Plectasin wildtype MIC;
 
3: &lt;50% of Plectasin wildtype MIC.
 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Relative MIC values; nd = not determined 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 SEQ ID 
                   
                   
                   
                   
                   
               
               
                 Mutation(s) 
                 NO: 
                 A 
                 B 
                 C 
                 D 
                 E 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 wildtype 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                   
                 (8 μg/mL) 
                 (22 μg/mL) 
                 (4 μg/mL) 
                 (15 μg/mL) 
                 (22 μg/mL) 
               
               
                 N5R + M13Y + N17R 
                 226 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9S + Q14K + V36L 
                 227 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5S + M13W + N17R 
                 228 
                 3 
                 nd 
                 3 
                 3 
                 3 
               
               
                 Q14R + K26R + K38R 
                 87 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9G + Q14R + K23R 
                 79 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 M13G + N17R + G33A 
                 229 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5S + D9S + M13L + Q14R + 
                 230 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N17V + A31S 
               
               
                 D9S + Q14L + K26R 
                 231 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5S + D9A + K26R 
                 232 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 Q14R + K20R 
                 221 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5G + M13L 
                 233 
                 2 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9A + K38R 
                 234 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 D11G + K32R 
                 81 
                 1 
                 3 
                 3 
                 2 
                 3 
               
               
                 Q14F 
                 46 
                 1 
                 nd 
                 3 
                 2 
                 3 
               
               
                 N5R + M13V 
                 235 
                 3 
                 nd 
                 3 
                 2 
                 3 
               
               
                 N5G + M13Y + N17K 
                 236 
                 3 
                 3 
                 3 
                 3 
                 2 
               
               
                 Q14K + K26R 
                 116 
                 3 
                 2 
                 3 
                 3 
                 3 
               
               
                 M13F + Q14K + K26R 
                 237 
                 3 
                 nd 
                 3 
                 3 
                 3 
               
               
                 M13K + K38R 
                 109 
                 1 
                 3 
                 3 
                 2 
                 3 
               
               
                 N5A + D9S + M13L + N17T 
                 238 
                 0 
                 3 
                 3 
                 2 
                 3 
               
               
                 N17Y 
                 96 
                 1 
                 3 
                 3 
                 2 
                 2 
               
               
                 M13T + Q14K + K26R 
                 239 
                 1 
                 3 
                 3 
                 2 
                 0 
               
               
                 D9S 
                 24 
                 2 
                 3 
                 3 
                 2 
                 nd 
               
               
                 N17R 
                 95 
                 nd 
                 3 
                 3 
                 3 
                 2 
               
               
                 Q14R 
                 92 
                 2 
                 3 
                 3 
                 3 
                 0 
               
               
                 N5S + Q14R + N17S + K23R 
                 77 
                 1 
                 3 
                 3 
                 3 
                 nd 
               
               
                 N5R 
                 3 
                 1 
                 2 
                 2 
                 2 
                 2 
               
               
                 D11G + K26R + Y40YR 
                 110 
                 3 
                 3 
                 3 
                 3 
                 nd 
               
               
                 N5S + Q14R + S21A + K23R 
                 76 
                 1 
                 3 
                 2 
                 2 
                 0 
               
               
                 M13L + Q14K + K26R 
                 240 
                 3 
                 nd 
                 3 
                 3 
                 nd 
               
               
                 D9N + M13L + Q14R 
                 241 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9A + Q14H + K26R + V36L 
                 242 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 Q14R + K23R + K26R 
                 220 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5S + D9S + M13V + N17R 
                 243 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N5G + D9S + M13L + 
                 244 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 N17Q + A31T 
               
               
                 M13V + N17T 
                 245 
                 nd 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9S + M13L + Q14H 
                 246 
                 3 
                 3 
                 3 
                 3 
                 3 
               
               
                 D9S + Q14L 
                 247 
                 3 
                 nd 
                 3 
                 3 
                 3 
               
               
                 D9N + Q14H + K38R 
                 248 
                 3 
                 3 
                 3 
                 3 
                 2 
               
               
                 M13Y + Q14K + K26R 
                 249 
                 2 
                 3 
                 3 
                 3 
                 3 
               
               
                 D11N 
                 40 
                 2 
                 3 
                 2 
                 2 
                 3 
               
               
                 N5S + D9S 
                 250 
                 0 
                 3 
                 3 
                 2 
                 3 
               
               
                 G24R 
                 169 
                 1 
                 2 
                 nd 
                 2 
                 2 
               
               
                 N5G + Q14K 
                 251 
                 2 
                 2 
                 3 
                 0 
                 2 
               
               
                 N5K + K26R 
                 74 
                 1 
                 2 
                 3 
                 2 
                 2 
               
               
                   
               
            
           
         
       
     
     Example 6 
     Evaluation of Antimicrobial Activity 
     A large number of antimicrobial peptides of the invention were tested against a panel of 6 different strains of  Staphylococcus aureus  listed below: 
       Staphylococcus aureus , ATCC29213, MSSA, NCCLS reference strain;
   Staphylococcus aureus , ATCC25923, MSSA, NCCLS reference strain;
   Staphylococcus aureus , ATCC29737, MSSA;
   Staphylococcus aureus , E33235, MSSA;
   Staphylococcus aureus,  698-01, MRSA ST5, Str, Kan, Oxa;
   Staphylococcus aureus,  566-03, MRSA ST80, Oxa, Tet, Fus, Kan.
 
       S. aureus  E33235,  S. aureus  698-01 and  S. aureus  566-03 are available from Statens Serum Institut, Denmark. 
     The staphylococci were exposed to the following peptide concentrations: 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.13; 0.6; and 0.03 microgram/mL. All peptides were purified, HPLC quantified and the concentrated peptides (&gt;160 micrograms/mL) were diluted to 160 micrograms/mL in peptide dilution buffer (0.1% BSA, 0.01% Acetic Acid). 
     The MIC determination was done essentially as described by NCCLS/CLSI guidelines using caMHB. The MICs were read after 18-24 hours of 37° C. incubations and recorded along with the CFU in the table below. 
     A total number of 95 antimicrobial peptides of the invention were evaluated in duplicate against the 6 bacterial strains described above. 
     In the majority of the double determinations (93%), the MIC varied &lt;2 fold. An average MIC is tabulated below. If the MIC was above 32 micrograms/mL, a value of 64 was used to calculate the average. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Average MIC values 
               
            
           
           
               
               
               
            
               
                   
                   
                 Average MIC 
               
               
                 Mutation(s) 
                 SEQ ID NO: 
                 (micrograms/mL) 
               
               
                   
               
            
           
           
               
               
               
            
               
                 N5R + M13Y + N17R 
                 226 
                 1 
               
               
                 D9N + M13L + Q14R 
                 241 
                 1 
               
               
                 D9S + Q14K + V36L 
                 227 
                 1 
               
               
                 D9A + Q14H + K26R + V36L 
                 242 
                 1 
               
               
                 D9G + Q14R + K23R 
                 79 
                 2 
               
               
                 Q14R + K23R + K26R 
                 220 
                 2 
               
               
                 M13G + N17R + G33A 
                 229 
                 2 
               
               
                 N5S + D9S + M13V + N17R 
                 243 
                 2 
               
               
                 N5S + M13W + N17R 
                 228 
                 2 
               
               
                 Q14R + K26R + K38R 
                 87 
                 2 
               
               
                 N5S + D9S + M13L + Q14R + 
                 230 
                 2 
               
               
                 N17V + A31S 
               
               
                 D9S + Q14L + K26R 
                 231 
                 3 
               
               
                 N5S + D9A + K26R 
                 232 
                 3 
               
               
                 N5G + D9S + M13L + N17Q + A31T 
                 244 
                 3 
               
               
                 M13V + N17T 
                 245 
                 3 
               
               
                 D9S + M13L + Q14H 
                 246 
                 3 
               
               
                 D9S + Q14L 
                 247 
                 3 
               
               
                 Q14R + K20R 
                 221 
                 3 
               
               
                 N5G + M13L 
                 233 
                 4 
               
               
                 D9A + K38R 
                 234 
                 4 
               
               
                 D9N + Q14H + K38R 
                 248 
                 4 
               
               
                 N17R 
                 95 
                 4 
               
               
                 M13R + Q14K + K26R 
                 252 
                 5 
               
               
                 M13Y + Q14K + K26R 
                 249 
                 5 
               
               
                 D11G + K32R 
                 81 
                 5 
               
               
                 Q14F 
                 46 
                 5 
               
               
                 N5R + M13V 
                 235 
                 5 
               
               
                 Q14R 
                 92 
                 5 
               
               
                 N5G + M13Y + N17K 
                 236 
                 5 
               
               
                 Q14R + K26R 
                 114 
                 5 
               
               
                 M13F + Q14K + K26R 
                 237 
                 6 
               
               
                 M13K + K38R 
                 109 
                 6 
               
               
                 D11N 
                 40 
                 6 
               
               
                 N5S + D9S 
                 250 
                 6 
               
               
                 Q14K + K26R 
                 116 
                 6 
               
               
                 N5A + D9S + M13L + N17T 
                 238 
                 6 
               
               
                 N17Y 
                 96 
                 6 
               
               
                 N17K + K32R 
                 253 
                 7 
               
               
                 M13T + Q14K + K26R 
                 239 
                 7 
               
               
                 D9K 
                 14 
                 7 
               
               
                 D9S 
                 24 
                 7 
               
               
                 M13K + N17R + Y40YR 
                 103 
                 8 
               
               
                 N5S + Q14R + N17S + K23R 
                 77 
                 8 
               
               
                 N5R 
                 3 
                 8 
               
               
                 G24R 
                 169 
                 8 
               
               
                 D11G + K26R + Y40YR 
                 110 
                 9 
               
               
                 N5S + Q14R + S21A + K23R 
                 76 
                 9 
               
               
                 N5G + M13W 
                 254 
                 9 
               
               
                 N5G + Q14K 
                 251 
                 9 
               
               
                 M13A + Q14K + K26R 
                 255 
                 9 
               
               
                 M13L + Q14K + K26R 
                 240 
                 9 
               
               
                 N5K + K26R 
                 74 
                 9 
               
               
                 A31N 
                 61 
                 9 
               
               
                 M13R 
                 43 
                 9 
               
               
                 N5K + M13L 
                 256 
                 9 
               
               
                 M13K + K26R 
                 108 
                 10 
               
               
                 N5G + M13W + N17S 
                 257 
                 10 
               
               
                 N17K 
                 258 
                 11 
               
               
                 S21N 
                 259 
                 11 
               
               
                 D11G + K26R 
                 101 
                 11 
               
               
                 K26C + Y40YRCG 
                 260 
                 11 
               
               
                 Q14V + K26R 
                 86 
                 12 
               
               
                 M13K + Y40YR 
                 107 
                 12 
               
               
                 M13K 
                 90 
                 12 
               
               
                 N5H + M13E + N17E 
                 261 
                 12 
               
               
                 N5K 
                 88 
                 12 
               
               
                 N17F 
                 262 
                 12 
               
               
                 D11G + M13K + K26R + Y40YR 
                 111 
                 12 
               
               
                 Q14L 
                 91 
                 13 
               
               
                 Wild-type 
                 1 
                 14 
               
               
                 N17I 
                 94 
                 14 
               
               
                 N17S 
                 263 
                 14 
               
               
                 N17S + K23R 
                 223 
                 14 
               
               
                 N5G + D9A + Q14S + K23T + A31T 
                 264 
                 14 
               
               
                 N17A 
                 265 
                 15 
               
               
                 K26R 
                 98 
                 15 
               
               
                 M13S + Q14K + K26R 
                 266 
                 15 
               
               
                 D11G + N17I 
                 267 
                 16 
               
               
                 N5S + M13V + N17T 
                 268 
                 16 
               
               
                 N5D + D9S + Q14R 
                 269 
                 16 
               
               
                 D11G + I22V 
                 83 
                 17 
               
               
                 S21A 
                 270 
                 17 
               
               
                 N17R + Y25R 
                 271 
                 18 
               
               
                 N5S + M13V + N17A 
                 272 
                 18 
               
               
                 Q14G 
                 47 
                 18 
               
               
                 D9G 
                 13 
                 21 
               
               
                 N17T 
                 273 
                 21 
               
               
                 Q14S 
                 49 
                 21 
               
               
                 N5G 
                 6 
                 24 
               
               
                 N5S 
                 7 
                 24 
               
               
                 M13S 
                 44 
                 24 
               
               
                 S21V 
                 274 
                 26 
               
               
                 M13T 
                 144 
                 26 
               
               
                 V36L 
                 71 
                 26 
               
               
                 N5A 
                 8 
                 29 
               
               
                 D9V 
                 25 
                 30 
               
               
                   
               
            
           
         
       
     
     Example 7 
     Using the HMM Files from the PFAM Database to Identify a Defensin 
     Sequence analysis using hidden markov model profiles (HMM profiles) may be carried out either online on the Internet or locally on a computer using the well-known HMMER freely available software package. The current version is HMMER 2.3.2 from October 2003. 
     The HMM profiles may be obtained from the well-known PFAM database. The current version is PFAM 16.0 from November 2004. Both HMMER and PFAM are available for all computer platforms from, e.g., Washington University in St. Louis (USA), School of Medicine (http://pfam.wustl.edu and http://hmmer.wustl.edu). 
     If a query amino acid sequence, or a fragment thereof, belongs to one of the following five PFAM families, the amino acid sequence is a defensin according to the present invention: 
     Defensin_beta or “Beta Defensin”, accession number: PF00711; 
     Defensin_propep or “Defensin propeptide”, accession number: PF00879; 
     Defensin — 1 or “Mammalian defensin”, accession number: PF00323; 
     Defensin — 2 or “Arthropod defensin”, accession number: PF01097; 
     Gamma-thionin or “Gamma-thionins family”, accession number: PF00304. 
     An amino acid sequence belongs to a PFAM family, according to the present invention, if it generates an E-value which is greater than 0.1, and a score which is larger 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 the sequence analysis is carried out locally using the hmmpfam program, it is necessary to obtain (download) the HMM profiles from the PFAM database. Two profiles exist for each family; xxx_ls.hmm for glocal searches, and xxx_fs.hmm for local searches (“xxx” is the name of the family). That makes a total of ten profiles for the five families mentioned above. 
     These ten profiles may be used individually, or joined (appended) into a single profile (using a text editor—the profiles are ASCII files) that could be named, e.g., defensin.hmm. A query amino acid sequence can then be evaluated by using the following command line:
         hmmpfam -E 0.1 defensin.hmm sequence_file       

     wherein “sequence_file” is a file with the query amino acid sequence in any of the formats recognized by the HMMER software package. 
     If the score is larger or equal to zero (0.0), and the E-value is greater than 0.1, the query amino acid sequence is a defensin according to the present invention. 
     The PFAM database is further described in Bateman et al. (2004) “The Pfam Protein Families Database”, Nucleic Acids Research, Vol. 32 (Database Issue) pp. D138-D141.