Efficient methods for producing antimicrobial cationic peptides in host cells

Abstract

Endogenously produced cationic antimicrobial peptides are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Cationic peptides are also effective when administered as therapeutic agents. A practical drawback in cationic peptide therapy, however, is the cost of producing the agents. The methods described herein provide a means to efficiently produce cationic peptides from recombinant host cells. These recombinantly-produced cationic peptides can be rapidly purified from host cell proteins using anion exchange chromatography.

1 . A fusion protein expression cassette, comprising a promoter operably linked to a nucleic acid molecule that encodes a fusion protein comprising a structure of (cationic peptide)-[(cleavage site)-(cationic peptide)] n  with n being an integer having a value between one and three, wherein the cationic peptides have antimicrobial activity and the cleavage site can be cleaved by low pH or by a reagent selected from the group consisting of 2-(2-nitrophenylsulphenyl)-3-methyl-3′-bromoindolenine, hydroxylamine, o-iodosobenzoic acid, Factor Xa, thrombin, enterokinase, collagenase,  Staphylococcus aureus  V8 protease, endoproteinase Arg-C, and trypsin.  
     
     
         2 . The expression cassette of  Claim 1  wherein said fusion protein comprises 3 cationic peptides.  
     
     
         3 . The expression cassette of  claim 1  wherein said fusion protein comprises 4 cationic peptides.  
     
     
         4 . The expression cassette according to  claim 1  wherein the fusion protein is cleaved by endoproteinase Lys-C.  
     
     
         5 . The expression cassette according to  claim 1  wherein the cationic peptide has up to 35 amino acids comprising the sequence of I L K K W P W W P W R R K (SEQ ID NO: 35) or 1 L R W P W W P W R R K (SEQ ID NO:36).  
     
     
         6 . The expression cassette according to  claim 1  wherein the cationic peptide is I L R W P W W P W R R K (SEQ ID NO:36).  
     
     
         7 . The expression cassette according to  claim 1  wherein said promoter is selected from the group consisting of lacP promoter, tacP promoter, trcp promoter, srpP promoter, SP6 promoter, T7 promoter, araP promoter, trpP promoter, and λ promoter.  
     
     
         8 . The expression cassette according to  claim 1  wherein said nucleic acid molecule also encodes a carrier protein.  
     
     
         9 . The expression cassette according to  claim 8  wherein the carrier protein is selected from cellulose binding domain, glutathione-S-transferase, outer membrane protein F, β-galactosidase, protein A, or IgG-binding domain.  
     
     
         10 . The expression cassette according to  claim 8  wherein said carrier protein is less than 100 amino acid residues in length.  
     
     
         11 . The expression cassette according to  claim 1  wherein said nucleic acid molecule also encodes an anionic spacer peptide component comprising a structure of (cationic peptide)-[(cleavage site)-(anionic spacer peptide)-(cleavage site)-(cationic peptide)] n .  
     
     
         12 . The expression cassette according to  claim 11  wherein said anionic spacer lacks a cysteine residue.  
     
     
         13 . A recombinant host cell comprising the expression cassette according to  claim 1 .  
     
     
         14 . The recombinant host cell of  claim 13  wherein the expression cassette is contained in an expression vector.  
     
     
         15 . The recombinant host cell of  claim 13  wherein said host cell is a yeast, fungi, bacterial or plant cell.  
     
     
         16 . The recombinant host cell of  claim 15  wherein said bacterial host cell is  Escherichia coli.    
     
     
         17 . A method of producing a fusion protein, comprising culturing the recombinant host cell of  claim 13  under conditions and for a time sufficient to produce the fusion protein.  
     
     
         18 . The method according to  claim 17  wherein the fusion protein is cleaved at the cleavage sites to release the cationic peptides.  
     
     
         19 . The method according to  claim 17  wherein the cationic peptide is I L R W P W W P W R R K (SEQ ID NO:36).