Claims for Patent: 6,946,261

Summary for Patent: 6,946,261

Title:	Efficient methods for producing anti-microbial 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.
Inventor(s):	Burian; Jan (Victoria, CA), Bartfeld; Daniel (Vancouver, CA)
Assignee:	Migenix Inc. (Vancouver, CA)
Application Number:	09/444,281
Patent Claims:	1. A multi-domain fusion protein expression cassette, comprising a promoter operably linked to a nucleic acid molecule that encodes a fusion protein, wherein the encoded fusion protein comprises a structure of [(cleavage site)-(cationic peptide)-(cleavage site)-(anionic spacer peptide)].sub.n with n being an integer having a value between 2 and 40, and wherein the cationic peptides have at least 30% tryptophan and have antimicrobial activity. 2. The expression cassette according to claim 1 wherein the 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 .lambda. promoter. 3. The expression cassette according to claim 1 wherein the anionic spacer has no cysteine residue. 4. The expression cassette according to claim 1 wherein the cumulative charge of the anionic spacer peptide reduces the cumulative charge of the cationic peptide. 5. The expression cassette according to claim 1 wherein n has a value of between 5 and 30. 6. The expression cassette according to claim 1 wherein n has a value of between 10 and 20. 7. The expression cassette according to claim 1 wherein the cationic peptide has up to 35 amino acids comprising the sequence of SEQ ID NO:35 or SEQ ID NO:36. 8. The expression cassette according to claim 1 wherein the cleavage site can be cleaved by low pH or by a reagent selected from cyanogen bromide, N-chlorosuccinimide, 2-(2-nitrophenylsulphenyl)-3-methyl-3'-bromoindolenine, hydroxylamine, o-iodosobenzoic acid, Factor Xa, Factor XIIa, thrombin, enterokinase, collagenase, Staphylococcus aureus V8 protease, endoproteinase Glu-C, endoproteinase Arg-C, endoproteinase Lys-C, chymotrypsin, trypsin, or a combination thereof. 9. The expression cassette according to claim 1 wherein the encoded fusion protein further comprises one additional cationic peptide or two additional cationic peptides, wherein the additional peptide or peptides are at the carboxy-terminus of the encoded fusion protein. 10. The expression cassette according to claim 1 or claim 9 wherein the expression cassette is contained in an expression vector. 11. The expression cassette according to claim 1 or claim 9 wherein the cationic peptide is SEQ ID NO:36. 12. The expression cassette according to claim 1 or claim 9 further comprising a carrier amino acid sequence wherein the carrier amino acid sequence is at the amino-terminus of the encoded fusion protein. 13. The expression cassette according to claim 12 wherein the carrier is selected from cellulose binding domain, glutathione-S-transferase, outer membrane protein F, .beta.-galactosidase, protein A, or IgG-binding domain. 14. The expression cassette according to claim 12 wherein the carrier is less than 100 amino acid residues in length. 15. The expression cassette according to claim 14 wherein the carrier is a truncated cellulose binding domain. 16. The expression cassette according to claim 12 wherein the cationic peptide is SEQ ID NO:36. 17. A recombinant host cell comprising the expression cassette according to claim 16 wherein the expression cassette is contained in an expression vector. 18. The recombinant host cell according to claim 17 wherein the encoded fusion protein is expressed as an insoluble protein. 19. A method of producing a fusion protein, comprising culturing a recombinant host cell according to claim 17 under conditions and for a time sufficient to produce said fusion protein. 20. The method according to claim 19 wherein the fusion protein is cleaved at the cleavage sites to release the cationic peptides from the anionic spacers. 21. The method according to claim 20 wherein the fusion protein is cleaved by endoproteinase Lys-C. 22. The method according to claim 20 wherein the released cationic peptides are amidated at the carboxy-terminus. 23. A recombinant host cell comprising the expression cassette according to any one of claims 1, 3, 5, and 6. 24. The recombinant host cell of claim 23 wherein the host cell is a yeast, a fungus, a bacteria or a plant cell. 25. The recombinant host cell of claim 24 wherein the bacteria is Escherichia coli. 26. A method of producing a fusion protein, comprising culturing the recombinant host cell of claim 23 under conditions and for a time sufficient to produce the fusion protein. 27. The recombinant host cell of claim 23 wherein the expression cassette is contained in an expression vector. 28. The recombinant host cell according to claim 27 wherein the encoded cationic peptide fusion protein is expressed as an insoluble protein. 29. A method of producing a fusion protein, comprising culturing a recombinant host cell according to claim 28 under conditions and for a time sufficient to produce said fusion protein.

Details for Patent 6,946,261

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2018-11-20