Protease for wound conditioning and skin care

Abstract

We have identified by molecular cloning a protease which originates from the larvae of Lucilia sericata and which was termed debrilase due to its activities useful for debridement of wounds. Described is a nucleic acid molecule encoding a serine protease having the ability to cleave fibrin and casein which is (a) a nucleic acid molecule encoding the serine protease comprising or consisting of the amino acid sequence of SEQ ID NO: 4 as well as to nucleic acid molecules encoding precursors or fragments of said serine protease; (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3; (c) a nucleic acid molecule encoding a serine protease the amino acid sequence of which is at least 80% identical to the amino acid sequence of (a), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 80% identical to the nucleotide sequence of (b), preferably at least 85% identical, more preferably at least 90% identical, and most preferred 95% identical; (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (b) or (d); or (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U.

The invention claimed is: 
     
       1. An isolated nucleic acid molecule encoding
 (i) a serine protease having the ability to cleave fibrin and casein, said serine protease encoded by
 (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 4; 
 (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3; 
 (c) a nucleic acid molecule encoding the amino acid sequence which is at least 90% identical to the amino acid sequence of (a); 
 (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence of (b); 
 (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or 
 (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U, 
 
 
       wherein the coding sequence of the isolated nucleic acid molecule is operably linked to a heterologous promoter. 
     
     
       2. A vector encoding the nucleic acid molecule of  claim 1 . 
     
     
       3. An isolated host cell transformed, transduced or transfected with the vector of  claim 2 . 
     
     
       4. A method of producing a serine protease comprising culturing the host cell of  claim 3  and isolating the serine protease, the propeptide or the pre-propeptide produced. 
     
     
       5. A serine protease, propeptide, or pre-propeptide encoded by the nucleic acid molecule of  claim 1  or produced by the method of  claim 4 , wherein the serine protease, propeptide, or pre-propeptide is provided in a pharmaceutical composition with a pharmaceutically acceptable sterile carrier. 
     
     
       6. A fusion protein comprising the serine protease, the propeptide, or the pre-propeptide of  claim 5 , wherein the fusion protein is a pre-propeptide having a heterologous signal sequence. 
     
     
       7. A composition comprising the nucleic acid of  claim 1 , the vector of  claim 2 , or the host cell of  claim 3  or combinations thereof. 
     
     
       8. The composition of  claim 7  which is a cosmetic composition. 
     
     
       9. The composition of  claim 7  which is a pharmaceutical composition. 
     
     
       10. A method for treatment of skin peeling, skin smoothening or the intervention with scar formation, comprising contacting skin with the nucleic acid of  claim 1 , the vector of  claim 2 , or the host cell of  claim 3 . 
     
     
       11. A method for treatment of wounds, comprising contacting a wound with the nucleic acid of  claim 1 , the vector of  claim 2 , or the host cell of  claim 3 . 
     
     
       12. The method of  claim 11  wherein the wounds are chronic or slow healing wounds. 
     
     
       13. A method for treatment of skin diseases accompanied by impaired wound healing comprising contacting a skin disease with the nucleic acid of  claim 1 , the vector of  claim 2 , or the host cell of  claim 3 . 
     
     
       14. The method  claim 10 , additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent. 
     
     
       15. The nucleic acid molecule of  claim 1 , wherein the nucleic acid encodes a propeptide of the serine protease of (i), and the propeptide is encoded by a nucleic acid molecule selected from:
 (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 6; 
 (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 5; 
 (c) a nucleic acid molecule encoding the amino acid sequence of which is at least 90% identical to the amino acid sequence of (a); 
 (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence which is at least 90% identical to the nucleotide sequence of (b); 
 (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or 
 (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U. 
 
     
     
       16. The nucleic acid molecule of  claim 1 , wherein the nucleic acid encodes a pre-propeptide of the serine protease of (i), wherein the pre-propeptide is encoded by a nucleic acid molecule selected from
 (a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2; 
 (b) a nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 1; 
 (c) a nucleic acid molecule encoding the amino acid sequence which is at least 90% identical to the amino acid sequence of (a); 
 (d) a nucleic acid molecule comprising or consisting of a nucleotide sequence at least 90% identical to the nucleotide sequence of (b); 
 (e) a nucleic acid molecule which is degenerate with respect to the nucleic acid molecule of (d); or 
 (f) a nucleic acid molecule corresponding to the nucleic acid molecule of any one of (a) to (d) wherein T is replaced by U. 
 
     
     
       17. The method of  claim 11 , additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent. 
     
     
       18. The method of  claim 12 , additionally comprising administering at least one component selected from the group of a further protease, nuclease, excipient, anti-microbial agent and pain-relieving agent. 
     
     
       19. The isolated nucleic acid molecule of claim 1, wherein the heterologous promoter is a yeast promoter. 
     
     
       20. The isolated nucleic acid molecule of claim 19, wherein the heterologous promoter is a methanol inducible promoter. 
     
     
       21. The isolated nucleic acid molecule of claim 19, wherein the heterologous promoter is a yeast promoter selected from an AOX1 promoter and a GAL1 promoter. 
     
     
       22. The isolated nucleic acid molecule of claim 1, wherein the serine protease is in the form of a propeptide or pre-propeptide. 
     
     
       23. The isolated host cell of claim 3, wherein the isolated host cell is a yeast cell. 
     
     
       24. The isolated host cell of claim 23, wherein the yeast cell is of a species selected from Saccharomyces cerevisiae, Hansenula polymorpha and Pichia sp. 
     
     
       25. The isolated host cell of claim 24, wherein the yeast cell is a Pichia pastoris cell. 
     
     
       26. The serine protease, propeptide, or pre-propeptide of claim 5 which is a propeptide. 
     
     
       27. The serine protease, propeptide, or pre-propeptide of claim 5 which is a propeptide of SEQ ID NO. 6. 
     
     
       28. The propeptide of claim 27, wherein the pharmaceutical composition is suitable for topical administration. 
     
     
       29. The propeptide of claim 27, wherein the pharmaceutical composition is a gel. 
     
     
       30. The propeptide of claim 29 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from cellulose derivatives, vinyl polymers, and carboxypoly-methylene derivatives. 
     
     
       31. The propeptide of claim 30 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from as methyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, polyvinyl alcohols, polyvinyl pyrrolidone, and carbopoline. 
     
     
       32. The propeptide of claim 29 wherein the pharmaceutical composition comprises at least one gel-forming agent selected from pectins, gums, alginates, agar and gelatin. 
     
     
       33. The propeptide of claim 29 wherein the pharmaceutical composition comprises one or more auxiliary agents selected from preservatives, antioxidants, stabilizers, colorants and perfumes. 
     
     
       34. The fusion protein of claim 6, wherein the heterologous sequence is a yeast signal sequence. 
     
     
       35. The fusion protein of claim 34, wherein the pre-propeptide comprises the debrilase propeptide sequence of SEQ ID No. 6 and wherein the native signal peptide of debrilase (“MFRFVALFAFVSCALA”) (SEQ. ID. NO 7) is substituted by the vector encoded-factor signal sequence from Saccharomyces cerevisiae.