Method of splitting polypeptides using protease ompt version

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly genetic engineering and can be used in biomedical industry. The invention discloses a method of obtaining a target peptide by splitting a polypeptide or a fused protein using a version of protease OmpT with substitution of the amino acid in position 97, where the amino acid is selected from alanine, leucine, phenylalanine, methionine, serine, threonine, cysteine, asparagine, glutamine, glutaminic acid and histidine, on a site whose structure is characterised by general formula Pn…P2-P1-P1'-P2'…Pn', where P1 is asparagine or lysine, P2 and P2' are an amino acid which is not glutamic or asparagic acid, and P1' is alanine, valine, isoleucine, phenylalanine, methionine, serine, threonine, cysteine, tyrosine or asparagine.

EFFECT: use of the invention can enable efficient and specific isolation of therapeutic peptides with any type of N-terminal sequence.

22 cl, 3 tbl, 20 dwg, 18 ex

 

The text descriptions are given in facsimile form.

1. The method of cleavage of the polypeptide, characterized in that the splitting is done according to the website, having the structure defined by the General formula Pn...P2-P1-P1'-P2'...Pn', where P1 is arginine or lysine, P2 and P2' amino acid, not a glutamic or aspartic acid, and P1', alanine, valine, isoleucine, phenylalanine, methionine, serine, threonine, cysteine, tyrosine or asparagine, by processing the specified polypeptide variant protease OmpT, in which aminosilica in position 97 replaced by an amino acid selected from the group including alanine, leucine, phenylalanine, methionine, serine, threonine, cysteine, asparagine, glutamine, glutamic acid and histidine.

2. The method of cleavage of the polypeptide according to claim 1, in which one basic amino acid or two or three consecutive basic amino acids located in any site amino acid sequence from position 10 to position P3 or from position P3 to position P5'.

3. A method of obtaining a target peptide, characterizes the same time, that transform cells of host-expression by plasmid having a gene that encodes a protein comprising the target peptide fused with a protective peptide through the desired cleavage site, which may be splitting variant protease OmpT on the 97th aminoxide, as defined in paragraph 1, Express named gene in these cells and get the target peptide of the fused protein by cleavage called protease in the specified site of cleavage.

4. The method of obtaining the target peptide according to claim 3, in which the end of the protective peptide is an arginine or lysine, and N-terminal target peptide is an amino acid other than arginine or lysine.

5. The method of obtaining the target peptide according to claim 4, where one basic amino acid or two or three consecutive basic amino acids located in any site amino acid sequence from position 10 to position P3 or from position P3 to position P5' of the desired cleavage site fused protein.

6. The method according to any one of claims 1 to 5, which in the case of the polypeptide or fused protein of the site on which it is undesirable to carry out the splitting of the variant protease OmpT on the 97th amino acid cleavage at a specific site inhibit, having an acidic amino acid at position P3 to the specified site.

7. The method according to any of claim 2 and 5, which includes aspolozhena two or three consecutive basic amino acids between positions P10 and P3 of the desired cleavage site in the polypeptide or fused protein.

8. The method according to claim 7, which includes the location of three consecutive basic amino acids between positions P5 and P3 of the desired cleavage site in the polypeptide or fused protein.

9. The method according to any of claim 2 and 5, in which the basic amino acids are arginine and/or lysine.

10. The method according to claim 9, where the basic amino acid is arginine.

11. The method according to claim 6, where the acidic amino acid is aspartic acid.

12. The method according to any of claim 2 and 5, where the amino acid sequence from position P5 to position P1 of the desired cleavage site in the polypeptide or fused protein is Arg-Arg-Arg-Ala-Arg.

13. The method according to any of claim 2 and 5, where the amino acid sequence from position P7 to the position P1 of the desired cleavage site in the polypeptide or fused protein is Asp-Ala-Arg-Arg-Arg-Ala-Arg.

14. The method according to any one of claims 1 to 5, where the 97th amino acid from the N-Terminus of the protease OmpT are leucine, methionine or histidine.

15. The method according to any one of claims 1 to 5, where the position P1' of the desired cleavage site of the polypeptide or fused protein or N-terminal target peptide represented by serine or alanine, and 97th amino acid used variant protease OmpT on the 97th amino acid is leucine.

16. The method according to any one of claims 1 to 5, where the position P1' of the desired cleavage site of the polypeptide or fused protein or N-terminal target peptide is redstavleny phenylalanine, by alanine, serine, cysteine or tyrosine, and 97th amino acid used variant protease OmpT on the 97th amino acid is methionine.

17. The method according to any one of claims 1 to 5, where the position P1' of the desired cleavage site of the polypeptide or fused protein or N-terminal target peptide represented by alanine, valine, isoleucine, methionine, serine, threonine, cysteine, or asparagine, and 97th amino acid used variant protease OmpT on the 97th amino acid is histidine.

18. The method according to claim 3, where the target peptide is a peptide consisting of amino acid residues 22-45.

19. The method according to p, where the target peptide is adrenocorticotropic hormone (1-24), motilium or a precursor of calcitonin.

20. The method according to claim 3, where the cells of the host cells are E. coli.

21. The method according to claim 3, which includes use as a digestive proteases of bacterial cells expressing the gene encoding a variant protease OmpT on the 97th amino acid, where the 97th amino acid from the N-Terminus of the protease OmpT is alanine, leucine, phenylalanine, methionine, serine, threonine, cysteine, asparagine, glutamine, glutamic acid or histidine.

22. The method according to claim 20, characterized in that the variant protease OmpT is the product of expression of the corresponding gene, included in the second expression plasmids, and the selection of the target PE the Chida is achieved by dissolving formed in the cell Taurus inclusion.



 

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