Method for enzymatic production of GLP-2(1-33) and GLP-2(1-34) peptides
View Patent ↗The invention provides methods for making peptides from a polypeptide containing at least one copy of the peptide using clostripain to excise the peptide from the polypeptide. The methods enable the use of a single, highly efficient enzymatic cleavage to produce any desired peptide sequence.
1. A method for producing a GLP-2(1-34) peptide comprising the peptide sequence of SEQ ID NO: 9, the method comprising the steps of
(a) obtaining a polypeptide of the Formula VI:
Tag-Linker-[GLP-2(1-34)] q Formula VI
wherein,
Tag is a translation initiation sequence having SEQ ID NO:17 or 18;
Linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
GLP-2(1-34) has SEQ ID NO:9; and
q is an integer of about 2 to about 20; and
(b) combining the polypeptide of Formula VI and clostripain.
2. A method for producing a GLP-2(1-34)NH 2 peptide comprising the peptide sequence of SEQ ID NO:10, the method comprising the steps of:
(a) obtaining a polypeptide of the Formula VII:
Tag-Linker-[GLP-2(1-34)-Linker 2 ] q VII
wherein:
Tag is an amino acid sequence comprising SEQ ID NO:17 or SEQ ID NO: 18;
Linker is a cleavable peptide linker, wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
Linker 2 is SEQ ID NO:23;
GLP-2(1-34) has the peptide sequence of SEQ ID NO:9;
q is an integer of about 2 to about 20;
(b) combining the polypeptide of Formula VII and clostripain in the presence of ammonia.
3. A method for producing a GLP-2(1-34)A2G-NH 2 peptide comprising the peptide sequence of SEQ ID NO:16, the method comprising:
(a) obtaining a polypeptide of the Formula VIII:
Tag-Linker-[GLP-2(1-34)A2G-Linker 2 ] q VIII
wherein:
Tag is an amino acid sequence comprising SEQ ID NO:17 or 18;
Linker is a cleavable peptide linker, wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
Linker 2 is SEQ ID NO:23;
GLP-2(1-34,A2G) has the peptide sequence of SEQ ID NO:15;
q is an integer of about 2 to about 20;
(b) combining the polypeptide of Formula VIII and clostripain in the presence of ammonia.
4. A method for cleaving a peptide bond of a polypeptide, comprising:
combining the polypeptide with clostripain;
wherein the polypeptide contains a GLP-2(1-33) amino acid sequence containing within it at least a fragment having an amino acid sequence of Formula I
-Xaa 1 -Xaa 2 -Xaa 3 - (I);
Xaa 1 is a residue of aspartic acid, glycine, proline or glutamic acid;
Xaa 2 is an arginine residue coupled to the N-terminus of the GLP-2(1-33) sequence;
Xaa 3 is histidine; and
wherein clostripain cleaves the peptide bond between amino acids Xaa 2 and Xaa 3 .
5. A method for producing a GLP-2(1-33) peptide comprising the peptide sequence of SEQ ID NO: 11, comprising the steps of
(a) obtaining a polypeptide of the Formula VI:
Tag-Linker-GLP-2(1-33) (VI)
wherein,
Tag is a translation initiation sequence having SEQ ID NO:17 or 18;
Linker is a cleavable peptide linker, wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36; GLP-2(1-33) has the peptide sequence of SEQ ID NO:11;
(b) combining the polypeptide of Formula VI and clostripain.
6. A method for producing a GLP-2(1-33)(A2G) peptide comprising the peptide sequence of SEQ ID NO:13, comprising:
(a) obtaining a polypeptide of the Formula VIII:
Tag-Linker-GLP-2(1-33)(A2G) (VIII)
wherein:
Tag is a translation initiation sequence comprising
SEQ ID NO:17 or 18;
Linker is a cleavable peptide linker, wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
GLP-2(1-33)(A2G) has SEQ ID NO:13;
(b) combining the polypeptide of Formula VIII and clostripain.
7. The method of any one of claims 1 - 4 , 5 and 6 , wherein the polypeptide is a soluble polypeptide.
8. The method of any one of claims 1 - 4 , 5 and 6 , wherein the cleavage is performed at about 18° C. to about 25° C.
9. The method of any one of claims 1 - 4 , 5 and 6 , wherein the cleavage is performed between a pH of about 5 to about 11.
10. The method of any one of claims 1 - 4 , 5 and 6 , wherein the concentration of clostripain is about 0.01 to about 3.0 units of clostripain per about 2 to about 5 mg polypeptide.
11. The method of any one of claims 1 - 4 , 5 and 6 , wherein the cleavage is performed in the presence of about 0.5 mM to about 10 mM CaCl 2 .
12. The method of any one of claims 1 or 5 wherein Tag has SEQ ID NO:17.
13. The method of claim 1 wherein q is 6.
14. The method of any one of claims 1 or 5 wherein the polypeptide of the Formula VI has SEQ ID NO:29 or 30.
15. The method of any one of claims 1 or 5 wherein the combining is performed between a pH of about 6.0 to about 6.9.
16. The method of any one of claims 1 or 5 wherein the combining is performed at about 40° C. to about 50° C.
17. The method of any one of claims 1 or 5 wherein the combining is performed between a pH of about 8.5 to about 9.7.
18. The method of any one of claims 1 or 5 wherein the concentration of clostripain is about 10 to about 30 units clostripain per about 1 mg polypeptide.
19. The method of any one of claims 1 or 5 wherein the concentration of polypeptide is about 1.5 to about 15 mg/mL.
20. A method of producing a GLP-2 peptide from a polypeptide comprising:
(a) obtaining bacterial inclusion bodies containing the polypeptide
(b) solubilizing polypeptide within the bacterial inclusion bodies using urea;
(c) combining the polypeptide and clostripain in the optional presence of up to about 8 M urea;
wherein the polypeptide contains a site of Formula I:
Xaa 1 -Xaa 2 -Xaa 3 (I)
Xaa 1 is aspartic acid, glycine proline or glutamic acid;
Xaa 2 is arginine; and
Xaa 3 is not an acidic amino acid; and
wherein the GLP-2 peptide is at least one of 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 and SEQ ID NO: 16.
21. A method for producing a GLP-2(1-34) peptide from a polypeptide comprising:
(a) obtaining bacterial inclusion bodies containing the polypeptide comprising Formula II
(Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 ) n -Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 (II)
wherein Xaa 3 -Peptide 1 -Xaa 1 -Xaa 2 is a GLP-2(1-34) peptide selected from SEQ ID NO: 9 or SEQ ID NO: 15;
n is an integer ranging from 0 to 50;
Xaa 1 is aspartic acid, glycine or glutamic acid;
Xaa 2 is arginine; and
Xaa 3 is a histidine;
(b) solubilizing the polypeptide within the bacterial inclusion bodies using urea;
(c) combining the polypeptide and clostripain in the optional presence of up to about 8 M urea.
22. A method for producing a GLP-2(1-34) peptide from a polypeptide, which comprises:
(a) obtaining bacterial inclusion bodies containing the polypeptide comprising Formula III
(Linker-Xaa 3 -Peptide 1 ) n -Linker-Xaa 3 -Peptide 1 (III)
wherein
n is an integer ranging from 0 to 50;
Xaa 3 -Peptide 1 is a GLP-2(1-34) peptide selected from SEQ ID NO: 9 or SEQ ID NO: 15:
Xaa 3 is histidine;
Linker is a cleavable peptide linker, wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36;
n is an integer ranging from 0 to 50;
(b) solubilizing the polypeptide within the bacterial inclusion bodies using urea;
(c) combining the polypeptide and clostripain in the optional presence of up to about 8 M urea.
23. A method for producing a GLP-2(1-33) peptide from a polypeptide comprising:
(a) obtaining bacterial inclusion bodies containing the polypeptide comprising Formula II
(Tag-IBFP-Xaa 1 -Xaa 2 ) n -Xaa 3 -Peptide 1 (II)
wherein
Xaa 3 -Peptide 1 is a GLP-2(1-33) peptide selected from SEQ ID NO: 11 or SEQ ID NO: 13;
Tag is a translation initiation sequence comprising SEQ ID NO:17 or 18;
IBFP is an inclusion body leader partner comprising any one of SEQ ID NO:19, 20, 21 or 22;
n is an integer ranging from 0 to 50;
Xaa 1 is aspartic acid, glycine, proline or glutamic acid;
Xaa 2 is arginine; and
Xaa 3 is histidine;
(b) solubilizing the polypeptide within the bacterial inclusion bodies using urea;
(c) combining the polypeptide and clostripain in the optional presence of up to about 8 M urea.
24. A method for producing a GLP-2(1-33) peptide from a polypeptide using clostripain, which comprises:
(a) obtaining bacterial inclusion bodies containing the polypeptide comprising Formula III
(Linker-Xaa 3 -Peptide 1 ) n -Linker-Xaa 3 -Peptide 1 (III)
wherein:
n is an integer ranging from 0 to 50;
Xaa 3 -Peptide 1 is SEQ ID NO: 11 or SEQ ID NO: 13;
Xaa 3 is histidine;
Linker is a cleavable peptide linker;
n is an integer ranging from 0 to 50;
wherein the linker is at least one of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, and wherein the linker comprises an inclusion body leader partner comprising any one of SEQ ID No: 19, 20, 21 or 22;
(b) solubilizing the polypeptide within the bacterial inclusion bodies using urea;
(c) combining the polypeptide and clostripain in the optional presence up to about 8 M urea.
25. The method of any one of claims 20 - 24 wherein the combining step is performed at about 40° C. to about 50° C.
26. The method of any one of claims 20 - 24 wherein the combining step is performed between a pH of about 8.5 to about 9.7.
27. The method of any one of claims 20 - 24 wherein the concentration of clostripain is about 10 to about 30 units clostripain per about 1 mg polypeptide.
28. The method of any one of claims 20 - 24 wherein the concentration of polypeptide is about 1.5 to about 15 mg/mL.
29. The method of any one of claims 20 - 24 wherein the combining step is performed in the presence of 0.5 mM to about 10 mM CaCl 2 .
30. The method of any one of claims 20 - 24 wherein the combining step is performed in the presence of about 0.5 to about 3.0 mM cysteine.
31. The method of any one of claims 20 - 24 wherein the combining step is performed in the presence of glycine thereby generating a peptide that has a C-terminal glycine.
32. The method of any one of claims 20 - 24 wherein the combining step is performed in the presence of Gly-Leu, to generate a peptide with Gly-Leu at the C-terminal end.
33. The method of any one of claims 20 - 24 wherein the combining step is performed in the presence of ammonia to generate a peptide with a C-terminal amide.
34. The method of claim 3 wherein the ammonia is present at about 1 M to about 5 M.
35. The method of any one of claims 1 - 3 , 5 , 6 and 20 - 24 wherein the peptide is continuously removed from the combining step.
36. The method of claim 35 wherein the peptide is continuously removed by performing the combining step in a chamber having a filtration membrane, wherein the membrane allows the peptide to pass through but does not permit the polypeptide or the clostripain to pass through.
37. A method of any one of claims 1 - 4 , 5 , 6 and 20 - 24 , wherein the polypeptide is obtained by recombinant production.
38. The method of any one of claims 1 - 4 , 5 , 6 and 20 - 24 wherein the combining is performed in the presence of about 0.5 to about 3.0 mM cysteine.