Methods and constructs for high yield expression of clostripain
View Patent ↗The invention provides methods and nucleic acid constructs to express clostripain. The source of the coding region for recombinantly expressed clostripain is Clostridium histolyticum.
1. A method to produce active clostripain comprising:
(a) incubating an Escherichia coli cell containing an expression cassette under conditions that cause the cell to produce clostripain inclusion bodies, wherein the expression cassette comprises a promoter operably linked to an open reading frame that encodes a peptide consisting of a tag linked to clostripain, wherein the tag is selected from the group consisting of SEQ ID NOs:17 and 25, and wherein the clostripain is selected from the group consisting of SEQ ID NO:28, amino acids 28-526 of SEQ NO:28, SEQ ID NO:29, and a variant of SEQ ID NO:29, wherein the variant of SEQ ID NO:29 has at least 98% sequence identity to SEQ II) NO:29;
(b) harvesting the clostripain inclusion bodies from the Escherichia coli cell; and
(c) solubilizing the clostripain inclusion bodies in an 8 M urea solution; and
(d) diluting said urea solution to a final solution to a final urea concentration of less than 3 M to produce active clostripain.
2. The method of claim 1 , wherein the promoter is a regulatable promoter or a constitutive promoter.
3. The method of claim 1 , wherein the promoter is a tac promoter, a T5 promoter, a T7 promoter, a trp promoter, a lac promoter, a lambda phage PL promoter, a heat shock promoter, or a Chlorella Virus promoter.
4. The method of claim 1 , wherein the tag is SEQ ID NO:17.
5. The method of claim 1 , wherein the tag is SEQ ID NO:25.
6. The method of claim 1 , wherein the expression cassette is chromosomally integrated.
7. The method of claim 1 , wherein the promoter is an inducible promoter.
8. The method of claim 1 , wherein the expression cassette further comprises an enhancer.
9. The method of claim 1 , wherein the expression cassette further comprises a signal sequence.
10. The method of claim 9 , wherein the tag is SEQ ID NO: 17 and the clostripain is SEQ ID NO: 29.
11. The method of claim 1 , wherein the diluting step includes addition of an activation buffer comprising Tris-HCl, DTT, and CaCl 2 .
12. The method of claim 11 wherein the activation buffer comprises 50 mM Tris-HCl (pH 7.6), 10mM DTT and 1 mM CaCl 2 .
13. The method of claim 1 , wherein the clostripain is solubilized in 8 M urea and the final urea concentration is 2M.
14. A method for producing an activated clostripain comprising:
(a) producing clostripain inclusion bodies in an Escherichia coli cell containing an expression cassette comprising a promoter operably linked to an open reading frame that encodes a peptide consisting of a tag linked to clostripain, wherein the tag is selected from the group consisting of SEQ ID NOs:17 and 25, and wherein the clostripain is selected from the group consisting of SEQ ID NO:28, amino acids 28-526 of SEQ ID NO:28, SEQ ID NO:29, and a variant of SEQ ID NO:29, wherein the variant of SEQ ID NO:29 has at least 98% sequence identity to SEQ ID NO:29;
(b) harvesting the clostripain inclusion bodies from a fermentation mixture comprising the Escherichia coli cell;
(c) solubilizing the clostripain inclusion bodies in a solution comprising 8M urea; and
(d) activating the solubilized clostripain by diluting the solution to a final urea concentration of less than 3 M in a metal ion containing buffer.
15. The method of claim 14 , wherein the buffer includes CaCl 2 .
16. The method of claim 14 , wherein upon solubilization of the clostripain inclusion bodies, the solution is centrifuged and the solubilized clostripain is thereafter recovered in the centrifugation supernatant.
17. The method of claim 14 , wherein the tag is SEQ ID NO: 17.
18. The method of claim 17 , wherein the clostripain is SEQ ID NO: 29.