C-peptides and proinsulin polypeptides comprising the same
A connecting polypeptide has SEQ ID NO:73. A proinsulin polypeptide includes a mature insulin A-chain, a mature insulin B-chain, and a connecting peptide comprising SEQ ID NO: 73 linking the mature A-chain and the mature B-chain, wherein the connecting peptide is not a native human proinsulin C-peptide. The proinsulin polypeptides according to the invention can be made in high titers and in high purity.
1. A connecting polypeptide comprising SEQ ID NO:73.
2. A proinsulin polypeptide comprising:
a mature insulin A-chain;
a mature insulin B-chain; and
a connecting peptide comprising SEQ ID NO:73 linking the mature A-chain and the mature B-chain, wherein the connecting peptide is not a native human proinsulin C-peptide.
3. The proinsulin polypeptide of claim 2 , wherein the mature insulin A-chain is selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:6.
4. The proinsulin polypeptide of claim 3 , wherein the mature insulin A-chain comprises SEQ ID NO:6.
5. The proinsulin polypeptide of claim 2 , wherein the mature insulin B-chain is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
6. The proinsulin polypeptide of claim 5 , wherein the mature insulin B-chain comprises SEQ ID NO:7.
7. The proinsulin polypeptide of claim 2 , wherein the proinsulin polypeptide lacks a signal peptide.
8. The proinsulin polypeptide of claim 2 , further comprising a carboxypeptidase B propeptide coupled to the N-terminal residue of the mature insulin B-chain, the carboxypeptidase B propeptide selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
9. The proinsulin polypeptide of claim 8 , wherein the carboxypeptidase B propeptide comprises SEQ ID NO:36.
10. The proinsulin polypeptide of claim 2 , further comprising a propeptide coupled to the N-terminal residue of the mature insulin B-chain, wherein the propeptide is SEQ ID NO:37.
11. An expression construct comprising a polynucleotide sequence encoding the insulin polypeptide of claim 2 .
12. A host cell comprising the expression construct of claim 11 .
13. A proinsulin polypeptide comprising:
a mature insulin A-chain comprising SEQ ID NO:6;
a mature insulin B-chain comprising SEQ ID NO:7; and
a connecting peptide comprising SEQ ID NO: 73 linking the mature A-chain and the mature B-chain, wherein the connecting peptide is not a native human proinsulin C-peptide.
14. The proinsulin polypeptide of claim 13 , wherein the proinsulin polypeptide lacks a signal peptide.
15. The proinsulin polypeptide of claim 13 , further comprising a carboxypeptidase B propeptide coupled to the N-terminal residue of the mature insulin B-chain, the carboxypeptidase B propeptide selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36.
16. The proinsulin polypeptide of claim 15 , wherein the carboxypeptidase B propeptide comprises SEQ ID NO:36.
17. An expression construct comprising a polynucleotide sequence encoding the insulin polypeptide of claim 13 .
18. A host cell comprising the expression construct of claim 17 .
19. A proinsulin polypeptide comprising:
a mature insulin A-chain comprising SEQ ID NO:6;
a mature insulin B-chain comprising SEQ ID NO:7;
a connecting peptide comprising SEQ ID NO:73 linking the mature A-chain and the mature B-chain, wherein the connecting peptide is not a native human proinsulin C-peptide; and
a carboxypeptidase B propeptide coupled to the N-terminal residue of the mature insulin B-chain, the carboxypeptidase B propeptide comprising SEQ ID NO:36.
20. The proinsulin polypeptide of claim 19 , wherein the proinsulin polypeptide lacks a signal peptide.