Claims
- 1. A method for synthesizing a peptide, comprising:
(a) ligating a protected amino acid carboxyl component to an amino acid nucleophilic component with a peptide-coupling enzyme active at a first temperature to produce a first peptide; (b) deprotecting the first peptide with a deprotection enzyme active at a second temperature; and (c) ligating a protected amino acid carboxyl component to the deprotected first peptide with the peptide-coupling enzyme active at the first temperature to produce a second peptide.
- 2. The method of claim 1, wherein steps (b) and (c) are repeated to obtain a peptide of a desired length.
- 3. The method of claim 1, wherein the protected amino acid carboxyl component is a N-acyl protected amino acid.
- 4. The method of claim 3, wherein the acyl is selected from the group consisting of acetyl, formyl, benzoyl, and carbamoyl.
- 5. The method of claim 1, wherein the protected amino acid carboxyl component consists of the formula,
- 6. The method of claim 5, wherein the R′ group of the protected amno acid carboxyl component is selected from the group consisting of hydrogen and alkyl.
- 7. The method of claim 1, wherein the amino acid nucleophilic component is a C-terminal protected amino acid.
- 8. The method of claim 1, wherein the amino acid nucleophilic component consists of the formula,
- 9. The method of claim 8, wherein the R″ group of the amino acid nucleophilic component is t-butyl.
- 10. The method of claim 1, wherein the peptide-coupling enzyme does not have protease activity.
- 11. The method of claim 1, wherein the peptide-coupling enzyme is selected from the group consisting of a peptidase, a ligase, a lipase, a protease, a carboxypeptidase, and an amidase.
- 12. The method of claim 1, wherein the deprotection enzyme is selected from the group consisting of a deacetylase, an aminoacylase, an amidase, a carbamate and a carboxypeptidase.
- 13. The method of claim 2, wherein the desired length is from about two to about forty amino acid residues in length.
- 14. The method of claim 13, wherein the desired length is from about three to about thirty amino acid residues in length.
- 15. The method of claim 1, wherein the method is performed in a single reaction vessel.
- 16. The method of claim 1, wherein the peptide-coupling enzyme and the deprotection enzyme are not active simultaneously.
- 17. The method of claim 16, wherein the first temperature is higher than the second temperature.
- 18. The method of claim 16, wherein the first temperature is lower than the second temperature.
- 19. The method of claim 16, wherein the peptide-coupling enzyme is not irreversibly inactivated at the second temperature.
- 20. The method of claim 16, wherein the deprotection enzyme is not-irreversibly inactivated at the first temperature.
- 21. The method of claim 1, wherein the peptide-coupling enzyme is derived from a thermophilic organism.
- 22. The method of claim 1, wherein the deprotection enzyme is derived from a thermophilic organism.
- 23. The method of claim 1, wherein the deprotection enzyme is inactive at the first temperature.
- 24. The method of claim 1, wherein the peptide-coupling enzyme is inactive at the second temperature.
- 25. A method for identifying a deprotection enzyme comprising culturing a plurality of host cells on a substrate comprising an acetylated peptide as a sole carbon source, wherein each host cell of the plurality comprises a nucleic acid molecule suspected of encoding a deprotection enzyme, and wherein a host cell that grows on the substrate is identified as a host cell that expresses a deprotection enzyme.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional application serial no. 60/292,852, filed May 23, 2001, the entire contents of which are hereby incorporated by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60292852 |
May 2001 |
US |