CPC C12P 21/02 (2013.01) [C07K 14/415 (2013.01); C12N 9/93 (2013.01)] | 12 Claims |
1. A method of forming a peptide of Formula (1)
P1-Asx-Xaa1-Xaa2-P2 (I),
wherein the method comprises:
ligating a first peptide of Formula (II
P1-Asx-Xaa3-Leu-COOH/CONH2 (II)
to a second peptide of Formula (III)
H2N-Xaa1-Xaa2-P2 (III),
wherein P1 and P2 are each independently any peptide, modified or unmodified; wherein Asx is Asp or Asn; wherein Xaa1 is any naturally occurring amino acid; wherein Xaa2 is any naturally occurring amino acid with the exception of Pro; and wherein Xaa3 is any naturally occurring amino acid;
enzymatically cleaving the bond between “Asx” and “Xaa3” in the first peptide of Formula (I); and
ligating the fragment P1-Asx of the first peptide via its C-terminus to the N-terminus of the second peptide of Formula (III) to form a ligated peptide of Formula (1), wherein the enzymatic cleavage between “Asx” and “Xaa3” and the ligation reaction of the fragment P1-Asx are catalyzed by a peptide ligase having the activity of Oldenlandia affinis asparaginyl endopeptidase 1 (OaAEP1) Cys247Ala (SEQ ID NO:1) under conditions suitable for said cleavage and ligation reaction, wherein the peptide ligase having the activity of OaAEP1 Cys247Ala (SEQ ID NO:1) comprises or consists of:
(a) the amino acid sequence set forth in SEQ ID NO:1;
(b) an amino acid sequence that shares at least 80% sequence identity with, the amino acid sequence as set forth in SEQ ID NO:1, provided that said peptide ligase comprises the amino acid sequence set forth in SEQ ID NO:2 at the positions corresponding to residues 247-264 of SEQ ID NO:1 and the amino acid at position 247 is alanine:
(c) a functional fragment of (a) or (b) provided that said functional fragment comprises the amino acid sequence set forth in SEQ ID NO: 2 at the positions corresponding to residues 247-264 of SEQ ID NO:1 and the amino acid at position 247 is alanine; or
(d) a polypeptide sequence containing the functional fragment of (c), wherein said peptide ligase is not the wild-type OaAEP1 having the amino acid sequence set forth in SEQ ID NO:3 or butelase 1 having the amino acid sequence set forth in SEQ ID NO: 4.
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