US 11,795,488 B2
Methods for enzymatic peptide ligation
Bin Wu, Singapore (SG); Renliang Yang, Singapore (SG); Yee Hwa Wong, Singapore (SG); Julien Lescar, Singapore (SG); Chuan Fa Liu, Singapore (SG); James P Tam, Singapore (SG); Kien Truc Giang Nguyen, Singapore (SG); and Ziqi Long, Singapore (SG)
Assigned to NANYANG TECHNOLOGICAL UNIVERSITY, Singapore (SG)
Appl. No. 16/336,023
Filed by NANYANG TECHNOLOGICAL UNIVERSITY, Singapore (SG)
PCT Filed Sep. 12, 2017, PCT No. PCT/SG2017/050458
§ 371(c)(1), (2) Date Mar. 22, 2019,
PCT Pub. No. WO2018/056899, PCT Pub. Date Mar. 29, 2018.
Claims priority of application No. 10201607951V (SG), filed on Sep. 23, 2016.
Prior Publication US 2019/0218586 A1, Jul. 18, 2019
Int. Cl. C12P 21/02 (2006.01); C12N 9/00 (2006.01); C07K 14/415 (2006.01)
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.