	US 12,466,861 B2
	Polypeptide with enhanced rate of spontaneous isopeptide bond formation with its peptide tag partner and uses thereof
Mark Howarth, Oxford (GB); and Anthony Keeble, Oxford (GB)
Assigned to OXFORD UNIVERSITY INNOVATION LIMITED, Oxford (GB)
Appl. No. 17/435,742
Filed by Oxford University Innovation Limited, Oxford (GB)
PCT Filed Mar. 13, 2020, PCT No. PCT/GB2020/050652 § 371(c)(1), (2) Date Sep. 2, 2021, PCT Pub. No. WO2020/183198, PCT Pub. Date Sep. 17, 2020.
Claims priority of application No. 1903479 (GB), filed on Mar. 14, 2019.
Prior Publication US 2022/0135628 A1, May 5, 2022
Int. Cl. C07K 14/315 (2006.01); C12N 15/70 (2006.01)

CPC C07K 14/315 (2013.01) [C12N 15/70 (2013.01)]

20 Claims

1. A polypeptide comprising:

i) an amino acid sequence as set forth in SEQ ID NO: 1, which comprises a lysine residue at position 31; or

ii) an amino acid sequence as set forth in SEQ ID NO: 2, which comprises a lysine residue at position 10; or

iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 1, wherein said amino acid sequence comprises a lysine at position 31, a glutamic acid at position 77 and one or more of the following specified amino acid residues:

1) A glutamic acid at position 91;

2) An aspartic acid at position 103; and/or

3) A glutamic acid at position 108;

wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 1; or

iv) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56 and one or more of the following specified amino acid residues:

1) A glutamic acid at position 70;

2) An aspartic acid at position 82; and/or

3) A glutamic acid at position 87;

wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 2,

and wherein said polypeptide is capable of spontaneously forming an isopeptide bond with a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, which comprises an aspartic acid residue at position 10, wherein said isopeptide bond forms between the aspartic acid residue at position 10 of SEQ ID NO: 3 and the lysine residue at position 31 of SEQ ID NO: 1 or position 10 of SEQ ID NO: 2 or at a position equivalent to position 31 in SEQ ID NO: 1 or position 10 of SEQ ID NO: 2.