IP Library › Granted Patent US 12,466,861

Granted Patent B2

US 12,466,861 · App. 17/435,742 · Granted Nov 11, 2025

Polypeptide with enhanced rate of spontaneous isopeptide bond formation with its peptide tag partner and uses thereof

Inventors: Mark Howarth (Oxford, GB); Anthony Keeble (Oxford, GB)

Assignee: OXFORD UNIVERSITY INNOVATION LIMITED

C07K14/315 C12N15/70

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Quick Facts

Patent No.: US 12,466,861
App. No.: 17/435,742
Granted: Nov 11, 2025
Kind: B2

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USPTO Patent Center↗ Full Patent PDF↗

Abstract

The present invention relates to a polypeptide that forms one part of a two-part linker in which the polypeptide spontaneously forms an isopeptide bond with a peptide tag, the second part of the two-part linker. Nucleic acid molecules encoding the polypeptide, vectors comprising said nucleic acid molecules, and host cells comprising said vectors and nucleic acid molecules are also provided. A kit comprising said two-part linker (i.e. peptide tag and polypeptide binding partner), and/or nucleic acid molecules/vectors is also provided. A method of producing the polypeptide (binding partner) and the uses of the polypeptide of the invention are also provided.

Claims (134)

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.

2 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 all of the following specified amino acid residues:

1) A glutamic acid at position 91;

2) An aspartic acid at position 103; and

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.

3 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 all of the following specified amino acid residues:

1) A proline at position 89;

2) A glutamic acid at position 91;

3) An aspartic acid at position 97;

4) An aspartic acid at position 103; and

5) A glutamic acid at position 108;

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

4 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 threonine at position 2;

2) A proline at position 13;

3) An arginine at position 37;

4) A histidine at position 62;

5) A glutamic acid at position 105; and/or

6) A threonine at position 113,

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

5 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 all of the following specified amino acid residues:

1) A threonine at position 2;

2) A proline at position 13;

3) An arginine at position 37;

4) A histidine at position 62;

5) A proline at position 89;

6) An aspartic acid at position 97;

7) A glutamic acid at position 105; and

8) A threonine at position 113,

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

6 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 all of the following specified amino acid residues:

1) A threonine at position 2;

2) A proline at position 13;

3) An arginine at position 37;

4) A histidine at position 62;

5) A proline at position 89;

6) An aspartic acid at position 97;

7) A glutamic acid at position 105; and

8) A threonine at position 113,

and two or more of the following specified amino acid residues:

9) a glutamic acid at position 91;

10) An aspartic acid at position 103; and

11) A glutamic acid at position 108,

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

7 . The polypeptide of claim 1 , wherein the polypeptide comprises 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 all of the following specified amino acid residues:

1) A threonine at position 2;

2) A proline at position 13;

3) An arginine at position 37;

4) A histidine at position 62;

5) A proline at position 89;

6) An aspartic acid at position 97;

7) A glutamic acid at position 105;

8) A threonine at position 113,

9) a glutamic acid at position 91;

10) An aspartic acid at position 103; and

11) A glutamic acid at position 108,

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

8 . The polypeptide of claim 1 , wherein the polypeptide comprises:

(A) one or both of the following specified amino acid residues:

1) A glycine at position 9; and

2) a threonine at position 19; and/or

(B) one or more of the following specified amino acid residues:

1) A glutamic acid at position 34;

2) A serine at position 50;

3) A tyrosine at position 69;

4) A glycine at position 83; and/or

5) a valine at position 86;

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

9 . The polypeptide of claim 1 , wherein the polypeptide comprises one or more of the following specified amino acid substitutions:

1) Substitution of a lysine with a glutamine at position 52;

2) Substitution of a valine with an aspartic acid at position 63;

3) Substitution of a tyrosine with a serine at position 69;

4) Substitution of a threonine with a leucine at position 88; and/or

5) Substitution of a glutamic acid with a proline at position 96;

wherein the specified amino acid substitutions are at positions equivalent to the positions in SEQ ID NO: 1.

10 . The polypeptide of claim 1 , wherein the polypeptide is conjugated to a nucleic acid molecule, protein, peptide, small-molecule organic compound, fluorophore, metal-ligand complex, polysaccharide, nanoparticle, 2D monolayer, nanotube, polymer, cell, virus, virus-like particle or a combination thereof.

11 . The polypeptide of claim 1 , wherein the polypeptide is immobilized on a solid substrate.

12 . A recombinant or synthetic polypeptide comprising a peptide or polypeptide linked to the polypeptide as defined in claim 1 .

13 . A nucleic acid molecule comprising a nucleotide sequence which encodes:

i) the polypeptide as defined in claim 1 ; or

ii) a recombinant or synthetic polypeptide comprising a peptide or polypeptide linked to the polypeptide as defined in claim 1 .

14 . A vector comprising the nucleic acid molecule of claim 13 .

15 . A cell comprising a nucleic acid comprising a nucleotide sequence which encodes the polypeptide as defined in claim 1 or a vector comprising said nucleic acid molecule.

16 . A process for producing or expressing the polypeptide of claim 1 or a recombinant polypeptide comprising a peptide or polypeptide linked to the polypeptide of claim 1 , the process comprising the steps of:

a) transforming or transfecting a host cell with a vector comprising a nucleic acid molecule comprising a nucleotide sequence which encodes (i) the polypeptide as defined in claim 1 or (ii) a recombinant polypeptide comprising a peptide or polypeptide linked to the polypeptide as defined in claim 1 ;

b) culturing the host cell under conditions which allow the expression of the polypeptide; and optionally

c) isolating the polypeptide.

17 . A process for conjugating two molecules or components via an isopeptide bond comprising:

a) providing a first molecule or component comprising the polypeptide of claim 1 ;

b) providing a second molecule or component comprising a peptide selected from:

(1) a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 5, which comprises an aspartic acid residue at position 7; and

(2) a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 6, which comprises an aspartic acid residue at position 10, wherein:

(i) Xaa at position 1 is an arginine or no amino acid;

(ii) Xaa at position 2 is a glycine or no amino acid;

(iii) Xaa at position 5 is a histidine or a threonine;

(iv) Xaa at position 11 is an alanine, a glycine or a valine; and

(v) Xaa at position 14 is an arginine or a lysine,

wherein when Xaa at position 1 is no amino acid, Xaa at position 2 is no amino acid,

and wherein said peptide is capable of spontaneously forming an isopeptide bond with a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, which comprises a lysine residue at position 31 or a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 which comprises a lysine residue at position 10, wherein said isopeptide bond forms between the aspartic acid residue at position 7 of SEQ ID NO: 5 or at position 10 of SEQ ID NO: 6 and the lysine residue at position 31 of SEQ ID NO: 1 or position 10 of SEQ ID NO: 2;

c) contacting said first and second molecules or components under conditions that enable the spontaneous formation of an isopeptide bond between the polypeptide and peptide, thereby conjugating said first molecule or component to said second molecule or component via an isopeptide bond to form a complex.

18 . A kit comprising:

(a) the polypeptide of claim 1 , optionally conjugated or fused to a molecule or component; and

(b) a peptide selected from:

(1) a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 5, which comprises an aspartic acid residue at position 7; and

(2) a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 6, which comprises an aspartic acid residue at position 10, wherein:

(i) Xaa at position 1 is an arginine or no amino acid;

(ii) Xaa at position 2 is a glycine or no amino acid;

(iii) Xaa at position 5 is a histidine or a threonine;

(iv) Xaa at position 11 is an alanine, a glycine or a valine; and

(v) Xaa at position 14 is an arginine or a lysine,

wherein when Xaa at position 1 is no amino acid, Xaa at position 2 is no amino acid,

and wherein said peptide is capable of spontaneously forming an isopeptide bond with a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 1, which comprises a lysine residue at position 31, or a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2 which comprises a lysine residue at position 10 wherein said isopeptide bond forms between the aspartic acid residue at position 7 of SEQ ID NO: 5 or at position 10 of SEQ ID NO: 6 and the lysine residue at position 31 of SEQ ID NO: 1 or position 10 of SEQ ID NO: 2, optionally conjugated or fused to a molecule or component; and/or

(d) a nucleic acid molecule encoding the peptide as defined in (b).

19 . The kit of claim 18 , wherein the peptide of (2) comprises one or more of the following specified amino acid residues:

1) A histidine at position 5;

2) An alanine at position 11; and/or

3) An arginine at position 14,

wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 6.

20 . The kit of claim 18 , wherein the peptide of (2) comprises an amino acid sequence as set forth in SEQ ID NO: 3 or 4.

Assignments (1)

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 11, 2021

From: HOWARTH, MARK; KEEBLE, ANTHONY

To: OXFORD UNIVERSITY INNOVATION LIMITED

Reel/Frame 057748/0216 →

Priority Claims (1)

GB 1903479 · Mar 14, 2019 · national

Continuity (1)

Related Publication 20220135628A1 · May 5, 2022

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