Oxford University Innovation Ltd

Isis Innovation Limited

Massachusetts Institute of Technology

Mark Howarth

Moungi G Bawendi

Wenhao Liu

Anthony Keeble

Irsyad Khairil Anuar

Can M Buldun

Alice Ting

The present invention relates to an affinity purification system that utilises a polypeptide comprising: (i) an amino acid sequence as set forth in SEQ ID NO: 1, wherein X at position 79 is selected from alanine, glycine, serine, asparagine, or threonine; (ii) a portion of (i) comprising an amino acid sequence as set forth in SEQ ID NO: 2, wherein X at position 56 is selected from alanine, glycine, serine, asparagine or threonine; (iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises alanine, glycine, serine, asparagine or threonine at a position equivalent to position 79 of SEQ ID NO: 1; or (iv) a portion of (iii) comprising an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein the polypeptide comprises alanine, glycine, serine, asparagine or threonine at a position equivalent to position 56 of SEQ ID NO: 2, wherein the polypeptide binds selectively and reversibly to a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, 4 or 5.

Polypeptide and its use in affinity purification

The present invention relates to a two-part linker comprising a peptide tag (peptide) and a polypeptide (protein) that is capable of spontaneously forming an isopeptide bond, particularly wherein: a) said peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1, wherein: (i) X at position 1 is arginine or no amino acid; (ii) X at position 2 is glycine or no amino acid; (iii) X at position 5 is histidine or threonine; (iv) X at position 11 is alanine, glycine or valine; and (v) X at position 14 is arginine or lysine, wherein when X at position 1 is no amino acid, X at position 2 is no amino acid; and b) said polypeptide comprises: i) an amino acid sequence as set forth in SEQ ID NO: 2; ii) a portion of (i) comprising an amino acid sequence as set forth in SEQ ID NO: 101; iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein said amino acid sequence comprises a lysine at position 34, a glutamic acid at position 80 and one or more of the following: 1) threonine at position 5; 2) proline at position 16; 3) arginine at position 40; 4) histidine at position 65; 5) proline at position 92; 6) aspartic acid at position 100: 7) glutamic acid at position 108; and 8) threonine at position 116, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 2; or iv) a portion of (iii) comprising an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 101, wherein the amino acid sequence comprises a lysine at position 10, a glutamic acid at position 56 and one or more of the following: 1) arginine at position 16; 2) histidine at position 41; 3) proline at position 68; and 4) aspartic acid at position 76, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 101, and wherein said peptide and polypeptide are capable of spontaneously forming an isopeptide bond between the aspartic acid residue at position 10 of SEQ ID NO: 1 and the lysine residue at position 34 of SEQ ID NO: 2 or position 10 of SEQ ID NO: 101.

Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof

The present invention relates to a polypeptide that is capable of promoting the covalent conjugation of two peptide tags or linkers and in particular to a polypeptide comprising: a) an amino acid sequence as set forth in SEQ ID NO: 1; or b) 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 glutamic acid at position 61 and one or more of the following: 1) proline at position 66; 2) proline at position 95; 3) glycine at position 96; and 4) valine at position 97, wherein the specified amino acid residues are at positions equivalent to the positions in SEQ ID NO: 1 and wherein said polypeptide is capable of promoting the formation of an isopeptide bond between the lysine residue at position 9 of SEQ ID NO: 2 and the asparagine residue at position 17 of SEQ ID NO: 3.

Peptide ligase and use thereof

A method of producing a fusion protein is provided. The method includes: a) contacting a first protein with a second protein under conditions that enable the formation of an isopeptide bond between said proteins to form a linked protein; and b) contacting the linked protein from (a) with a third protein under conditions that enable the formation of an isopeptide bond between said third protein and said linked protein to form a fusion protein. Peptide linkers and the use of orthogonal pairs of said linkers in the synthesis of fusion proteins are also provided. Recombinant proteins comprising said linkers, nucleic acid molecules encoding said proteins and linkers, vectors comprising said nucleic acid molecules and host cells comprising said vectors and nucleic acid molecules are also contemplated.

Methods and products for fusion protein synthesis

Described herein is a protein capable of spontaneously forming an isopeptide bond for the development of a peptide tag and binding partner pair wherein the peptide tag and binding partner are capable of covalently binding to each other via an isopeptide bond. Also provided is a method for developing a peptide tag and binding partner pair which are capable of covalently binding to each other based on a protein which is capable of spontaneously forming an isopeptide bond. Additionally provided are peptide tag and binding partner pairs which are obtainable from isopeptide proteins. Further, specifically developed peptide tags and binding partners are described, together with nucleic acid molecules and vectors which encode those peptides or proteins.

Peptide tag systems that spontaneously form an irreversible link to protein partners via isopeptide bonds

Described herein is-a protein capable of spontaneously forming an isopeptide bond for the development of a peptide tag and binding partner pair wherein the peptide tag and binding partner are capable of covalently binding to each other via an isopeptide bond. Also provided is a method for developing a peptide tag and binding partner pair which are capable of covalently binding to each other based on a protein which is capable of spontaneously forming an isopeptide bond. Additionally provided are peptide tag and binding partner pairs which are obtainable from isopeptide proteins. Further, specifically developed peptide tags and binding partners are described, together with nucleic acid molecules and vectors which encode those peptides or proteins.

Peptide tag system that spontaneously form an irreversible link to protein partners via isopeptide bonds

The present invention provides a mutant streptavidin subunit which comprises one or more amino acid substitutions compared to a wildtype streptavidin subunit at any one of residue positions equivalent to positions (50, 51, 52, 53) and (54) of SEQ ID NO. 2 and wherein amino acid residues at positions equivalent to positions (23, 27, 43, 45, 49, 79, 88, 90, 92, 108, 110) and (128) of SEQ ID NO. 2, in said mutant streptavidin subunit, are wildtype, wherein (i) when said mutant streptavidin subunit unit is comprised in streptavidin, said streptavidin has a lower off rate for biotin or for a biotin conjugate than wildtype streptavidin or (ii) when said mutant streptavidin subunit is in monomelic form said mutant monomelic streptavidin has a lower off rate for biotin or a biotin conjugate than monomelic streptavidin. The invention also encompasses nucleic acid molecules comprising a nucleotide sequence encoding the mutant streptavidin subunit and vectors and cells comprising the nucleic acid. Further, methods of capturing biotinylated molecules and cells and therapeutic methods are encompassed.

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Oxford, United Kingdom

Mark Howarth