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

Abstract

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 isno 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 80and 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 glutamicacid 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.

Description

technical field [0001] The present invention relates to a two-part linker comprising a peptide tag and a polypeptide (protein) capable of spontaneously forming isopeptide bonds. In particular, the two-part linkers of the invention can be viewed as pairs of peptide tag and polypeptide binding partner homologues that, when contacted under conditions that allow the spontaneous formation of isopeptide bonds between the peptide tag and its polypeptide binding partner, Pairs can be conjugated via covalent bonds. Also provided are nucleic acid molecules encoding said each part of said two-part linker (ie, peptide tag and polypeptide binding partner), vectors comprising said nucleic acid molecules, and host cells comprising said vectors and nucleic acid molecules. Kits comprising said two-part linker (ie peptide tag and polypeptide binding partner) and / or nucleic acid molecule / carrier are also provided. Also provided are methods of producing said two-part linkers (ie peptide tag and...

AI Technical Summary

Problems solved by technology

In this regard, it is hypothesized that the rate at which the spy-tag peptide and the spy-capture peptide interact may limit the rate of the reaction

Therefore, the development of a suitable screening system requires the selection of reaction conditions under which the reaction rate between the spy tag peptide and the spy capture polypeptide is not optimal

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