Synthesis of Site Specifically-Linked Ubiquitin
AssigneeMedical Research Council
InventorsJason CHIN, Satpal Virdee
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Abstract
The invention relates to a method of modifying a specific lysine residue in a polypeptide comprising at least two lysine residues, said method comprising (a) providing a polypeptide comprising a target lysine residue protected by a first protecting group, and at least one further lysine residue; (b) treating the polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue; (c) selectively deprotecting the target lysine residue; and (d) modifying the deprotected lysine residue of (c).
Claims18
A method of modifying a specific lysine residue in a ubiquitin polypeptide comprising at least two lysine residues, said method comprising:
- (a) providing a ubiquitin polypeptide comprising a target lysine residue protected by a first protecting group, and at least one further lysine residue, wherein the ubiquitin polypeptide comprising the target lysine group is provided by genetically incorporating the target lysine residue protected by the first protecting group into the ubiquitin polypeptide chain during its translation by
- (i) providing a nucleic acid encoding the ubiquitin polypeptide which nucleic acid comprises an orthogonal codon encoding the target lysine;
- and
- (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said target lysine residue protected by a first protecting group into the ubiquitin polypeptide chain;
- (b) treating the ubiquitin polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue;
- (c) selectively deprotecting the target lysine residue;
- and
- (d) modifying the deprotected lysine residue of (c).
A method of modifying a specific lysine residue in a ubiquitin polypeptide comprising at least two lysine residues, said method comprising:
- (a) providing a ubiquitin polypeptide comprising a target lysine residue protected by a first protecting group, and at least one further lysine residue, wherein the ubiquitin polypeptide comprising the target lysine group is provided by genetically incorporating the target lysine residue protected by the first protecting group into the ubiquitin polypeptide chain during its translation by
- (i) providing a nucleic acid encoding the ubiquitin polypeptide which nucleic acid comprises an orthogonal codon encoding the target lysine;
- and
- (ii) translating said nucleic acid in the presence of an orthogonal tRNA synthetase/tRNA pair capable of recognising said orthogonal codon and incorporating said target lysine residue protected by a first protecting group into the ubiquitin polypeptide chain;
- (b) treating the ubiquitin polypeptide to protect said further lysine residue(s), wherein the protecting group for said further lysine residues is different to the protecting group for the target lysine residue;
- (c) selectively deprotecting the target lysine residue;
- and
- (d) modifying the deprotected target lysine by activating thioester by conversion to N-hydroxysuccinimidyl ester in the presence of Ag(I), adding a polypeptide to be joined to the target lysine;
- and incubating to allow formation of a specific isopeptide bond.
A ubiquitin polypeptide comprising at least two lysine residues, wherein a single target lysine residue in the ubiquitin polypeptide lacks a protecting group and the remaining lysine residues in the ubiquitin polypeptide comprise a protecting group.
A ubiquitin polypeptide according to claim 15 produced according to claim 1.
A ubiquitin polypeptide according to claim 17, wherein the target lysine residue is K6 or K29.
Dependencies are inferred from the claim text.