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US 2005/0233389 A1Published application
Methods and compositions for peptide and protein labeling
AssigneeMassachusetts Institute of Technology
InventorsAlice Ting, Irwin Chen
Life of this patent
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Abstract
The invention provides compositions and methods of use thereof for labeling peptide and proteins in vitro or in vivo. The methods described herein employ biotin ligase and biotin analogs.
Claims23
A composition comprising a benzophenone-biotin hydrazide having the structure
- or a derivative thereof.
A composition comprising a fluorescein hydrazide having the structure
- or a derivative thereof.
A method for labeling a target protein comprising
- contacting a fusion protein of the target protein and an acceptor peptide with a biotin analog in the presence of a biotin ligase, and
- allowing sufficient time for the biotin analog to be conjugated to the fusion protein via the acceptor peptide in the presence of a biotin ligase, and
- contacting the biotin analog with a detectable hydrazide and allowing sufficient time for the hydrazide to react with the biotin analog to form a hydrazone.
16-42. (canceled)
A method for identifying a biotin ligase mutant having specificity for a biotin analog conjugated to a detectable hydrazide comprising
- contacting a biotin analog conjugated to a detectable hydrazide with an acceptor peptide in the presence of a candidate biotin ligase mutant, and
- detecting the biotin analog conjugated to the detectable hydrazide that is bound to the acceptor peptide,
- wherein the presence of the biotin analog conjugated to the detectable hydrazide bound to the acceptor peptide indicates that the candidate biotin ligase mutant is a biotin ligase mutant having specificity for the biotin analog conjugated to the detectable hydrazide.
46-162. (canceled)
A composition comprising
- a biotin analog that binds to a biotin ligase mutant, wherein the biotin analog is ketone biotin analog or NBD-GABA.
165-215. (canceled)
Dependencies are inferred from the claim text.