Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2005-5-3
pubmed:abstractText
We report a highly specific, robust and rapid new method for labeling cell surface proteins with biophysical probes. The method uses the Escherichia coli enzyme biotin ligase (BirA), which sequence-specifically ligates biotin to a 15-amino-acid acceptor peptide (AP). We report that BirA also accepts a ketone isostere of biotin as a cofactor, ligating this probe to the AP with similar kinetics and retaining the high substrate specificity of the native reaction. Because ketones are absent from native cell surfaces, AP-fused recombinant cell surface proteins can be tagged with the ketone probe and then specifically conjugated to hydrazide- or hydroxylamine-functionalized molecules. We demonstrate this two-stage protein labeling methodology on purified protein, in the context of mammalian cell lysate, and on epidermal growth factor receptor (EGFR) expressed on the surface of live HeLa cells. Both fluorescein and a benzophenone photoaffinity probe are incorporated, with total labeling times as short as 20 min.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
1548-7091
pubmed:author
pubmed:issnType
Print
pubmed:volume
2
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
99-104
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
pubmed:affiliation
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Room 18-496, Cambridge, Massachusetts 02139, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't, Evaluation Studies