Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase.
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.
Chen
Irwin
I
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Room 18-496, Cambridge, Massachusetts 02139, USA.
Howarth
Mark
M
Lin
Weiying
W
Ting
Alice Y
AY
D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D012097:Repressor Proteins; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D014157:Transcription Factors
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D066246:ErbB Receptors; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli