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Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase

Nature Methods volume 2, pages 99–104 (2005)Cite this article

Abstract

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.

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Figure 1: Site-specific protein labeling using biotin ligase and a ketone analog of biotin.
Figure 2: BirA-catalyzed ligation of ketone 1 to the AP.
Figure 3: Labeling of recombinant CFP-AP with fluorescein hydrazide.
Figure 4: Site-specific labeling of proteins expressed on the surface of live HeLa cells with BP.

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Acknowledgements

Funding was provided by the US National Institutes of Health (K22-HG002671-01), EJLB Foundation and Massachusetts Institute of Technology. I.C. was supported by a National Science Foundation predoctoral fellowship and a Wyeth Pharmaceuticals fellowship, and M.H. was supported by an MIT-Merck postdoctoral fellowship. We thank Tanabe USA for biotin, D. Beckett for the BirA plasmid and helpful advice, K.D. Wittrup for the EGFR plasmid, J.M. Baskin, E. McNeill and K.H. Riesenburger for assistance, and J.E. Wilson and G.C. Fu for use of their chiral HPLC. We also thank S.S. Licht and R.Y. Tsien for helpful advice.

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  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave., Room 18-496, Cambridge, 02139, Massachusetts, USA

    Irwin Chen, Mark Howarth, Weiying Lin & Alice Y Ting

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Correspondence to Alice Y Ting.

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Supplementary information

Supplementary Fig. 1

Comparison of our labeling method (ketone 1 followed by FH) to antibody detection (anti-pentahistidine mouse monoclonal followed by fluorescein-conjugated secondary antibody) for visualization of CFP-AP in lysate. (PDF 88 kb)

Supplementary Fig. 2

Analysis of the effect of the AP tag on EGFR distribution and function. (PDF 4787 kb)

Supplementary Methods (PDF 172 kb)

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Chen, I., Howarth, M., Lin, W. et al. Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods 2, 99–104 (2005). https://doi.org/10.1038/nmeth735

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