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Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin

Nature Protocols volume 3, pages 534–545 (2008)Cite this article

Abstract

This protocol describes a simple and efficient way to label specific cell surface proteins with biophysical probes on mammalian cells. Cell surface proteins tagged with a 15-amino acid peptide are biotinylated by Escherichia coli biotin ligase (BirA), whereas endogenous proteins are not modified. The biotin group then allows sensitive and stable binding by streptavidin conjugates. This protocol describes the optimal use of BirA and streptavidin for site-specific labeling and also how to produce BirA and monovalent streptavidin. Streptavidin is tetravalent and the cross-linking of biotinylated targets disrupts many of streptavidin's applications. Monovalent streptavidin has only a single functional biotin-binding site, but retains the femtomolar affinity, low off-rate and high thermostability of wild-type streptavidin. Site-specific biotinylation and streptavidin staining take only a few minutes, while expression of BirA takes 4 d and expression of monovalent streptavidin takes 8 d.

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Figure 1: Schematic of protein imaging with BirA and streptavidin.
Figure 2: Monovalent streptavidin structure and purification.
Figure 3: Specificity of cell surface labeling with BirA and streptavidin.
Figure 4: Monovalent streptavidin expression and purification.

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Acknowledgements

We thank all past and current members of the Ting lab who have contributed to methods described here, particularly Marta Fernandez Suarez for cloning pET21a-BirA and Yi Zheng for supplying Figure 4b. Funding was provided by the National Institutes of Health (R01 GM072670-01 and P20 GM072029-01), the McKnight Foundation, the Dreyfus Foundation and the Massachusetts Institute of Technology. M.H. was supported by a Computational and Systems Biology Initiative MIT-Merck postdoctoral fellowship. We thank Tanabe USA for biotin.

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  1. Mark Howarth

    Present address: Present address: Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK.,

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  1. Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, 02139, Massachusetts, USA

    Mark Howarth & Alice Y Ting

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Correspondence to Mark Howarth or Alice Y Ting.

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Howarth, M., Ting, A. Imaging proteins in live mammalian cells with biotin ligase and monovalent streptavidin. Nat Protoc 3, 534–545 (2008). https://doi.org/10.1038/nprot.2008.20

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