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Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes


Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function1,2, or dissociate from proteins after internalization3,4. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA)5 to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne6 conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling7,8, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

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Figure 1: Redirecting LplA for site-specific protein labeling with fluorescent probes.
Figure 2: LplA labels the LAP peptide without modifying endogenous mammalian proteins.
Figure 3: Site-specific labeling of LAP fusion proteins with fluorophores.
Figure 4: Simultaneous labeling and imaging of two receptors in polarized cells in a wound healing assay.


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The authors thank Mark Howarth, John Cronan, Irwin Chen, Chi-Wang Lin, Robin Prince and Martin Lackmann for their assistance and advice. This work was supported by the National Institutes of Health (R01 GM072670-01 to A.Y.T. and GM58867 to C.R.B.), the Sloan Foundation, the Dreyfus Foundation, a La Caixa Foundation predoctoral fellowship (to M.F.-S.), and National Science Foundation and National Science Defense and Engineering predoctoral fellowships (to J.M.B.).

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M.F.-S., H.B., L.M.-H. and A.Y.T. designed the research; M.F.-S., H.B., L.M.-H. and K.T.X. performed the research; J.M.B. and C.R.B. provided cyclo-octyne starting material; M.F.-S., H.B. and A.Y.T. analyzed data; M.F.-S. and A.Y.T. wrote the paper.

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Correspondence to Alice Y Ting.

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Massachusetts Institute of Technology is seeking to file a patent application covering part of the information contained in this article.

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Supplementary Figures 1–6; Supplementary Methods (PDF 405 kb)

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Fernández-Suárez, M., Baruah, H., Martínez-Hernández, L. et al. Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. Nat Biotechnol 25, 1483–1487 (2007).

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