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Nat Biotechnol. 2007 Dec;25(12):1483-7. Epub 2007 Dec 2.

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

Author information

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

Abstract

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 function, or dissociate from proteins after internalization. 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) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne 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 labeling, 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.

PMID:
18059260
PMCID:
PMC2654346
DOI:
10.1038/nbt1355
[Indexed for MEDLINE]
Free PMC Article

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