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Methods Mol Biol. 2011;705:295-307. doi: 10.1007/978-1-61737-967-3_18.

Fluorescent site-specific labeling of Escherichia coli expressed proteins with Sfp phosphopantetheinyl transferase.

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  • 1New England BioLabs, 01938 Ipswich, MA, USA. zhang@neb.com

Abstract

Fluorescent tagging of proteins has become a critical step in optical analysis of protein function in vitro and in living cells. Here we describe a two-tag system for expression and isolation of a protein of interest from Escherichia coli and subsequent site-specific fluorescent labeling with Sfp phosphopantetheinyl transferase (Sfp synthase). In the example presented, adenoviral protein E3-14.7 K (E14.7) was expressed as a tripartite fusion protein with a fluorophore-targeting peptide tag and a chitin-binding domain. This system allows for rapid isolation of the recombinant fusion protein from crude bacterial cell lysate via a single chitin column. Sfp synthase-mediated labeling with fluorophore conjugated to coenzyme A-SH (CoA-SH) resulted in covalent attachment of a fluorescent dye to a specific residue of the peptide tag via a phosphopantetheinyl linker. The fluorescently labeled E14.7 fusion protein was analyzed with a fluorescence imager and subsequently transfected into mammalian cells for imaging with a fluorescence microscope.

PMID:
21125394
[PubMed - indexed for MEDLINE]
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