Heterologous Gene Expression in E.coli pp 295-307
Fluorescent Site-Specific Labeling of Escherichia coli Expressed Proteins with Sfp Phosphopantetheinyl Transferase
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.
Key wordsProtein labeling Sfp synthase ACP synthase ACP tag ybbR S6 tag fluorescent labeling
- 17.Spector, D., Goldman, R. (eds.) (2004). Live Cell Imaging: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar