Heterologous Gene Expression in E.coli pp 295-307

Part of the Methods in Molecular Biology book series (MIMB, volume 705)

Fluorescent Site-Specific Labeling of Escherichia coli Expressed Proteins with Sfp Phosphopantetheinyl Transferase

  • Aihua Zhang
  • Luo Sun
  • John Buswell
  • Nancy Considine
  • Inca Ghosh
  • Anastasiya Masharina
  • Christopher Noren
  • Ming-Qun Xu
Protocol

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.

Key words

Protein labeling Sfp synthase ACP synthase ACP tag ybbR S6 tag fluorescent labeling 

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Aihua Zhang
    • 1
  • Luo Sun
    • 1
  • John Buswell
    • 1
  • Nancy Considine
    • 1
  • Inca Ghosh
    • 1
  • Anastasiya Masharina
    • 1
  • Christopher Noren
    • 1
  • Ming-Qun Xu
    • 2
  1. 1.New England BioLabsIpswichUSA
  2. 2.Chemical Biology DivisionNew England BioLabsIpswichUSA

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