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.
New England BioLabs, 01938 Ipswich, MA, USA. zhang@neb.com
Aihua
A
Zhang
Luo
L
Sun
John
J
Buswell
Nancy
N
Considine
Inca
I
Ghosh
Anastasiya
A
Masharina
Christopher
C
Noren
Ming-Qun
MQ
Xu
D017503:Adenovirus E3 Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017503:Adenovirus E3 Proteins; D001426:Bacterial Proteins; C509184:E3 14.7K protein, Adenovirus; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)