Site-specific protein labeling by Sfp phosphopantetheinyl transferase.
Sfp phosphopantetheinyl transferase covalently attaches small-molecule probes including biotin and various organic fluorophores to a specific serine residue in the peptidyl carrier protein (PCP) or a short 11-residue peptide tag ybbR through a phosphopantetheinyl linker. We describe here a protocol for site-specific protein labeling by Sfp-catalyzed protein post-translational modification that includes (i) expression and purification of Sfp, (ii) synthesis of small-molecule probe-CoA conjugates, (iii) construction of target protein fusions with PCP or the ybbR tag, (iv) labeling PCP- or ybbR-tagged target protein fusions in cell lysates and on live cell surfaces and (v) imaging fluorophore-labeled cell surface receptors by fluorescence microscopy. To follow this protocol, we advise that you allow 3 d for the expression and purification of Sfp phosphopantetheinyl transferase, 1 d for the synthesis and purification of the small-molecule probe-CoA conjugates as the substrates of Sfp, 3 d for the cloning of target protein genes as fusions to the PCP or the ybbR tag in the appropriate plasmids and another 3 d for transfecting cell lines with the plasmids and the expression of PCP- or ybbR-tagged proteins. Labeling of the PCP- or the ybbR-tagged proteins in cell lysates or on cell surfaces should require only 15-30 min.
Yin
Jun
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.
Lin
Alison J
AJ
Golan
David E
DE
Walsh
Christopher T
CT
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D015335:Molecular Probes / Q000737:chemistry*; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; C082662:phosphopantetheinyl transferase; D012694:Serine; D001710:Biotin; D017855:Transferases (Other Substituted Phosphate Groups)