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.