Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems
Fig 1
Live-cell labeling strategies described in this work.
(A) Structure of a CoA-reporter conjugate [7], and examples of possible reporter dyes, easily attachable through maleimide chemistry to the thiol group of CoA. (B) Labeling of small peptide tags, genetically fused to the protein of interest (POI), mediated through 4′-phosphopantetheinyl transferase (PPTase) enzymes. (C) A HaloTag ligand (HTL), comprising a chloroalkane, polyethylene glycol (PEG) linker units for better access to the catalytic center of the enzyme as well as for improved solubility [8], and a reporter, in this example again coupled via thiol chemistry as in (A).The HaloTag, which can be genetically fused to the protein of interest, is a haloalkane dehalogenase engineered to stably form a reaction intermediate, allowing covalent incorporation of labeled substrates.