One-pot chemo-enzymatic synthesis of reporter-modified proteins.
To meet recent advancements in the covalent reporter labeling of proteins, we propose a flexible synthesis for reporter analogs. Here we demonstrate a one-pot chemo-enzymatic synthesis of reporter-labeled proteins that allows the covalent tethering of any amine-terminal fluorescent or affinity label to a carrier protein or fusion construct. This two-reaction sequence consists of activated panthothenate coupling, biosynthetic conversion to the coenzyme A (CoA) analog, and enzymatic carrier protein modification via phosphopantetheinyltransferase (PPTase). We also probe substrate specificity for CoAA, the first enzyme in the pathway. With this approach CoA analogs may be rapidly prepared, thus permitting the regiospecific attachment of reporter moieties from a variety of molecular species.
Worthington
Andrew S
AS
Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0358, USA.
Burkart
Michael D
MD
D000345:Affinity Labels / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry; D003065:Coenzyme A; D004798:Enzymes / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry*; D015336:Molecular Probe Techniques; D010205:Pantothenic Acid; D011506:Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D014166:Transferases
D000345:Affinity Labels; D002352:Carrier Proteins; D004798:Enzymes; D005456:Fluorescent Dyes; D011506:Proteins; D010205:Pantothenic Acid; D014166:Transferases; D003065:Coenzyme A