One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca2+ to th e inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase compon ent (E2) of the mammalian pyruvate dehydrogenase complex. The affinity colu mn consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl2, 5 mM MgCl2, 150 m M NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and 1 M urea was passed thr ough the affinity column, and the column was washed extensively with this b uffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl2, 0.5 mM dithiothreitol, and 1 mM EGTA, Approximately 22 mg of hig hly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc, The c leavage is between Arg-394 and Arg-395. (C) 2000 Academic Press.