title: One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase.
abstract: 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 Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column 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 CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly 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 cleavage is between Arg-394 and Arg-395.
authors.affiliation: Department of Chemistry and Biochemistry, Biochemical Institute, University of Texas, Austin, Texas 78712, USA.
authors.forename: W
authors.initials: W
authors.lastname: Soo Choi
authors.forename: J
authors.initials: J
authors.lastname: Yan
authors.forename: D B
authors.initials: DB
authors.lastname: McCarthy
authors.forename: S
authors.initials: S
authors.lastname: Hee Park
authors.forename: L J
authors.initials: LJ
authors.lastname: Reed
mesh_terms: D000818:Animals; D002384:Catalysis; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011994:Recombinant Proteins; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase