Overexpression and Characterization of Human Tetrameric Pyruvate Dehydrogenase and Its Individual Subunits

• Published in: Protein expression and purification 1995-02, Vol.6 (1), p.79-90
• Main Authors: Korotchkina, L.G., Tucker, M.M., Thekkumkara, T.J., Madhusudhan, K.T., Pons, G., Kim, H.J., Patel, M.S.
• Format: Article
• Language: English
• Subjects: Base Sequence; Cloning, Molecular; DNA Primers; Escherichia coli - genetics; Humans; Molecular Sequence Data; Phosphorylation; Phosphotransferases - pharmacology; Protein Conformation; Pyruvate Dehydrogenase Complex - chemistry; Pyruvate Dehydrogenase Complex - genetics; Pyruvate Dehydrogenase Complex - isolation & purification; Recombinant Proteins - genetics; Thiamine Pyrophosphate - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1995.1011

Pyruvate dehydrogenase (E1), an α 2 β 2 tetramer, is the first component of the pyruvate dehydrogenase complex which catalyzes a two-step oxidative decarboxylation of pyruvic acid. To overexpress human E1 and its subunits individually, cDNAs for the mature forms of human E1α and E1β were subcloned either individually or together into a plasmid pQE-9 and expressed in Escherichia coli M15. A polyhistidine extension was added at the NH 2-termini of the recombinant E1α and E1β for the rapid purification of the proteins by Ni-nitrilotriacetic-agarose chromatography, The polyhistidine extension on either E1α or E1β subunit did not affect the activity of the recombinant tetrameric E1. Highly purified recombinant human E1 catalyzed the partial reactions of the oxidative and nonoxidative conversion of pyruvic acid with the same efficiency as E1 purified from bovine kidney, Recombinant human E1 interacted with thiamin pyrophosphate by forming a charge transfer complex band at 330 nm that changed during the catalytic cycle, Recombinant human E1 was phosphorylated by E1-kinase (with concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1. When expressed individually, E1α and E1βsubunits lacked any catalytic activity in the oxidative or nonoxidative reactions, Spectral studies demonstrated that there was no thiamin pyrophosphate binding to either recombinant E1α or E1β subunit, The E1α subunit retained the ability to be phosphorylated; however, the incorporation of phosphoryl groups into recombinant E1α alone was only about 12% of that observed with the tetrameric E1. These findings show that both subunits are required for formation of the active center and catalysis.