title: Component co-expression and purification of recombinant human pyruvate dehydrogenase complex from baculovirus infected SF9 cells.
abstract: The mammalian pyruvate dehydrogenase complex (PDC) is a multi-component mitochondrial enzyme that plays a key role in the conversion of pyruvate to acetyl-CoA connecting glycolysis to the citric acid cycle. Recent studies indicate that targeting the regulation of PDC enzymatic activity might offer therapeutic opportunities by inhibiting cancer cell metabolism. To facilitate drug discovery in this area, a well defined PDC sample is needed. Here, we report a new method of producing functional, recombinant, high quality human PDC complex. All five components were co-expressed in the cytoplasm of baculovirus-infected SF9 cells by deletion of the mitochondrial localization signal sequences of all the components and E1a was FLAG-tagged to facilitate purification. The protein FLAG tagged E1a complex was purified using FLAG-M2 affinity resin, followed by Superdex 200 sizing chromatography. The E2 and E3BP components were then Lipoylated using an enzyme based in vitro process. The resulting PDC is over 90% pure and homogenous. This non-phosphorylated, lipoylated human PDC was demonstrated to produce a robust detection window when used to develop an enzyme coupled assay of PDHK.
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States. Electronic address: yong.2.jiang@gsk.com.
authors.forename: Yong
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Juan
authors.initials: J
authors.lastname: Wang
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Guofeng
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Khyati
authors.initials: K
authors.lastname: Oza
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Linda
authors.initials: L
authors.lastname: Myers
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Marc A
authors.initials: MA
authors.lastname: Holbert
authors.affiliation: Department of Biological Sciences, Molecular Discovery Research, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA 19426, United States.
authors.forename: Sharon
authors.initials: S
authors.lastname: Sweitzer
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D061987:Sf9 Cells / Q000378:metabolism*
chemical_list: D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases
keywords: Component co-expression; Pyruvate dehydrogenase; Pyruvate dehydrogenase kinase