title: Expression, purification and characterization of Solanum tuberosum recombinant cytosolic pyruvate kinase.
abstract: The cDNA encoding for a Solanum tuberosum cytosolic pyruvate kinase 1 (PKc1) highly expressed in tuber tissue was cloned in the bacterial expression vector pProEX HTc. The construct carried a hexahistidine tag in N-terminal position to facilitate purification of the recombinant protein. Production of high levels of soluble recombinant PKc1 in Escherichia coli was only possible when using a co-expression strategy with the chaperones GroES-GroEL. Purification of the protein by Ni(2 +) chelation chromatography yielded a single protein with an apparent molecular mass of 58kDa and a specific activity of 34unitsmg(-1) protein. The recombinant enzyme had an optimum pH between 6 and 7. It was relatively heat stable as it retained 80% of its activity after 2min at 75°C. Hyperbolic saturation kinetics were observed with ADP and UDP whereas sigmoidal saturation was observed during analysis of phosphoenolpyruvate binding. Among possible effectors tested, aspartate and glutamate had no effect on enzyme activity, whereas α-ketoglutarate and citrate were the most potent inhibitors. When tested on phosphoenolpyruvate saturation kinetics, these latter compounds increased S0.5. These findings suggest that S. tuberosum PKc1 is subject to a strong control by respiratory metabolism exerted via citrate and other tricarboxylic acid cycle intermediates.
authors.affiliation: Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, Qc H1X 2B2, Canada.
authors.forename: Evgenia L
authors.initials: EL
authors.lastname: Auslender
authors.affiliation: Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, Qc H1X 2B2, Canada.
authors.forename: Sonia
authors.initials: S
authors.lastname: Dorion
authors.affiliation: Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, Qc H1X 2B2, Canada.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Dumont
authors.affiliation: Institut de Recherche en Biologie Végétale, Université de Montréal, 4101 Sherbrooke Est, Montréal, Qc H1X 2B2, Canada. Electronic address: jean.rivoal@umontreal.ca.
authors.forename: Jean
authors.initials: J
authors.lastname: Rivoal
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D019343:Citric Acid / Q000737:chemistry; D003001:Cloning, Molecular; D003600:Cytosol / Q000737:chemistry* / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011198:Solanum tuberosum / Q000737:chemistry* / Q000201:enzymology; D014530:Uridine Diphosphate / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; C028715:GroE protein, E coli; D006360:Heat-Shock Proteins; D007656:Ketoglutaric Acids; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019343:Citric Acid; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase
keywords: Chaperone; Glycolysis; Plant; Pyruvate kinase; Recombinant protein; Respiratory metabolism