Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae

Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was emp loyed to obtain, through the polymerase chain reaction, the Trypanosoma bru cei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then clone d into the yeast expression plasmid pYES2. The cloned gene was completely s equenced and the expression plasmid transformed into Saccharomyces cerevisi ae PUK-3B (MAT alpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant I: brucei PEP c arboxykinase was purified to near homogeneity. The basic molecular and cata lytic characteristics of the recombinant enzyme were determined, and they s howed to be essentially similar to those reported for wild type I: brucei P EP carboxykinase (Hunt and Kohler, Biochim. Biophys. Acta 1249 (1995) 15-22 ). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase. (C) 2000 Societe francaise de biochimie et biologie moleculaire / Editions scientifiques et medicales Elsevier SAS.