Abstract

Plasmid pTbp60B (Kueng et al., J. Biol. Chem. 264 (1989) 5203-5209) was employed to obtain, through the polymerase chain reaction, the Trypanosoma brucei gene coding for phosphoenolpyruvate (PEP) carboxykinase, and then cloned into the yeast expression plasmid pYES2. The cloned gene was completely sequenced and the expression plasmid transformed into Saccharomyces cerevisiae PUK-3B (MAT alpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant I: brucei PEP carboxykinase was purified to near homogeneity. The basic molecular and catalytic characteristics of the recombinant enzyme were determined, and they showed to be essentially similar to those reported for wild type I: brucei PEP 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.