Expression of the Trypanosoma brucei phosphoenolpyruvate carboxykinase gene in Saccharomyces cerevisiae.
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 (MATalpha pck1 ura3 ade1) competent cells. Gene expression took place upon induction with 2% galactose, and the recombinant T. 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 T. brucei PEP carboxykinase (Hunt and Köhler, Biochim. Biophys. Acta 1249 (1995) 15-22). The expression system here described is a reliable non-pathogenic source of T. brucei PEP carboxykinase.
Yévenes
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Departamento de Ciencias Químicas, Facultad de Química y Biología, Universidad de Santiago de Chile, Casilla 40, Santiago, Chile.
Cardemil
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D000818:Animals; D003001:Cloning, Molecular / Q000379:methods; D015870:Gene Expression; D017125:Genes, Protozoan; D005822:Genetic Vectors; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D019764:Phosphoenolpyruvate Carboxykinase (ATP)