OVEREXPRESSION IN ESCHERICHIA-COLI AND PURIFICATION OF THE 6-PHOSPHOGLUCONATE DEHYDROGENASE OF TRYPANOSOMA-BRUCEI

The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system. Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28°C. The enzyme was rapidly degraded at temperatures higher than 30°C. The T. brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2′ AMP-agarose affinity chromatography. The purified protein crystallized readily. A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding. © 1993 Academic Press. All rights reserved.