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 degrees C. The enzyme was rapidly degraded at temperatures higher than 30 degrees 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.
Barrett
M P
MP
University of Cambridge, Department of Pathology, United Kingdom.
Phillips
C
C
Adams
M J
MJ
Le Page
R W
RW
D000818:Animals; D001051:Apoenzymes / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003198:Computer Simulation; D003460:Crystallization; D004790:Enzyme Induction; D004926:Escherichia coli; D005822:Genetic Vectors; D008958:Models, Molecular; D010734:Phosphogluconate Dehydrogenase / Q000096:biosynthesis* / Q000302:isolation & purification; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012756:Sheep; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics
D001051:Apoenzymes; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D010734:Phosphogluconate Dehydrogenase