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Combined use of strain construction and affinity chromatography in the rapid, high-yield purification of 6-phosphogluconate dehydrogenase from Escherichia coli.

ABSTRACT

A rapid, high-yield method for purification of 6-phosphogluconate dehydrogenase from Escherichia coli K-12 is described. Sonic extracts prepared from heat-induced cultures of strain RW184, doubly lysogenic for the specialized transducing bacteriophage lambdacI857St68h80dgndhis and bearing a deletion of the gene for glucose 6-phosphate dehydrogenase, contained levels of 6-phosphogluconate dehydrogenase 15- to 20-fold higher than cultures of wild-type cells. Affinity chromatography on blue dextran-Sepharose with batchwise elution with 1 mM nicotinamide adenine dinucleotide phosphate affected a further 10-fold purification. Enzyme prepared in this manner was homogeneous according to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and immunoelectrophoresis using antiserum directed against it. Fructose 1,6-diphosphate is an inhibitor of enzyme activity.

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• Lundberg, B. E., Wolf, R. E. Jr., Dinauer, M. C., Xu, Y., Fang, F. C. (1999). Glucose 6-Phosphate Dehydrogenase Is Required for Salmonella typhimurium Virulence and Resistance to Reactive Oxygen and Nitrogen Intermediates. Infect. Immun. 67: 436-438 [Abstract] [Full Text]