Improved methods for the purification of enzymes of the folate pathway in Escherichia coli. I. Chromatographic methods

A sequence of chromatographic procedures is described for the isolation of three consecutive enzymes of the folate pathway in Escherichia coli: hydroxymethyldihydropteridine pyrophosphokinase (E.C. 2.7.6.3) (I), 7,8-dihydropteroate synthase (E.C. 2.5.1.15) (II) and 7,8-dihydrofolate reductase, (E.C. 1.5.1.3) (III). Starting with the crude extract, ion-exchange chromatography on a DEAE-Sepharose CL-6B column with a salt gradient completely separated I, II and III. I and II were further purified by hydrophobic-interaction chromatography on Phenyl-Sepharose CL-4B, followed by size-exclusion chromatography on Ultrogel AcA 54. For III only size-exclusion chromatography was used. The overall enrichment factors, on the basis of protein, were 13,700-fold for I, 280-fold for II and 500-fold for III. Bacterial batches of more than 500 g were handled.