Purification of dihydrofolate reductase amplified in Escherichia coli K-12.
The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.
D001482:Base Composition; D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005784:Gene Amplification; D009154:Mutation; D010957:Plasmids; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification
D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes