PMID:6386050
| Citation |
Frost, JW, Bender, JL, Kadonaga, JT and Knowles, JR (1984) Dehydroquinate synthase from Escherichia coli: purification, cloning, and construction of overproducers of the enzyme. Biochemistry 23:4470-5 |
|---|---|
| Abstract |
Dehydroquinate synthase has been purified 9000-fold from Escherichia coli K-12 (strain MM294). The synthase is encoded by the aroB gene, which is carried by plasmid pLC29-47 from the Carbon-Clarke library. Construction of an appropriate host bearing pLC29-47 results in a strain that produces 20 times more enzyme than strain MM294. Subcloning of the aroB gene behind a tac promoter results in E. coli transformants that produce 1000 times more enzyme than MM294: the synthase constitutes 5% of the soluble protein of the cell. A laborious isolation from 50 g of wild-type E. coli cells yields 80 micrograms of impure enzyme, whereas 50 g of cells containing the subcloned gene yields 150 mg of homogeneous enzyme in a two-column purification. Dehydroquinate synthase is a monomeric protein of Mr 40 000-44 000. The chromosomal enzyme from E. coli K-12, the cloned enzyme encoded by the plasmid pLC29-47, and the subcloned inducible enzyme encoded by pJB14 all comigrate on polyacrylamide gel electrophoresis under denaturing conditions. |
| Links | |
| Keywords |
Chromatography, Gel; Cloning, Molecular; Escherichia coli/enzymology; Lyases/genetics; Lyases/isolation & purification; Molecular Weight; Phosphorus-Oxygen Lyases |
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