Characterization of the cloned Escherichia coli dihydrofolate reductase

Julian I. Rood, Jeffrey W. Williams

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3 Citations (Scopus)

Abstract

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was purified from Escherichia coli strains that carried derivatives of the multicopy recombinant plasmid, pJFM8. The results of enzyme kinetic and two-dimensional gel electrophoresis experiments showed that the cloned enzyme is indistinguishable from the chromosomal enzyme. Therefore it can be concluded that these strains are ideal for use as a source of enzyme for further studies on the biochemistry and regulation of this important enzyme. The plasmid derivatives were constructed by recloning experiments that utilized several restriction endonucleases. From the analysis both of these plasmids and the purified dihydrofolate reductase enzymes it was possible to deduce the location and orientation of the dihydrofolate reductase structural gene on the parent plasmid, pJFM8.

Original languageEnglish
Pages (from-to)214-218
Number of pages5
JournalBBA - Enzymology
Volume660
Issue number2
DOIs
Publication statusPublished - 13 Aug 1981
Externally publishedYes

Keywords

  • (E. coli)
  • Cloning
  • Dihydrofolate reductase
  • Gene localization

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