Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme

Y. Wang, J. A. Bruenn, Sherry Queener, V. Cody

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

Original languageEnglish (US)
Pages (from-to)2517-2523
Number of pages7
JournalAntimicrobial Agents and Chemotherapy
Volume45
Issue number9
DOIs
StatePublished - 2001
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Folic Acid Antagonists
Enzymes
Genes
Recombinant Proteins
Amino Acids
Liver
Gene Library
Open Reading Frames
Catalytic Domain
Bacteria

ASJC Scopus subject areas

  • Pharmacology (medical)

Cite this

Isolation of rat dihydrofolate reductase gene and characterization of recombinant enzyme. / Wang, Y.; Bruenn, J. A.; Queener, Sherry; Cody, V.

In: Antimicrobial Agents and Chemotherapy, Vol. 45, No. 9, 2001, p. 2517-2523.

Research output: Contribution to journalArticle

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