Expression and site-directed mutagenesis of human dihydrofolate reductase

Journal Article · Tue May 17 04:00:00 EDT 1988 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00410a022· OSTI ID:7191254

Prendergast, N J; Delcamp, T J; Smith, P L; Freisheim, J H

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-sate kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6 ..-->.. Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the k/sub cat/ for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the K/sub m/ values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by ..cap alpha..-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.

• Research Organization: Medical College of Ohio, Toledo (USA)
• OSTI ID: 7191254
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 27:10; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

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59 BASIC BIOLOGICAL SCIENCES
62 RADIOLOGY AND NUCLEAR MEDICINE
ANIMALS
AUTORADIOGRAPHY
BACTERIA
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
DNA
DNA SEQUENCING
ENZYMES
ESCHERICHIA COLI
GENE REPRESSORS
KINETICS
MAMMALS
MAN
MICROORGANISMS
MUTAGENESIS
NUCLEIC ACIDS
NUCLEOPROTEINS
OLIGONUCLEOTIDES
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PLASMIDS
PRIMATES
PROTEINS
REACTION KINETICS
RECOMBINANT DNA
STRUCTURAL CHEMICAL ANALYSIS
VERTEBRATES