Expression, characterization and site-directed mutagenesis of a human dihydrofolate reductase gene

Thesis/Dissertation · Thu Jan 01 04:00:00 EST 1987

OSTI ID:6152738

Prendergast, Jr, N J

A procaryotic expression vector for human dihydrofolate reductase (DHFR) has been constructed and the protein characterized as a first step towards structure function studies on this enzyme. A vector bearing the tac promoter, four synthetic oligonucleotides and a restriction fragment from the DHFR cDNA were aligned in a manner which optimized the transcriptional and translational frequency of the DHFR mRNA. The enzyme was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by its amino acid composition, partial amino acid sequence, and steady-state kinetic properties. The expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. This protocol circumvents shuttling mutant copies of the DHFR gene between vectors allowing for the immediate expression of altered enzyme forms. Functional studies on a Cysteine-6 to Serine mutant support the contention that Cysteine-6 is obligatory for mercurial activation of human DHFR.

• Research Organization: Medical Coll. of Ohio, Toledo (USA)
• OSTI ID: 6152738
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
AMINO ACID SEQUENCE
ANIMALS
CHEMICAL COMPOSITION
ELECTROPHORESIS
ENZYMES
GENE REGULATION
GENES
MAMMALS
MAN
MESSENGER-RNA
MOLECULAR STRUCTURE
MUTAGENESIS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PRIMATES
RNA
STRUCTURE-ACTIVITY RELATIONSHIPS
TRANSCRIPTION
VERTEBRATES