Synthesis and expression of a gene for a mini type II dihydrofolate reductase.
The Type II dihydrofolate reductases (DHFR) are resistant to the folate analogs, trimethoprim and methotrexate. The monomer is very small (MW 9,000) and has no structural homology with other known DHFR types. A dhfr structural gene was synthesized which incorporates many unique restriction sites (Nco I, Nhe I, Pvu I, Hind III, Sma I, Bgl II, Xho I, and Ban I) within the coding sequence. This gene encodes a small DHFR (68 amino acids) which is 10 amino acids shorter at the amino-terminus than natural Type II DHFRs. The last 60 residues of the synthetically encoded protein are identical in sequence to R388 DHFR. The enzyme is functional and relatively stable, as evidenced by trimethoprim resistance conferred to cells expressing the synthetic gene. The gene was cloned onto a high-copy-number plasmid, pPV7SYN5, in which a trp-lac promoter drives transcription of both the dhfr gene and the primer for plasmid replication (RNA II). High levels of the small DHFR are accumulated in stationary phase cultures of MC1061(p3) containing pPV7SYN5 without the addition of IPTG.
Vermersch
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PS
Department of Biochemistry, Rice University, Houston, TX 77001.
Bennett
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GN
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D013762:Tetrahydrofolate Dehydrogenase; D004262:DNA Restriction Enzymes