Vermersch P S, Bennett G N
Department of Biochemistry, Rice University, Houston, TX 77001.
DNA. 1988 May;7(4):243-51. doi: 10.1089/dna.1988.7.243.
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.
II型二氢叶酸还原酶（DHFR）对叶酸类似物甲氧苄啶和甲氨蝶呤具有抗性。该单体非常小（分子量9000），与其他已知的DHFR类型没有结构同源性。合成了一个dhfr结构基因，其编码序列中包含许多独特的限制性酶切位点（Nco I、Nhe I、Pvu I、Hind III、Sma I、Bgl II、Xho I和Ban I）。该基因编码一种小的DHFR（68个氨基酸），其氨基末端比天然II型DHFR短10个氨基酸。合成编码蛋白的最后60个残基在序列上与R388 DHFR相同。该酶具有功能且相对稳定，这通过赋予表达合成基因的细胞甲氧苄啶抗性得以证明。该基因被克隆到高拷贝数质粒pPV7SYN5上，其中trp-lac启动子驱动dhfr基因和质粒复制引物（RNA II）的转录。在不添加IPTG的情况下，含有pPV7SYN5的MC1061(p3)的稳定期培养物中积累了高水平的小DHFR。
