Iwakura M, Tanaka T
Research Institute for Polymers and Textiles, Ibaraki.
J Biochem. 1992 Jan;111(1):31-6. doi: 10.1093/oxfordjournals.jbchem.a123714.
The Escherichia coli dihydrofolate reductase (DHFR) gene has been used as a genetic marker specifying trimethoprim resistance (TmpR). In order to use the DHFR gene as a versatile expression marker, we have constructed three types of plasmids: promoter cloning vector, terminator cloning vector, and the plasmid containing the DHFR gene cassette. In these systems, the selection of recombinant plasmids was carried out just by examining the TmpR phenotype of the transformed cells. Then, levels of the enzymatic activity of DHFR were measured to evaluate the efficiency of promoters and terminators in the fused DNA fragment. An expression plasmid which resulted in the E. coli host cells being able to produce DHFR up to 20% of total cellular proteins was also constructed by changing the promoter and Shine-Dalgarno sequences of the DHFR gene.
大肠杆菌二氢叶酸还原酶（DHFR）基因已被用作指定甲氧苄啶抗性（TmpR）的遗传标记。为了将DHFR基因用作通用的表达标记，我们构建了三种类型的质粒：启动子克隆载体、终止子克隆载体以及包含DHFR基因盒的质粒。在这些系统中，仅通过检测转化细胞的TmpR表型来进行重组质粒的筛选。然后，测量DHFR的酶活性水平，以评估融合DNA片段中启动子和终止子的效率。通过改变DHFR基因的启动子和Shine-Dalgarno序列，还构建了一种表达质粒，该质粒能使大肠杆菌宿主细胞产生的DHFR高达总细胞蛋白的20%。
