Iwakura M, Shimura Y, Sakai T, Tsuda K
J Biochem. 1983 Sep;94(3):1021-4. doi: 10.1093/oxfordjournals.jbchem.a134400.
The Escherichia coli strain carrying pTP 6-10 which was constructed in our previous work (Iwakura, M., et al. (1983) J. Biochem. 93, 927-930) produces more than 400-fold dihydrofolate reductase as compared with the strain without the plasmid. Dihydrofolate reductase was highly purified from the cell-free extract of the plasmid strain simply by two steps; ammonium sulfate fractionation and ion-exchange chromatography. By 10-fold purification, the enzyme was essentially homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The restriction map of pTP 6-10 was also determined and the plasmid was shown to have an Ava I, an EcoR I, a Pst I, a Pvu I, and a Pvu II site. Our results indicate that the plasmid strain is suitable as a source of the enzyme and that plasmid pTP 6-10 is promising as a versatile plasmid vector for efficiently yielding the product of the cloned gene.
携带pTP 6 - 10的大肠杆菌菌株是我们在之前的研究中构建的（岩仓，M.等人（1983年）《生物化学杂志》93卷，927 - 930页），与不含该质粒的菌株相比，它产生的二氢叶酸还原酶多出400多倍。通过两步简单操作，即硫酸铵分级分离和离子交换色谱法，从该质粒菌株的无细胞提取物中高度纯化了二氢叶酸还原酶。经过10倍的纯化，通过十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳判断，该酶基本达到了均一状态。还确定了pTP 6 - 10的限制性图谱，结果表明该质粒具有一个Ava I、一个EcoR I、一个Pst I、一个Pvu I和一个Pvu II位点。我们的结果表明，该质粒菌株适合作为该酶的来源，并且质粒pTP 6 - 10有望成为一种通用的质粒载体，用于高效产生克隆基因的产物。
