Ordu Emel Biçakçi, Karagüler Nevin Gül
Faculty of Science and Letters, Department of Molecular Biology and Genetics, Istanbul Technical University, Istanbul, Turkey.
Prep Biochem Biotechnol. 2007;37(4):333-41. doi: 10.1080/10826060701593233.
The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.
已将甲基假丝酵母（cm）重组野生型甲酸脱氢酶（FDH）基因克隆到pQE - 2 TAGZyme表达载体中，并且通过使用外肽酶TAGZyme纯化系统，使带有6xHis标签的FDH基因在JM105细胞中过表达，从而更有效地纯化FDH蛋白，该系统能够完全去除小的N端His标签。经过纯化过程后，获得了纯度>95%、浓度为1.2 mg/mL的cmFDH蛋白。通过观察340 nm处烟酰胺辅酶的氧化来测定cmFDH的动力学参数。还将结果与FDH标准纯化和亲和纯化的产量进行了比较。
