Wilkinson M C, Potter P M, Cawkwell L, Georgiadis P, Patel D, Swann P F, Margison G P
Department of Carcinogenesis, Christie Hospital and Holt Radium Institute, Manchester, UK.
Nucleic Acids Res. 1989 Nov 11;17(21):8475-84. doi: 10.1093/nar/17.21.8475.
The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.
大肠杆菌基因ogt编码DNA修复蛋白O6-烷基鸟嘌呤-DNA-烷基转移酶（O6-烷化鸟嘌呤ATase）。该基因的蛋白质编码区被克隆到一个多拷贝表达载体中，以获得高产率的酶（约占总蛋白的0.2%），通过亲和、分子排阻和反相色谱法将其纯化至表观均一。测定的氨基酸组成与预测的氨基酸组成之间存在良好的相关性。将纯化后的蛋白作用于自身互补的十二聚脱氧核糖核苷酸中O6-甲基鸟嘌呤（O6-MeG）、O6-乙基鸟嘌呤（O6-EtG）和O4-甲基胸腺嘧啶（O4-MeT）的能力，与相关ada蛋白的19 kDa片段的能力进行了比较。对于这两种蛋白，反应速率顺序均为O6-MeG＞O6-EtG＞O4-MeT，然而，发现ogt蛋白修复O6-MeG、O6-EtG和O4-MeT的速度分别比ada蛋白快1.1倍、173倍和84倍。
