Shiota S, von Wronski M A, Tano K, Bigner D D, Brent T P, Mitra S
University of Tennessee, Oak Ridge Graduate School of Biomedical Sciences 37831.
Biochemistry. 1992 Feb 25;31(7):1897-903. doi: 10.1021/bi00122a001.
A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.
从λgt11文库中克隆出一个编码O6-甲基鸟嘌呤-DNA甲基转移酶（MGMT）的小鼠cDNA克隆，该酶负责修复DNA中诱变的O6-烷基鸟嘌呤。根据cDNA中的开放阅读框，小鼠蛋白含有211个氨基酸，分子量为22 kDa。通过实验证实了小鼠蛋白的大小和预测的N端序列。推导的小鼠MGMT氨基酸序列与人类MGMT的序列同源性为70%。已证明半胱氨酸-149是小鼠蛋白中唯一的烷基接受残基，这证实了基于不同MGMT保守序列的预测。小鼠MGMT蛋白可被一些针对人类MGMT的单克隆抗体识别。利用定点诱变技术将小鼠cDNA重新克隆到基于T7启动子的载体中，以便在大肠杆菌中过量表达天然修复蛋白。小鼠蛋白在第56 - 59位有一个四肽序列Pro-Glu-Gly-Val，人类蛋白中没有该序列。删除这个四肽或用丙氨酸替代缬氨酸-169均不影响突变蛋白的活性。
