Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells.
Applied molecular evolution of O6-benzylguanine-resistant DNA alkyltransferases in human hematopoietic cells.
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人类造血细胞中 O6-苄基鸟嘌呤抗性 DNA 烷基转移酶的应用分子进化。
DOI：
10.1073/pnas.091601198
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发表时间：
2001
影响因子：
11.1
通讯作者：
Gerson,SL
中科院分区：
文献类型：
--
作者：
Davis,BM;Encell,LP;Zielske,SP;Christians,FC;Liu,L;Friebert,SE;Loeb,LA;Gerson,SL
应用分子进化是一项快速发展的技术，可用于创造和鉴定自然界未选择的新酶。这项技术的一个重要应用是为癌症基因治疗创造高度耐药的酶。通过在大肠杆菌中进行遗传互补，筛选了800万个高抗性too6 -苄基鸟嘌呤（BG）突变体，鉴定出17个6-烷基鸟嘌呤- dna烷基转移酶（AGT）突变体。为了研究这些突变体在人类中的应用潜力，将AGT克隆亚文库引入人类造血细胞，并严格选择其抵抗BG和1,3-双（2-氯乙基）-1-亚硝基脲联合杀伤。人类细胞中突变体之间的竞争分析显示，三种AGT突变体对BG和1,3-双（2-氯乙基）-1-亚硝基脲的组合具有显著的抗性。其中，一种恢复的频率明显高于其他。经过进一步分析，该突变体在人造血细胞中显示出比以往任何报道的突变体更高的BG抵抗水平。
Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. SeventeenO6-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant toO6-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation inEscherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.