Christians F C, Scapozza L, Crameri A, Folkers G, Stemmer W P
Maxygen, Inc., Santa Clara, CA 95051, USA.
Nat Biotechnol. 1999 Mar;17(3):259-64. doi: 10.1038/7003.
The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.
单纯疱疹病毒1型和2型的胸苷激酶（TK）基因通过一种称为DNA家族改组的技术在体外进行重组。一种高通量机器人筛选方法鉴定出了对叠氮胸苷（AZT）磷酸化能力增强的嵌合体。将改良的克隆体进行合并、重新改组，并在越来越低浓度的AZT上进行筛选。经过四轮改组和筛选后，分离出了两个克隆体，与单纯疱疹病毒1型TK相比，它们使大肠杆菌对AZT的敏感性降低了32倍，与单纯疱疹病毒2型TK相比降低了16000倍。这两个克隆体都是源自两个亲本基因之间多次交叉事件的杂种，并且携带了在任何一个亲本中都未发现的几个额外氨基酸取代，包括活性位点突变。动力学测量表明，嵌合酶对AZT的米氏常数（K(M)）降低，对胸苷的特异性也降低。与动力学数据一致，分子建模表明，两种进化酶的活性位点以牺牲胸苷为代价，更好地容纳了AZT的叠氮基团。尽管两种嵌合酶总体相似，但每种酶在影响底物亲和力的位置上都包含来自不同亲本的关键贡献。此类突变体可能对抗HIV基因治疗有用，类似的定向进化方法可能会改善其他酶-前药组合。
