Black M E, Loeb L A
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, School of Medicine, University of Washington, Seattle 98195.
Biochemistry. 1993 Nov 2;32(43):11618-26. doi: 10.1021/bi00094a019.
Random sequence mutagenesis in conjunction with genetic complementation was used to map the function of amino acid residues within the putative nucleoside binding site of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK). Six codons of the putative nucleoside binding site of the HSV-1 tk were substituted by a duplex of extended oligonucleotides containing 20% random sequences. Approximately 260 mutants were screened for the ability to genetically complement a TK-deficient Escherichia coli. Of those screened, 32% conferred TK activity. Approximately 60% of the TK positive clones contained single amino acid changes, 23% contained double changes, and 13.4% encoded the wild-type TK amino acid sequence. A small percentage of clones, 2.4% and 1.2%, contained triple or quadruple alterations, respectively. Three residues (D162, H163, and R164) appeared to be highly conserved especially with regard to the type of residues able to substitute. Secondary screening results indicated that several of the mutants had higher affinities for acyclovir and/or 3'-azido-3'-deoxythymidine than thymidine in complementation assays. In addition, a number of clones were unable to form colonies on selection medium at elevated temperatures (42 degrees C). Eight selected mutants were subcloned into an in vitro transcription vector and the derived transcripts used to program a rabbit reticulocyte lysate cell-free translation system. Biologically active translation products were then analyzed in vitro for thymidine kinase activity, for thermal stability, and for the ability to phosphorylate selected nucleoside analogues. Two of the eight mutants had an elevated thymidine kinase activity, two were significantly thermolabile, and three exhibited enhanced efficiency in phosphorylation of nucleoside analogues.
结合随机序列诱变和基因互补技术来定位单纯疱疹病毒1型（HSV-1）胸苷激酶（TK）假定核苷结合位点内氨基酸残基的功能。HSV-1 tk假定核苷结合位点的六个密码子被含有20%随机序列的延伸寡核苷酸双链体取代。对大约260个突变体进行了基因互补缺陷型大肠杆菌能力的筛选。在筛选出的突变体中，32%具有TK活性。大约60%的TK阳性克隆含有单个氨基酸变化，23%含有双氨基酸变化，13.4%编码野生型TK氨基酸序列。一小部分克隆，分别为2.4%和1.2%，含有三重或四重改变。三个残基（D162、H163和R164）似乎高度保守，特别是在能够替代的残基类型方面。二次筛选结果表明，在互补试验中，一些突变体对阿昔洛韦和/或3'-叠氮-3'-脱氧胸苷的亲和力高于胸苷。此外，许多克隆在高温（42℃）的选择培养基上无法形成菌落。将八个选定的突变体亚克隆到体外转录载体中，并将得到的转录本用于编程兔网织红细胞裂解物无细胞翻译系统。然后在体外分析具有生物活性的翻译产物的胸苷激酶活性、热稳定性以及磷酸化选定核苷类似物的能力。八个突变体中有两个胸苷激酶活性升高，两个显著热不稳定，三个在核苷类似物磷酸化方面表现出增强的效率。
