Generation of thermostable Moloney murine leukemia virus reverse transcriptase variants using site saturation mutagenesis library and cell-free protein expression system

We attempted to increase the thermostability of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). The eight-site saturation mutagenesis libraries corresponding to Ala70−Arg469 in the whole MMLV RT (Thr24−Leu671), in each of which 1 out of 50 amino acid residues was replaced with other amino acid residue, were constructed. Seven-hundred and sixty eight MMLV RT clones were expressed using a cell-free protein expression system, and their thermostabilities were assessed by the temperature of thermal treatment at which they retained cDNA synthesis activity. One clone D200C was selected as the most thermostable variant. The highest temperature of thermal treatment at which D200C exhibited cDNA synthesis activity was 57ºC, which was higher than for WT (53ºC). Our results suggest that a combination of site saturation mutagenesis library and cell-free protein expression system might be useful for generation of thermostable MMLV RT in a short period of time for expression and selection. Site saturation mutagenesis library of MMLV RT was constructed and expressed using cell-free protein expression system.

本研究旨在提升莫洛尼鼠白血病病毒（Moloney murine leukemia virus, MMLV）反转录酶（reverse transcriptase, RT）的热稳定性。针对完整MMLV RT（Thr24−Leu671）中的Ala70−Arg469区段，我们构建了8个位点饱和突变文库，每个文库内的50个氨基酸残基中的任意1个均可被替换为其他氨基酸残基。本研究采用无细胞蛋白表达系统（cell-free protein expression system）表达了768株MMLV RT克隆，并通过各克隆保留cDNA合成活性（cDNA synthesis activity）的最高热处理温度，评估其热稳定性。最终筛选得到一株热稳定性最优的突变体D200C，该突变体保留cDNA合成活性的最高热处理温度为57℃，高于野生型（wild type, WT）的53℃。本研究结果表明，结合位点饱和突变文库与无细胞蛋白表达系统，可在较短时间内获得热稳定性提升的MMLV RT，用于后续表达与筛选。本研究通过无细胞蛋白表达系统完成了MMLV RT位点饱和突变文库的构建与表达。