Martinez M A, Pezo V, Marlière P, Wain-Hobson S
Unité de Rétrovirologie Moléculaire, Institut Pasteur, Paris, France.
EMBO J. 1996 Mar 15;15(6):1203-10.
The evolution of natural proteins is thought to have occurred by successive fixation of individual mutations. In vitro protein evolution seeks to accelerate this process. RNA hypermutagenesis, cDNA synthesis in the presence of biased dNTP concentrations, delivers elevated mutant and mutation frequencies. Here lineages of active enzymes descended from the homotetrameric 78 residue dihydrofolate reductase (DHFR) encoded by the Escherichia coli R67 plasmid were generated by iterative RNA hypermutagenesis, resulting in >20% amino acid replacement. The 22 residue N-terminus could be deleted yielding a minimum functional entity refractory to further changes, designating it as a determinant of R67 robustness. Complete substitution of the segment still allowed fixation of mutations. By the facile introduction of multiple mutations, RNA hypermutagenesis allows the generation of active proteins derived from extant genes through a mode unexplored by natural selection.
天然蛋白质的进化被认为是通过单个突变的连续固定而发生的。体外蛋白质进化旨在加速这一过程。RNA超诱变，即在有偏向性的dNTP浓度存在下进行cDNA合成，可提高突变体和突变频率。在这里，通过迭代RNA超诱变产生了由大肠杆菌R67质粒编码的同四聚体78个残基的二氢叶酸还原酶（DHFR）衍生的活性酶谱系，导致超过20%的氨基酸替换。22个残基的N端可以被删除，产生一个对进一步变化具有抗性的最小功能实体，将其指定为R67稳健性的决定因素。该片段的完全替换仍然允许突变的固定。通过轻松引入多个突变，RNA超诱变允许通过自然选择未探索的模式从现有基因产生活性蛋白。
