Ito Yoichiro, Kawama Toshihiro, Urabe Itaru, Yomo Tetsuya
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita City, Osaka 565-0871, Japan.
J Mol Evol. 2004 Feb;58(2):196-202. doi: 10.1007/s00239-003-2542-2.
We have investigated the evolvability of an insoluble random polypeptide, RP3-34, to a soluble form through iterative mutation and selection with the aid of the green fluorescent protein (GFP) folding reporter. To assess the solubility of the polypeptides in the selected clones of each generation, the polypeptide genes were detached from the GFP fusions and expressed with a His(6) tag. The solubility of the variant random polypeptides increased in each generation within the scope of the evolutionary process, and the polypeptides assumed a soluble form from the fourth generation. Analysis of the synonymous and nonsynonymous mutations found in the deduced amino acid sequence of the selected polypeptides revealed that selection had accelerated the evolutionary rate. The solubility and hydrophobicity of the polypeptides and the 25 arbitrarily chosen random polypeptides found in a previously prepared library were determined, analyzed, and interpreted from the landscape on the protein sequence space. This study showed the evolvability of an insoluble arbitrary sequence toward a soluble one, hence, it provides a new perspective on the field of artificial evolution.
我们借助绿色荧光蛋白（GFP）折叠报告基因，通过迭代突变和筛选，研究了一种不溶性随机多肽RP3 - 34向可溶性形式的进化能力。为评估各代所选克隆中多肽的溶解性，将多肽基因从GFP融合体中分离出来，并带有His(6)标签进行表达。在进化过程范围内，变异随机多肽的溶解性在每一代中都有所增加，并且多肽从第四代开始呈现出可溶形式。对所选多肽推导氨基酸序列中发现的同义突变和非同义突变的分析表明，筛选加速了进化速率。从蛋白质序列空间的格局出发，对多肽以及先前制备文库中发现的25种任意选择的随机多肽的溶解性和疏水性进行了测定、分析和解释。这项研究展示了不溶性任意序列向可溶性序列的进化能力，因此，它为人工进化领域提供了一个新的视角。
