Strategies for the in vitro evolution of protein function: Enzyme evolution by random recombination of improved sequences
Strategies for the in vitro evolution of protein function: Enzyme evolution by random recombination of improved sequences
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DOI：
10.1006/jmbi.1997.1252
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发表时间：
1997-09-26
影响因子：
5.6
通讯作者：
Arnold, FH
中科院分区：
文献类型：
--
作者：
Moore, JC;Jin, HM;Arnold, FH
通过定向进化改进的基因集可以在体外重组，以进一步改善蛋白质功能。当有改进的序列可用时，重组特别有用；然而，产生这种序列的成本必须与通过顺序随机突变进一步进化的成本进行权衡。四个编码对硝基苯酯酶（pNB）变异的基因表现出增强的活性，在两个高保真DNA改组和筛选循环中重组。分析活性进一步提高的酶编码基因，以阐明DNA水平上的进化过程，并开始为选择体外进化策略和设置重组关键参数提供实验依据。来自两轮DNA洗牌的改进变体的DNA测序证实了重组过程的重要特征：从多个亲本序列中快速固定和积累有益突变，以及去除沉默和有害突变。通过重组多个亲本序列的突变和新的点突变，对氯螨贝对对硝基苯基（pNP）酯的总活性进一步提高了5 ~ 6倍。重组和筛选的计算机模拟说明了重组较少亲本序列（为了减少筛选需求）和降低进一步进化潜力之间的权衡。描述了在某些情况下可能大大减少筛选要求的搜索策略。(C) 1997学术出版社有限公司
Sets of genes improved by directed evolution can be recombined in vitro to produce further improvements in protein function. Recombination is particularly useful when improved sequences are available; costs of generating such sequences, however, must be weighed against the costs of further evolution by sequential random mutagenesis. Four genes encoding para-nitrobenzyl (pNB) esterase variants exhibiting enhanced activity were recombined in two cycles of high-fidelity DNA shuffling and screening. Genes encoding enzymes exhibiting further improvements in activity were analyzed in order to elucidate evolutionary processes at the DNA level and begin to provide an experimental basis for choosing in vitro evolution strategies and setting key parameters for recombination. DNA sequencing of improved variants from the two rounds of DNA shuffling confirmed important features of the recombination process: rapid fixation and accumulation of beneficial mutations from multiple parent sequences as well as removal of silent and deleterious mutations. The five to sixfold further enhancement of total activity towards the para-nitrophenyl (pNP) ester of loracarbef was obtained through recombination of mutations from several parent sequences as well as new point mutations. Computer simulations of recombination and screening illustrate the trade-offs between recombining fewer parent sequences (in order to reduce screening requirements) and lowering the potential for further evolution. Search strategies which may substantially reduce screening requirements in certain situations are described. (C) 1997 Academic Press Limited.