Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.
Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme.
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通过缺陷溶菌酶的补偿突变描绘进化挽救途径。
DOI：
10.1002/pro.5560071018
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Poteete,AR
中科院分区：
文献类型：
--
作者：
Jucovic,M;Poteete,AR
无模型方法（随机诱变，DNA改组）结合更“合理”的三维信息引导随机化已用于缺陷型T4溶菌酶突变体中溶菌酶活性的定向进化。来源于噬菌体λ的特化溶菌酶克隆载体噬菌体依赖于T4溶菌酶功能以形成噬斑。T4溶菌酶中的W138P取代完全消除了其空斑形成能力。选择在全基因的顺序随机诱变后以及在Trp138附近的残基的靶向随机化后W138PT4溶菌酶中的补偿突变。在第二阶段，这些突变通过DNA改组的重组PCR方法随机重组。改组和选择的W138P T4溶菌酶变体为杂合λ噬菌体提供了足够的溶菌酶活性，即使在升高的温度（42 °C）下也能产生正常大小的噬斑。对W138 P修复具有最高补偿信息的单个突变是取代A146 F和A146 M，它们是通过组合突变对Trp 138附近的三个残基进行靶向随机化后选择的。然而，进化最好的W138P T4溶菌酶积累了来自随机诱变和靶向随机化变体的突变。
Model‐free approaches (random mutagenesis, DNA shuffling) in combination with more “rational,” three‐dimensional information‐guided randomization have been used for directed evolution of lysozyme activity in a defective T4 lysozyme mutant. A specialized lysozyme cloning vector phage, derived from phage λ, depends upon T4 lysozyme function for its ability to form plaques. The substitution W138P in T4 lysozyme totally abolishes its plaque‐forming ability. Compensating mutations in W138PT4 lysozyme after sequential random mutagenesis of the whole gene as well as after targeted randomization of residues in the vicinity of Trp138 were selected. In a second stage, these mutations were randomly recombined by the recombinatorial PCR method of DNA shuffling. Shuffled and selected W138P T4 lysozyme variants provide the hybrid λ phage with sufficient lysozyme activity to produce normal‐size plaques, even at elevated temperature (42 °C). The individual mutations with the highest compensatory information for W138P repair are the substitutions A146F and A146M, selected after targeted randomization of three residues in the neighborhood of Trp138 by combinatorial mutagenesis. The best evolved W138P T4 lysozymes, however, accumulated mutations originating from both randomly mutagenized as well as target‐randomized variants.