Rapid evolution of β-glucuronidase specificity by saturation mutagenesis of an active site loop
Rapid evolution of β-glucuronidase specificity by saturation mutagenesis of an active site loop
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DOI：
10.1074/jbc.m401447200
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发表时间：
2004-06-18
影响因子：
4.8
通讯作者：
Matsumura, I
中科院分区：
文献类型：
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作者：
Geddie, ML;Matsumura, I
蛋白质工程师已广泛采用定向进化作为设计算法，但从业者还没有达成共识的最佳方法来进化蛋白质分子识别。我们以前使用DNA改组指导进化的大肠杆菌β-葡萄糖醛酸酶（GUS）的变种，增加β-半乳糖苷酶的活性。在该实验中鉴定了作为活性位点环的一部分的氨基酸557、566和568中的上位（协同）突变（Matsumura，I.，和Ellington，A. D.等人（2001）J. Mol. 305，331-339）。在这里，我们表明，这些残基的位点饱和诱变，在E。高通量筛选导致克隆的快速进化，这些克隆在与对硝基苯基-β-D-吡喃木糖苷（pNP-xyl）的反应中表现出增加的活性。14个最适克隆的木糖苷酶活性平均比野生型GUS高30倍。将相应的14个质粒合并，通过长PCR扩增，用T4 DNA连接酶自连接，并转化到E.杆菌在第二轮筛选中分离出13个表现出木糖苷酶活性平均提高80倍的克隆。其中一种进化的蛋白质在与pNP-xyl的反应性（k（cat）/K-m）方面表现出比野生型高出200倍的相似改进，具有290，000倍的特异性反转。对13个第2轮分离株的序列分析表明，所有分离株都是全质粒PCR过程中发生的分子间重组事件的产物。使用DNA改组和交错延伸过程（StEP）的进一步进化导致适度的改善。这些结果强调了上位相互作用的重要性，并表明它们可以通过简单的全质粒PCR技术的变化进行优化。
Protein engineers have widely adopted directed evolution as a design algorithm, but practitioners have not come to a consensus about the best method to evolve protein molecular recognition. We previously used DNA shuffling to direct the evolution of Escherichia coli beta-glucuronidase (GUS) variants with increased beta-galactosidase activity. Epistatic ( synergistic) mutations in amino acids 557, 566, and 568, which are part of an active site loop, were identified in that experiment (Matsumura, I., and Ellington, A. D. (2001) J. Mol. Biol. 305, 331-339). Here we show that site saturation mutagenesis of these residues, overexpression of the resulting library in E. coli, and high throughput screening led to the rapid evolution of clones exhibiting increased activity in reactions with p-nitrophenyl-beta-D-xylopyranoside (pNP-xyl). The xylosidase activities of the 14 fittest clones were 30-fold higher on average than that of the wild-type GUS. The 14 corresponding plasmids were pooled, amplified by long PCR, self-ligated with T4 DNA ligase, and transformed into E. coli. Thirteen clones exhibiting an average of 80-fold improvement in xylosidase activity were isolated in a second round of screening. One of the evolved proteins exhibited a similar to200-fold improvement over the wild type in reactivity (k(cat)/K-m) with pNP-xyl, with a 290,000-fold inversion of specificity. Sequence analysis of the 13 round 2 isolates suggested that all were products of intermolecular recombination events that occurred during whole plasmid PCR. Further rounds of evolution using DNA shuffling and staggered extension process (StEP) resulted in modest improvement. These results underscore the importance of epistatic interactions and demonstrate that they can be optimized through variations of the facile whole plasmid PCR technique.