Geddie Melissa L, Matsumura Ichiro
Department of Biochemistry, Center for Fundamental and Molecular Evolution, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
J Biol Chem. 2004 Jun 18;279(25):26462-8. doi: 10.1074/jbc.M401447200. Epub 2004 Apr 6.
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 approximately 200-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.
蛋白质工程师已广泛采用定向进化作为一种设计算法，但从业者对于进化蛋白质分子识别的最佳方法尚未达成共识。我们之前利用DNA改组来指导具有增强β-半乳糖苷酶活性的大肠杆菌β-葡萄糖醛酸酶（GUS）变体的进化。在该实验中，鉴定出了位于活性位点环中的氨基酸557、566和568中的上位性（协同）突变（松村，I.，和埃林顿，A. D.（2001年）《分子生物学杂志》305卷，331 - 339页）。在此我们表明，对这些残基进行位点饱和诱变、在大肠杆菌中对所得文库进行过表达以及高通量筛选，导致了在与对硝基苯基-β-D-木糖苷（pNP-xyl）反应中表现出活性增强的克隆的快速进化。14个最适配克隆的木糖苷酶活性平均比野生型GUS高30倍。将这14个相应的质粒汇集，通过长PCR进行扩增，用T4 DNA连接酶进行自我连接，然后转化到大肠杆菌中。在第二轮筛选中分离出了13个木糖苷酶活性平均提高了80倍的克隆。其中一种进化后的蛋白质在与pNP-xyl的反应性（k(cat)/K(m)）方面比野生型提高了约200倍，特异性发生了290,000倍的反转。对13个第二轮分离株的序列分析表明，它们都是在全质粒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）进行进一步的进化轮次导致了适度的改进。这些结果强调了上位性相互作用的重要性，并证明它们可以通过简便的全质粒PCR技术的变体来进行优化。
