A general strategy for the evolution of bond-forming enzymes using yeast display
A general strategy for the evolution of bond-forming enzymes using yeast display
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DOI：
10.1073/pnas.1101046108
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发表时间：
2011-07-12
影响因子：
11.1
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Chen, Irwin;Dorr, Brent M.;Liu, David R.
由研究人员而不是自然选择的常规生成成键反应的有效蛋白质催化剂的能力是分子生命科学的长期目标。在这里，我们描述了一个定向进化策略的酶催化，在原则上，任何键形成反应。该系统集成了酵母展示，酶介导的生物缀合和荧光激活细胞分选，以分离表达催化研究人员选择的两种底物偶联的蛋白质的细胞。我们使用金黄色葡萄球菌分选酶A催化的转肽活性的模型筛选来验证该系统，在单轮筛选后产生6，000倍的富集因子。我们应用该系统进化分选酶A以提高催化活性。经过八轮筛选，我们分离出了分选酶A的变体，与起始野生型酶相比，LPETG偶联活性增加了140倍。与野生型分选酶相比，进化的分选酶变体能够更有效地标记HeLa细胞表面上表达的LPETG标记的人CD154。因为这里开发的方法不依赖于底物或产物的任何特定的可筛选或可选择的性质，所以它代表了现有酶进化方法的有力替代方案。
The ability to routinely generate efficient protein catalysts of bond-forming reactions chosen by researchers, rather than nature, is a long-standing goal of the molecular life sciences. Here, we describe a directed evolution strategy for enzymes that catalyze, in principle, any bond-forming reaction. The system integrates yeast display, enzyme-mediated bioconjugation, and fluorescence-activated cell sorting to isolate cells expressing proteins that catalyze the coupling of two substrates chosen by the researcher. We validated the system using model screens for Staphylococcus aureus sortase A-catalyzed transpeptidation activity, resulting in enrichment factors of 6,000-fold after a single round of screening. We applied the system to evolve sortase A for improved catalytic activity. After eight rounds of screening, we isolated variants of sortase A with up to a 140-fold increase in LPETG-coupling activity compared with the starting wild-type enzyme. An evolved sortase variant enabled much more efficient labeling of LPETG-tagged human CD154 expressed on the surface of HeLa cells compared with wild-type sortase. Because the method developed here does not rely on any particular screenable or selectable property of the substrates or product, it represents a powerful alternative to existing enzyme evolution methods.