A Genetic Assay for Protein Evolution and Proteomics

定向进化有望创造出在生物医学研究中用作试剂的设计酶， 医学诊断学，甚至可能是治疗学。定向进化已被成功地用于 选择具有从头结合和催化性质的RNA分子，以及最近具有从头结合和催化性质的蛋白质 来自大分子文库(&gt；108)的结合特性。研究人员还未能将 然而，定向进化的力量与酶催化有关，因为没有合适的高活性基因。 对于大多数酶催化反应的功能的吞吐量选择。虽然定向进化现在已经 通常用于提高来自小蛋白质文库(103-104)的已知酶的活性 通过传统酶分析的自动化，酶的常规定向进化 底物专一性的规定变化还有待实现。酶的从头进化 活动从来没有实现过。为了解决定向进化领域的这一瓶颈，我的实验室 在前一个授权期创造了“化学互补”？高通量的酶检测 成键和裂解反应的催化作用。我们在下一个授权期的目标是使用化学物质 在蛋白质引导的进化领域中跨越这一下一个里程碑的互补 底物专一性有规定变化的酶的进化。具体地说，我们建议实现 这一目标是通过TIM桶的定向进化实现的，TIM桶是一种酶催化的“特权”支架，具有 改变底物专一性以催化两个合成上重要的转化，碳水化合物 合成(目标1)和羟醛反应(目标2)。定向进化有两个步骤？第一个DNA 第二，选择最合适的变异体。拥有 工程酵母，以扩大化学范围，使细胞具有选择性优势(化学 互补)，在目标3中，我们进一步工程酵母也在体内进行DNA突变。我们的 长期目标是TIM Barrel酶用于各种化学品的常规从头指导的进化 变形。

Directed evolution holds promise for creating designer enzymes for use as reagents in biomedical research, medical diagnostics, and perhaps even as therapeutics. Directed evolution has been used successfully to select RNA molecules with de novo binding and catalytic properties and most recently proteins with de novo binding properties from large libraries (>108) of molecules. Researchers have not been able to bring the power of directed evolution to bear on enzyme catalysis, however, because there are no suitable high- throughput selections for function for most enzyme-catalyzed reactions. While directed evolution has now been used routinely to increase the activity of known enzymes from small libraries (103-104) of protein variants by automation of traditional enzyme assays, the routine directed evolution of enzymes with prescribed changes in substrate specificity is yet to be achieved. The de novo directed evolution of enzyme activity has never been achieved. To address this bottleneck in the directed evolution field, my laboratory created "chemical complementation" in the previous granting period¿a high-throughput assay for enzyme catalysis of bond formation and cleavage reactions. Our goal in this next granting period is to use chemical complementation to cross this next milestone in the protein directed evolution field¿the routine directed evolution of enzymes with prescribed changes in substrate specificity. Specifically, we propose to achieve this goal through the directed evolution of TIM barrels, a "privileged" scaffold for enzyme catalysis, with altered substrate specificities for catalysis of two synthetically important transformations, carbohydrate synthesis (Aim 1) and the aldol reaction (Aim 2). There are two steps to directed evolution¿first DNA mutagenesis to create large libraries of protein variants; and second, selection of the fittest variants. Having engineered yeast to expand the range of chemistry that can give the cell a selective advantage (chemical complementation), in Aim 3 we further engineer yeast to also carry out the DNA mutagenesis in vivo. Our long-term goal is the routine de novo directed evolution of TIM barrel enzymes for myriad chemical transformations.