Entirely in vivo enzyme evolution and engineering of Cre recombinase

描述（由申请人提供）：定向进化是一种创造具有新特性的蛋白质的通用方法。酶的进化已经提供了更高的稳定性、更高的效率和改变的特异性或对映体选择性的变体。随着酶在工业过程、药物合成、消费者保健和清洁产品中的应用趋势不断增长，将需要更新、更好的定向进化技术来解决更复杂的问题。当前定向进化方法的一个主要限制是用于搜索改进蛋白质的随机文库。目前的技术可以使平均库在10'^8-10'^9的量级上，而研究表明，要发现具有显著改变的特异性或全新功能的酶，需要更大的库（bbb10 '^13）。该提案描述了酵母中的一个新系统，旨在克服当前库大小的限制。目的1描述了一种在出芽酵母酿酒酵母内进行靶基因的体内诱变的方法。该方法依赖于酵母中高效的同源重组机制。在质粒上引入诱变文库，使用高度特异的内切酶将其释放。由此产生的线性DNA链将通过同源重组靶向并诱变感兴趣的基因。目的2概述了一种在酵母之间共享诱变文库以增加潜在文库大小的方法。通过允许两个具有互补随机文库的酵母集合交配，这两个文库可以在整个种群中共享。将施加选择压力，因此只传播最好的变体，而实际上搜索所有可能的突变体。这种协议具有搜索大型虚拟库的潜力，远远超出了现有技术的能力。目标3提出了一个定向进化实验，使用目标1和目标2中开发的方法。Cre是一种广泛应用于小鼠遗传学的位点特异性重组酶，将通过体内诱变和交配过程进化出一种新的变体。这个实验应该能令人信服地证明这些新方法的效用。公共卫生相关性：本提案描述了一种酶定向进化的新技术。利用这种技术产生的新酶可以用于发现和合成新药或作为药物本身。所提出的方法可以提供多种用途的蛋白质，如清洁产品，环境修复和人造器官。

DESCRIPTION (provided by applicant): Directed evolution is a versatile method for the creation of proteins with novel properties. The evolution of enzymes has already provided variants with higher stability, increased efficiency, and altered specificity or enantioselectivity. As the trend of using enzymes in industrial processes, the synthesis of pharmaceuticals, and consumer health and cleaning products continues to grow, newer and better directed evolution techniques will be required to solve more complex problems. One major limitation of current directed evolution approaches is the random library used to search for improved proteins. Current technology enables average libraries on the order of 10'^8-10'^9 variants, while research has suggested that much larger libraries (>10'^13) will be necessary to discover enzymes with significantly altered specificities or entirely new functions. This proposal describes a new system in yeast that aims to overcome current limitations on library size. Aim 1 describes a method for performing in vivo mutagenesis of a target gene within the budding yeast S. cerevisiae. This method relies on the highly efficient homologous recombination machinery in yeast. Mutagenic libraries, introduced on plasmids, will be liberated using a highly specific endonuclease. The resulting linear DNA strands will target and mutagenize a gene of interest through homologous recombination. Aim 2 outlines a method for sharing mutagenic libraries between yeast to increase potential library size. By allowing two collections of yeast with complementary random libraries to mate, those two libraries can be shared among the entire population. A selective pressure will be applied, thus propagating only the best variants while essentially searching all possible mutants. Such a protocol has the potential to search very large virtual libraries well beyond the ability of existing technology. Aim 3 proposes a directed evolution experiment that uses the methods developed in Aims 1 and 2. A new variant of Cre, a site-specific recombinase widely used in mouse genetics, will be evolved using the in vivo mutagenesis and mating procedures. This experiment should convincingly demonstrate the utility of these new methods. PUBLIC HEALTH RELEVANCE: This proposal describes a new technique for directed evolution of enzymes. New enzymes generated using this technique could be used in the discovery and synthesis of new drugs or as drugs themselves. The proposed methodology could provide access to proteins with uses as varied as cleaning products, environmental remediation, and artificial organs.