Protein Evolution by in Vitro Compartmentalization

具有新的、高度专业化的或优化功能的蛋白质的定向进化 需要基因、基因产物及其效应/活性之间的物理关联 产品.尽管这种结合通过体内细胞区室化来维持， 基因型-表型连锁也可以通过PCR文库的转录/翻译来实现 在油：水乳液中。后一种方法，称为体外区室化 (IVC)，消除了与隔室进入相关的一些实际困难，系统 复杂性和与体内进化相关的遗传选择，并已成功地 用于进化具有改进功能或改变特异性的酶。本项目4使用 IVC进化出对RNase H抑制剂耐药的HIV-1 RT变体，我们通过 但在细胞培养中是有毒的。IVC的第二个适应性调整旨在 不断进化的锌指蛋白，其识别PPT-U3连接并基于质量 光谱和核磁共振研究表明，在这个交界处， 可能是逆转录病毒和LTR-逆转录转座子共有的。[对应于2004年的Le Grice项目4] 2007年4月艾滋病毒耐药性方案实地访问报告]

Directed evolution of proteins with novel, highly specialized, or optimized function(s) requires physical association among genes, gene products, and the effects/activities of these products. Whereas this association is maintained by cellular compartmentalization in vivo, genotype-phenotype linkage can also be achieved via transcription/translation of PCR libraries in an oil:water emulsion. The latter approach, referred to as in vitro compartmentalization (IVC), obviates some of the practical difficulties related to compartmental access, system complexity, and genetic selection associated with in vivo evolution, and has been successfully used to evolve enzymes with improved functionality or altered specificity. This project 4 uses IVC to evolve HIV-1 RT variants resistant to RNase H inhibitors that we identified by high-throughput screening but are toxic in cell culture. A second adaptation of IVC aims at evolving zinc finger proteins that recognize the PPT-U3 junction and is based on mass spectrometry and NMR investigations indicating that abnormal duplex geometry at this junction may be common to retroviruses and LTR-retrotransposons. [Corresponds to Le Grice Project 4 in the April 2007 site visit report of the HIV Drug Resistance Program]