Protein Evolution by in Vitro Compartmentalization

具有新颖、高度特化或优化功能的蛋白质的定向进化需要基因、基因产物以及这些产物的作用/活性之间的物理关联。尽管这种关联在体内是通过细胞区隔化来维持的，但基因型-表型连锁也可以通过油：水乳液中PCR文库的转录/翻译来实现。后一种方法被称为体外区室化（IVC），它消除了与区室通路、系统复杂性和与体内进化相关的遗传选择相关的一些实际困难，并已成功地用于进化具有改进功能或改变特异性的酶。该项目4使用IVC进化对RNase H抑制剂具有抗性的HIV-1 RT变体，我们通过高通量筛选确定了这些变体，但在细胞培养中是有毒的。IVC的第二个适应性旨在进化识别PPT-U3连接的锌指蛋白，并基于质谱和核磁共振研究表明，该连接的异常双工几何结构可能是逆转录病毒和ltr -逆转录转座子的共同特征。[对应于2007年4月艾滋病耐药项目实地考察报告中的Le Grice Project 4]

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]