Protein Evolution by in Vitro Compartmentalization

通过体外区室化进行蛋白质进化

• 批准号: 7592918
• 负责人: Stuart F. J. Le Grice
• 金额: $ 41.36万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592918
• 关键词: Antiviral Agents; Attention; Biochemical; Class; Condition; Cultured Cells; Development; Drug resistance; Emulsions; Enzymes; Evolution; Genes; Genetic Transcription; Genotype; Goals; HIV Drug Resistance Program; HIV-1 Reverse Transcriptase; In Vitro; Investigation; Libraries; Mass Spectrum Analysis; Oils; Phenotype; Polymerase Chain Reaction; Proteins; RNA-Directed DNA Polymerase; Reporting; Resistance; Retrotransposon; Retroviridae; Ribonuclease H; Site Visit; Specificity; Standards of Weights and Measures; System; Techniques; Toxic effect; Translations; Variant; Virus Replication; Water; Zinc Fingers; base; directed evolution; genetic selection; high throughput screening; improved; improved functioning; in vivo; infancy; inhibitor/antagonist; novel; prevent; small molecule; tool

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]

具有新颖、高度专业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。虽然这种关联是通过体内细胞区室化维持的，但基因型-表型联系也可以通过油:水乳液中 PCR 文库的转录/翻译来实现。后一种方法称为体外区室化（IVC），消除了与区室进入、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，并已成功用于进化具有改进功能或改变的酶。特异性。该项目 4 使用 IVC 进化出对 RNase H 抑制剂具有抗性的 HIV-1 RT 变体，我们通过高通量筛选鉴定出这种变体，但在细胞培养物中具有毒性。 IVC 的第二个改进旨在进化识别 PPT-U3 连接的锌指蛋白，并基于质谱和 NMR 研究，表明该连接处的异常双链体几何形状可能是逆转录病毒和 LTR 逆转录转座子所共有的。 [对应于 2007 年 4 月 HIV 耐药项目实地考察报告中的 Le Grice 项目 4]