Scanning amino acid mutagenesis for protein engineering

用于蛋白质工程的扫描氨基酸诱变

• 批准号: 8264277
• 负责人: THOMAS ASHTON CROPP
• 金额: $ 19.8万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8264277
• 关键词: Scanning; amino; acid; mutagenesis; protein

DESCRIPTION (provided by applicant): Traditional protein site-directed mutagenesis has served biochemists for many years but is lacking when one seeks to construct libraries of mutants such as in alanine scanning. In protein engineering experiments, protein libraries are redundant mixtures requiring more screening or selection than would be necessary from a "perfect" mixture. This proposal seeks to create a simple, fast, and effective way to perform scanning codon mutagenesis throughout any protein coding sequence. Coupled with a reading frame selection, this will allow the rapid production of collections of proteins that contain substitutions of a defined set of amino acids, including those that contain unnatural functional groups. The proposed studies will i)develop a Mu transposon-based method for the random insertion and deletion of in-frame codon mutations from an open reading frame, ii) verify this methodology by performing whole-gene alanine scanning on an a counterselectable enzyme expressed in E. coli iii) generate a series of "smart" 2-lactamase enzyme libraries which are rich in disulfide bonds or charged amino acids and screen those libraries for improved thermostablity, and iv)use this technology to singly incorporate photo-crosslinking amino acids at every possible position throughout a multi-protein complex allowing subsequent analysis by mass-spectroscopy. PUBLIC HEALTH RELEVENCE: This proposal will develop new protein engineering technology that provides access to improved biocatalysts and therapeutic proteins. Furthermore it will provide a tool for structure-function studies on large protein complexes and membrane proteins, both of which are difficult to study using currently available methods.

描述（由申请人提供）：传统的蛋白质定点诱变已经为生物化学家服务了很多年，但当人们试图构建突变体文库（例如丙氨酸扫描）时，却缺乏这种方法。在蛋白质工程实验中，蛋白质文库是多余的混合物，需要比“完美”混合物更多的筛选或选择。该提案旨在创建一种简单、快速且有效的方法来在任何蛋白质编码序列中进行扫描密码子诱变。与阅读框选择相结合，这将允许快速生产含有一组定义的氨基酸（包括含有非天然官能团的氨基酸）的取代的蛋白质集合。拟议的研究将i）开发一种基于Mu转座子的方法，用于从开放阅读框中随机插入和删除框内密码子突变，ii）通过对表达于其中的反选择酶进行全基因丙氨酸扫描来验证该方法。大肠杆菌 iii) 生成一系列富含二硫键或带电氨基酸的“智能”2-内酰胺酶文库，并筛选这些文库以提高热稳定性，以及 iv) 使用该技术单独掺入多蛋白复合物中每个可能位置的氨基酸光交联，允许随后通过质谱进行分析。公共健康相关性：该提案将开发新的蛋白质工程技术，提供改进的生物催化剂和治疗性蛋白质。此外，它将为大型蛋白质复合物和膜蛋白的结构功能研究提供工具，这两种蛋白质很难使用目前可用的方法进行研究。