Scanning amino acid mutagenesis for protein engineering

描述(申请人提供)：传统的蛋白质定点突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的文库时，例如在丙氨酸扫描中，就缺乏了。在蛋白质工程实验中，蛋白质文库是多余的混合物，需要比从“完美”混合物中进行更多的筛选或选择。这一建议旨在创造一种简单、快速和有效的方法来在任何蛋白质编码序列中进行密码子扫描突变。与阅读框选择相结合，这将允许快速产生包含一组特定氨基酸的替代的蛋白质集合，包括那些包含非天然官能团的蛋白质。拟议的研究将：i)开发一种基于Mu转座子的方法，用于从开放阅读框架中随机插入和删除框内密码子突变；ii)通过对在大肠杆菌III中表达的可逆选择的酶进行全基因丙氨酸扫描来验证该方法；iii)产生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)使用该技术在整个多蛋白质复合体中的每个可能位置单一地引入光交联氨基酸，以便随后通过质谱分析。公共卫生进展：这项提案将开发新的蛋白质工程技术，提供获得改进的生物催化剂和治疗性蛋白质的途径。此外，它还将为大型蛋白质复合体和膜蛋白的结构-功能研究提供工具，这两者都很难用现有的方法进行研究。

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.