Scanning amino acid mutagenesis for protein engineering

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多年，但当人们寻求构建突变文库时，如丙氨酸扫描，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“完美”混合物更多的筛选或选择。本提案旨在创建一种简单、快速、有效的方法，在任何蛋白质编码序列中执行扫描密码子突变。再加上阅读框选择，这将允许快速生产包含替换一组确定的氨基酸的蛋白质集合，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，用于从开放阅读框中随机插入和删除框内密码子突变；ii)通过对大肠杆菌中表达的一种反选择酶进行全基因丙氨酸扫描来验证该方法；iii)生成一系列“智能”2-内酰胺酶文库，这些文库富含二硫键或带电氨基酸，并筛选这些文库以提高热稳定性。iv)使用该技术在整个多蛋白复合物的每个可能位置单独加入光交联氨基酸，从而允许随后的质谱分析。公共卫生相关性：该提案将开发新的蛋白质工程技术，为改进生物催化剂和治疗性蛋白质提供途径。此外，它将为大蛋白复合物和膜蛋白的结构-功能研究提供工具，这两者都是目前难以使用现有方法研究的。

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.