Genetic Method for Evaluating Solubility of Recombinant Proteins in E. coli

描述(申请人提供)：对于大多数应用，重组蛋白质必须以可溶形式纯化；然而，当在大肠杆菌中表达时，许多蛋白质折叠异常并在细胞中形成不容溶解的聚集体。我们建议创造一种遗传方法，使科学家能够利用大肠杆菌表达重组蛋白，立即确定他们的目标是可溶的还是不可溶的。这是直接完成的，而不需要培养这些细胞和分离蛋白质。此外，这种方法还将包括一种基因选择，用于识别不可溶蛋白质的结构域或突变体，使其适用于下游应用。7.项目叙述了解人类、动物和所有细胞生物学的核心部分是研究有机体编码的蛋白质。我们建议开发一种新的方法来加速蛋白质的研究，方法是创建一种快速方法来评估是否可以立即纯化和研究感兴趣的蛋白质，或者在开始这项工作之前是否需要对其进行修饰。

DESCRIPTION (provided by applicant): For most applications, recombinant proteins must be purified in soluble form; however, when expressed in E. coli, many proteins fold aberrantly and become an insoluble aggregate in the cell. We propose to create a genetic method to allow scientists using E. coli for recombinant protein expression to immediately ascertain whether their target is soluble or insoluble. This is done directly without having to grow cultures of these cells and isolate protein. In addition, this method would also comprise a genetic selection for identifying domains or mutants of insoluble proteins that are soluble, making them amenable to downstream applications. 7. PROJECT NARRATIVE A central component towards the understanding of human, animal, and all cell biology is the study of the proteins encoded by the organism. We propose to develop a novel method to accelerate the study of proteins by creating a rapid way to evaluate whether the protein of interest can be immediately purified and studied or whether it needs to be modified before this work can begin.