NOVEL SOLUBILITY-ENHANCING PROTEIN EXPRESSION TECHNOLOGY

描述(申请人提供)：后基因组时代的一个主要目标是表达大量的蛋白质编码序列，最终导致对蛋白质相互作用的更好理解和新疗法的发展。表达异源重组蛋白最受欢迎的宿主是大肠杆菌。尽管有许多改进，但在大肠杆菌中生产可溶蛋白质仍然是结构基因组学的主要瓶颈：通常，50%的重组蛋白质以不可溶的形式表达。优化可溶性蛋白表达的方法是劳力和试剂密集型的。它们包括筛选生长条件、宿主菌株和溶解性增强融合伙伴，并通过细胞裂解、分级和凝胶电泳法评估溶解性。这项研究的目标是开发一个整合表达载体和宿主菌的系统，以提高重组蛋白的可溶性表达。该系统将包括一个新的黄色荧光蛋白标签，它将作为重组蛋白表达和溶解性的在体报告。这种简单的视觉读数将有助于对个体和高通量的表达进行筛选。此外，我们将利用这一报告系统对促进困难靶标可溶性表达的新型蛋白融合伙伴进行基因筛选。我们将验证得到的“溶解度标签”，并将它们整合到一套用于蛋白质表达和纯化的产品中。所得到的系统将通过对融合伙伴、宿主菌株和表达条件的并行筛选，实现对可溶性蛋白表达的高通量优化。这对于单个蛋白质的表达也同样是一个巨大的优势，最大限度地减少了劳动力和试剂的使用。这些努力的成功预计将对生物医学产生重大影响，无论是出于学术目的还是蛋白质疗法的发展。 公共卫生相关性：用于结构和功能研究的蛋白质表达通常在细菌宿主大肠杆菌中进行。重要的是，在151种生物药用蛋白质中，有30%是在大肠杆菌中生产的，2005年的产品销售额为530亿美元。然而，由于许多蛋白质不能折叠成可溶的构象，细菌中外源蛋白质的生产受到阻碍。目前这项研究计划的目标是开发一种在大肠杆菌中增强可溶性蛋白表达的系统。

DESCRIPTION (provided by applicant): A major goal in the post-genomic era is to express the vast collection of protein- coding sequences, eventually resulting in a better understanding of protein interactions and the development of novel therapeutics. The most favored host for heterologous recombinant protein expression is Escherichia coli. Despite many improvements, producing soluble proteins in E. coli is still a major bottleneck for structural genomics: typically, >50% of recombinant proteins are expressed in an insoluble form. Methods to optimize soluble protein expression are labor- and reagent-intensive. They involve screening for growth conditions, host strains, and solubility enhancing fusion partners and assessing solubility by cell lysis, fractionation and gel electrophoresis. The goal of the proposed research is to develop an integrated system of expression vectors and host strains to improve soluble expression of recombinant proteins. The system will include a novel yellow fluorescent protein tag that will function as an in vivo reporter of expression and solubility of the recombinant protein. This simple visual readout will facilitate individual and high-throughput expression screening. Further, we will exploit this reporter system to conduct genetic screens for novel protein fusion partners that promote soluble expression of difficult targets. We will validate the resulting "solubility tags" and incorporate them into a suite of products for protein expression and purification. The resulting system will enable high-throughput optimization of soluble protein expression through parallel screening of fusion partners, host strains, and expression conditions. It will likewise be a great advantage for expression of individual proteins, minimizing the use of labor and reagents. The success of these efforts is expected to have a major impact on biomedicine, both for academic purposes and for development of protein therapeutics. PUBLIC HEALTH RELEVANCE: Expression of proteins for structural and functional studies is usually undertaken in the bacterial host Escherichia coli. Importantly, 30% of the 151 biopharmaceutical proteins, worth $53B in product sales in 2005, were produced in E. coli. However, production of foreign proteins in bacteria is hampered by the inability of many proteins to fold into a soluble conformation. The goal of the current research proposal is to develop a system for enhanced expression of soluble protein in E. coli.