NOVEL SOLUBILITY-ENHANCING PROTEIN EXPRESSION TECHNOLOGY

新型增强溶解度的蛋白质表达技术

• 批准号: 8250969
• 负责人: DAVID Alan MEAD
• 金额: $ 39.41万
• 依托单位: LUCIGEN CORPORATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8250969
• 关键词: Acids; Bacteria; Biological; Biological Products; Categories; Cell membrane; Cells; Chimera organism; Chimeric Proteins; Cloning; Code; Collaborations; Collection; Cytolysis; Cytoplasmic Protein; Detection; Development; Drug Delivery Systems; Environment; Enzymes; Escherichia coli; Evaluation; Expression Library; Fluorescence; Foxes; Fractionation; Gene Fusion; Genes; Genetic Screening; Genome; Genomics; Goals; Green Fluorescent Proteins; Growth; High temperature of physical object; Human; Individual; Integral Membrane Protein; Ion Channel; Letters; Libraries; Membrane Proteins; Methods; Molecular Biology; Molecular Conformation; Open Reading Frames; Organism; Phase; Photobleaching; Preparation; Production; Property; Proteins; Proteomics; Reagent; Recombinant Proteins; Reporter; Research; Research Proposals; Rhamnose; Sales; Screening procedure; Services; Shotguns; Solubility; Solutions; Speed; Structural Protein; System; Technology; Therapeutic; Universities; Visual; Wisconsin; design; expression cloning; expression vector; gel electrophoresis; genome wide association study; high throughput screening; improved; in vivo; metagenome; microbial genome; novel; novel therapeutics; polypeptide; progenitor; promoter; protein B; protein expression; protein purification; protein structure function; quantum; research study; structural genomics; success; therapeutic protein; tool; vector

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.

描述（由申请人提供）：后基因组时代的主要目标是表达大量蛋白质编码序列，最终导致更好地理解蛋白质相互作用和开发新的治疗方法。异源重组蛋白表达的最有利宿主是大肠杆菌。尽管有许多改进，但在E.大肠杆菌仍然是结构基因组学的主要瓶颈：通常，>50%的重组蛋白以不溶性形式表达。优化可溶性蛋白表达的方法是劳动密集型和试剂密集型的。它们涉及筛选生长条件、宿主菌株和溶解度增强融合伴侣，并通过细胞裂解、分级分离和凝胶电泳评估溶解度。本研究的目的是建立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。该系统将包括一种新的黄色荧光蛋白标签，其将作为重组蛋白的表达和溶解度的体内报告物。这种简单的视觉读数将有助于个体和高通量表达筛选。此外，我们将利用这一报告系统进行基因筛选的新蛋白融合伙伴，促进可溶性表达的困难的目标。我们将验证所得的“溶解度标签”，并将其纳入一套产品的蛋白质表达和纯化。所得到的系统将通过融合伴侣、宿主菌株和表达条件的平行筛选来实现可溶性蛋白表达的高通量优化。这对于单个蛋白质的表达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，2005年价值530亿美元的151种生物制药蛋白质中有30%是在E。杆菌然而，细菌中外源蛋白质的产生受到许多蛋白质不能折叠成可溶性构象的阻碍。本研究的目的是建立一个可溶性蛋白在大肠杆菌中的高效表达系统。杆菌