SBIR Phase I: Production of Properly-Folded Recombinant Proteins in Eschericihia coli

SBIR 第一阶段：在大肠杆菌中生产正确折叠的重组蛋白

• 批准号: 1248151
• 负责人: Donald O'Keefe
• 金额: $ 14.5万
• 依托单位: Da Yu Enterprises, LLC
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1248151&HistoricalAwards=false
• 关键词: SBIR; Phase; Production; Properly; Folded

This Small Business Innovation Research (SBIR) Phase I project proposes a new expression platform in Escherichia coli applicable to both drug discovery and biopharmaceutical manufacturing. It solves and leverages recombinant protein misfolding in E. coli. Small molecular weight compounds are used in conjunction with high recombinant protein expression in E. coli to produce proteins with their native structure. In the area of drug discovery this allows one to obtain properly-folded proteins for structural and functional genomics, high-throughput screening, and to identify specific protein stabilizers for protein-misfolding diseases. For biopharmaceutical manufacturing, recombinant protein inclusion body formation is prevented and refolding steps are eliminated. Cultures of E. coli containing a plasmid encoding a gene for the recombinant protein of interest are allowed to grow to a desired cell density and then an innovative condition-dependent change is made that allows the passive influx of small molecular weight compounds into the E. coli cytoplasm during protein synthesis that support proper protein folding. This proposal will examine the feasibility of this condition-dependent change and the alteration of the cell?s cytoplasm in a prescribed way to produce properly-folded proteins by monitoring the soluble and functional expression of selected recombinant proteins.The broader impact/commercial potential of this project will be a gene expression platform that is an enabling technology that has applications wherever properly-folded recombinant proteins are utilized. The described system can produce recombinant proteins in E. coli that would otherwise be toxic such as membrane proteins, which are highly desirable drug targets but are recalcitrant proteins for functional and structural genomics research. It is the first E. coli system described that can be leveraged to screen for drugs directed against protein-misfolding diseases. It is a new avenue for identifying drugs to treat devastating and costly diseases including p53-mediated cancers, Alzheimer's, Parkinson's, Tay-Sachs, and amyotrophic lateral sclerosis. The proposed expression platform can potentially make these proteins more research accessible. Inclusion body formation in E. coli may also benefit from this technology. This is particularly applicable to recombinant proteins intended as biopharmaceuticals. Preventing inclusion bodies increases process yields, shortens process development timelines and reduces commercial manufacturing costs. Hence, this proposal describes a new tool to discover drugs for unmet medical needs, expedite drug discovery, and streamline biopharmaceutical development and manufacturing; all of which will ultimately provide economical and societal benefits.

这个小型企业创新研究(SBIR)第一阶段项目提出了一种新的在大肠杆菌中的表达平台，适用于药物开发和生物制药制造。它解决并利用了重组蛋白在大肠杆菌中的错误折叠。小分子化合物与重组蛋白在大肠杆菌中的高效表达相结合，产生具有天然结构的蛋白质。在药物发现领域，这使人们能够获得正确折叠的蛋白质，用于结构和功能基因组学、高通量筛选，并识别蛋白质错误折叠疾病的特定蛋白质稳定剂。对于生物制药制造，防止了重组蛋白包涵体的形成，并消除了复性步骤。含有编码目的重组蛋白基因的质粒的大肠杆菌培养物被允许生长到所需的细胞密度，然后进行创新的条件依赖的改变，允许在支持适当蛋白质折叠的蛋白质合成期间，小分子化合物被动地流入大肠杆菌细胞质。这项提案将通过监测选定重组蛋白的可溶性和功能性表达来检验这种条件依赖的变化和细胞-S细胞质以规定方式产生正确折叠蛋白的可行性。该项目的更广泛的影响/商业潜力将是一个基因表达平台，这是一种使能技术，在利用正确折叠的重组蛋白的任何地方都有应用。所描述的系统可以在大肠杆菌中产生原本有毒的重组蛋白，如膜蛋白，这些蛋白是非常理想的药物靶标，但对于功能和结构基因组学研究来说却是顽固的蛋白。这是第一个被描述的可以用来筛选针对蛋白质错误折叠疾病的药物的大肠杆菌系统。它是确定治疗毁灭性和昂贵疾病的药物的新途径，包括p53介导的癌症、阿尔茨海默氏症、帕金森氏症、泰-萨克斯和肌萎缩侧索硬化症。拟议的表达平台可能会使这些蛋白质的研究更容易获得。在大肠杆菌中形成包涵体也可能受益于这项技术。这尤其适用于打算用作生物制药的重组蛋白。防止包含体增加了工艺产量，缩短了工艺开发时间，降低了商业制造成本。因此，这项建议描述了一种新的工具，用于发现未得到满足的医疗需求的药物，加快药物发现，并简化生物制药的开发和制造；所有这些最终都将提供经济和社会效益。