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

这个小企业创新研究（SBIR）第一阶段项目提出了一个新的表达平台，在大肠杆菌适用于药物发现和生物制药制造。它解决并利用了重组蛋白在大肠杆菌中的错误折叠。杆菌小分子量化合物与在E.大肠杆菌产生具有天然结构的蛋白质。在药物发现领域，这使得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，并确定蛋白质错误折叠疾病的特定蛋白质稳定剂。对于生物制药生产，防止了重组蛋白包涵体的形成，并消除了重折叠步骤。E.使含有编码目的重组蛋白基因的质粒的大肠杆菌生长到所需的细胞密度，然后进行创新的条件依赖性改变，使小分子量化合物被动流入大肠杆菌。在蛋白质合成过程中支持蛋白质正确折叠的大肠杆菌细胞质。这项建议将研究这种条件依赖性的变化和细胞的改变的可行性？的细胞质中以规定的方式产生正确折叠的蛋白质，通过监测选定的重组蛋白的可溶性和功能性表达。更广泛的影响/商业潜力，这个项目将是一个基因表达平台，这是一个使能技术，具有应用程序，只要正确折叠的重组蛋白被利用。所述系统可以在E.大肠杆菌中，否则将是有毒的，如膜蛋白，这是非常理想的药物靶点，但功能和结构基因组学研究是不确定的蛋白质。这是第一个E。大肠杆菌系统描述，可以利用筛选针对蛋白质错误折叠疾病的药物。这是一个新的途径，用于确定药物来治疗破坏性和昂贵的疾病，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使这些蛋白质更容易获得研究。E.大肠杆菌也可能受益于这项技术。这特别适用于打算作为生物药物的重组蛋白。防止包涵体增加了工艺产率，缩短了工艺开发时间表并降低了商业制造成本。因此，该提案描述了一种新的工具，用于发现未满足医疗需求的药物，加快药物发现，并简化生物制药开发和制造;所有这些最终都将提供经济和社会效益。

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.