Development of an Algae-based Membrane Protein Expression System

基于藻类的膜蛋白表达系统的开发

• 批准号: 7614567
• 负责人: Alexander M Aravanis
• 金额: $ 10万
• 依托单位: SAPPHIRE ENERGY, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7614567
• 关键词: Address; Algae; Area; Asthma; Biology; Biotechnology; Cells; Chimeric Proteins; Chlamydomonas reinhardtii; Chloroplasts; Collaborations; Communities; Congestive Heart Failure; Data; Deposition; Development; Diabetes Mellitus; Disease; Drug Design; Economics; Engineering; Enzymes; Goals; Green Algae; Green Fluorescent Proteins; Hypersensitivity; Hypertension; Integral Membrane Protein; Lead; Malignant Neoplasms; Membrane; Membrane Fusion; Membrane Proteins; Phase; Phase I Clinical Trials; Phase II Clinical Trials; Process; Production; Proteins; Protocols documentation; Reagent; Recombinant Proteins; Recombinants; Reporter; Research; Research Institute; Sampling; Sapphire; Small Business Innovation Research Grant; Structure; System; Technology; Testing; Thylakoid Membranes; Thylakoids; United States National Institutes of Health; Vaccines; Validation; Work; base; commercialization; cost; design; design and construction; engineering design; microbial; novel; numb protein; overexpression; professor; protein expression; public health relevance; receptor; three dimensional structure; tool; web site

DESCRIPTION (provided by applicant): Sapphire Energy is a biotechnology company developing algae as a scalable low-cost platforms for producing a broad range of proteins with biomedical and biofuels applications. The overall goal of this SBIR application is the development of "enabling technology" capable of producing relatively large amounts of highly purified prokaryotic and eukaryotic integral membrane proteins. Completion of Phase II work would represent validation of a new expression system that uses the chloroplast of the eukaryotic green algae Chlamydomonas reinhardtii as a cost-effective platform for the production of these biologically and pharmaceutically important molecules. Commercialization opportunities include the use of the system for production of important IMP samples for structure-based drug design (SBDD) studies. Production of sufficient amounts of IMPs' remains the most significant bottleneck in efforts to structurally characterize these biologically important molecules, particularly eukaryotic IMPs. The new expression system could also serve as a platform for the industrial scale production of proteins (e.g. microbial membrane proteins) used as vaccines. The immediate goal of Phase I studies is to show that chloroplast thylakoid membranes are suitable substrates for the deposition of recombinant membrane proteins, particularly important mammalian proteins such as transmembrane receptors. Chloroplast thylakoid membranes make up a large portion of algal cells and are quite elastic, capable of harboring large quantities of membrane proteins. Previous studies had generated fusion proteins that contain chloroplast trans-membrane (TM) domains from the photosynthetic proteins, D1 and D2 fused to green fluorescent protein as a reporter. These chimeric recombinant proteins accumulate to relatively high levels in thylakoids, demonstrating that these membranes are capable of harboring engineered recombinant proteins. In this project we will generate recombinant IMPs using a panel of test proteins. We will attempt to understand processes leading to their accumulation in thylakoid membranes with the goal of developing tools, reagents, and protocols for the routine high-throughput over-expression of IMPs. To attain this goal, we propose to achieve three specific objectives; a) Design and Engineer chimeric chloroplast integral membrane fusion proteins, b) Overexpress integral membrane proteins using constructs designed in Aim1, and c) Attempt preliminary purification of a number of these membrane protein that are observed to have high level of expression. Public Health Relevance: The long term goal of this study is the development of a cost-effective platform using the chloroplast of the green algae Chlamydomonas reinhardtii as an expression engine for integral membrane proteins. Successful completion of the project will provide a new and powerful tool to the Structure- Based Drug Design community as it will provide highly purified material for functional and structural studies. Application of the technology will have impact on disease areas such as diabetes, congestive heart failure, cancer, asthma, allergies, high blood pressure and others.

描述（由申请人提供）：蓝宝石能源是一家生物技术公司，开发藻类作为可扩展的低成本平台，用于生产生物医学和生物燃料应用的多种蛋白质。该SBIR应用的总体目标是开发能够生产相对大量高纯度原核和真核整体膜蛋白的“使能技术”。II期工作的完成将代表一种新的表达系统的验证，该系统使用真核绿藻莱茵衣藻的叶绿体作为生产这些生物学和药学上重要分子的成本效益平台。商业化机会包括使用该系统生产用于基于结构的药物设计（SBDD）研究的重要IMP样品。在研究这些重要的生物分子，特别是真核imp分子的结构特征时，产生足够数量的imp仍然是最大的瓶颈。新的表达系统还可以作为工业规模生产用作疫苗的蛋白质（例如微生物膜蛋白）的平台。第一阶段研究的直接目标是表明叶绿体类囊体膜是沉积重组膜蛋白的合适底物，特别是重要的哺乳动物蛋白，如跨膜受体。叶绿体类囊体膜构成了藻类细胞的很大一部分，具有相当的弹性，能够容纳大量的膜蛋白。以前的研究已经从光合蛋白中产生了含有叶绿体跨膜（TM）结构域的融合蛋白，D1和D2作为报告基因融合到绿色荧光蛋白上。这些嵌合重组蛋白在类囊体中积累到相对较高的水平，表明这些膜能够容纳工程重组蛋白。在这个项目中，我们将使用一组测试蛋白生成重组imp。我们将尝试了解导致它们在类囊体膜中积累的过程，目的是开发常规高通量过表达imp的工具、试剂和方案。为实现这一目标，我们建议实现三个具体目标：a)设计和工程嵌合叶绿体完整膜融合蛋白，b)使用Aim1设计的构建体过表达完整膜蛋白，c)尝试初步纯化观察到具有高表达水平的这些膜蛋白。公共卫生相关性：本研究的长期目标是开发一种具有成本效益的平台，利用绿藻莱茵衣藻的叶绿体作为整体膜蛋白的表达引擎。该项目的成功完成将为基于结构的药物设计界提供一个新的有力工具，因为它将为功能和结构研究提供高纯度的材料。该技术的应用将对糖尿病、充血性心力衰竭、癌症、哮喘、过敏、高血压等疾病领域产生影响。