Development of an Algae-based Membrane Protein Expression System

描述(申请人提供)：蓝宝石能源是一家生物技术公司，开发藻类作为一种可扩展的低成本平台，用于生产各种生物医学和生物燃料应用的蛋白质。这项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完整膜蛋白的“使能技术”。第二阶段工作的完成将意味着验证一种新的表达系统，该系统使用真核绿藻衣藻的叶绿体作为生产这些具有生物和药学意义的重要分子的具有成本效益的平台。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。生产足够数量的IMP‘仍然是对这些生物重要分子，特别是真核IMP进行结构表征的最大瓶颈。新的表达系统还可以作为工业化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证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细胞的很大一部分，具有很强的弹性，能够容纳大量的膜蛋白。以前的研究已经从光合作用蛋白中获得了含有叶绿体跨膜结构域的融合蛋白，D1和D2作为报告与绿色荧光蛋白融合。这些嵌合重组蛋白在类囊体中积累到相对较高的水平，表明这些膜能够携带工程重组蛋白。在这个项目中，我们将使用一组测试蛋白质来生成重组IMP。我们将试图了解导致它们在类囊体膜中积累的过程，目标是开发工具、试剂和方案，用于常规的高通量过量表达IMP。为了实现这一目标，我们提出了三个具体的目标：a)设计和设计嵌合的叶绿体完整膜融合蛋白，b)使用Aim1中设计的构建体过表达完整膜蛋白，以及c)尝试对一些观察到高水平表达的这些膜蛋白进行初步纯化。公共卫生相关性：这项研究的长期目标是开发一个成本效益高的平台，使用绿藻衣藻的叶绿体作为完整膜蛋白的表达引擎。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强大工具，因为它将为功能和结构研究提供高度纯化的材料。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。

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.