Baculovirus expression modified for enhanced membrane protein production

描述（由申请方提供）：本研究的目的是确定阻碍使用杆状病毒表达系统常规高水平表达完整膜蛋白的潜在机制。在这项研究中，一个快速克隆系统将被用来产生重组杆状病毒表达荧光蛋白标记的模型膜蛋白作为工具，用于确定可能的原因表达差，并评估有效性的表达系统的变化，以减轻这些瓶颈。荧光标签将有助于通过荧光显微镜、流式细胞术和凝胶荧光分析蛋白表达、表达动力学和定位。微阵列分析和RNA沉默将用于鉴定阻碍或增强膜蛋白表达的候选宿主和病毒基因。这些数据将被分析，以推断哪些细胞途径和过程可能受到影响。基于这些分析，将测试宿主细胞和病毒基因表达的修饰作为改善膜蛋白生产和质量的手段。生物化学和分子技术将与荧光蛋白监测结合使用，以评估这些变化对适用于结构研究的膜蛋白生产的影响。这些研究的结果将用于设计新一代杆状病毒表达载体，并开发用于膜蛋白合成的优化宿主细胞。公共卫生相关性：该项目与人类健康的相关性是开发用于结构研究的膜蛋白的生产方法，以更好地了解当膜蛋白突变，错误折叠或错误表达时引起高血压，癌症，阿尔茨海默病和慢性疼痛等疾病的分子机制，并帮助设计针对这些疾病的有效和更安全的药物。

DESCRIPTION (provided by applicant): The goal of this study is to identify underlying mechanisms, which hinder routine high-level expression of integral membrane proteins using the baculovirus expression system. In this study a rapid cloning system will be employed to generate recombinant baculoviruses expressing fluorescent-protein-tagged model membrane proteins as tools for identifying possible causes for poor expression and evaluating the effectiveness of changes in the expression system made to alleviate those bottlenecks. The fluorescent-tag will facilitate analysis of protein expression, expression kinetics, and localization by fluorescent microscopy, flow cytometry, and in gel fluorescence. Microarray analyses and RNA-silencing will be used to identify candidate host and virus genes that either impede or enhance membrane protein expression. These data will be analyzed to deduce what cellular pathways and processes may be affected. Based on these analyses, modifications in host cell and virus gene expression will be tested as means for improving membrane protein production and quality. Biochemical and molecular techniques will be used in conjunction with fluorescent protein monitoring to assess the effects of these changes on production of membrane proteins suitable for structural studies. Results from these studies will be used to design a new generation of baculovirus expression vectors and develop host cells optimized for membrane protein synthesis. PUBLIC HEALTH RELEVANCE: The human health relevance of this project is the development methods to produce membrane proteins for structural studies in order to better understand the molecular mechanisms responsible for conditions such as hypertension, cancer, Alzheimer's disease, and chronic pain that arise when membrane proteins are mutated, misfolded, or misexpressed and to aid in the design of effective and safer drugs for these conditions.