Assembly of Cyanobacterial Carboxysomes

描述（由申请人提供）：许多细菌具有涉及微室的复杂代谢调节机制。这些细菌微室由独特的蛋白质外壳组成，结构类似于病毒衣壳，并在特定的反应途径中包裹两种或多种酶。来自蓝藻和化学营养不良的羧小体是最著名的细菌微室。在过去的几年中，已经解决了几个壳组件的晶体结构，并在亚基组装和单个组件功能的知识方面取得了显着进展。到目前为止，还没有真核生物中细菌微室的异源组装的报道。该项目将针对多种羧基体蛋白进入叶绿体。本研究将采用农业渗透技术将外源蛋白与荧光标记融合瞬时表达。任何由表达蛋白组装的分子复合物将用共聚焦显微镜监测，形成的结构将用电子显微镜表征。此外，这项工作将产生稳定的转基因系，其中羧基体蛋白从细胞核或叶绿体基因组中表达。通过表达所有必需的羧基体结构成分，本项目将尝试在真核生物中组装羧基体微室壳。此外，本研究将确定是否有可能将外源蛋白靶向到羧基体外壳上。该研究是构建新型微室的重要一步，在药物输送、代谢工程和生物技术等领域具有潜在的应用前景。

DESCRIPTION (provided by applicant): Many bacteria have a sophisticated mechanism for metabolic regulation that involves microcompartments. These bacterial microcompartments are made up of unique protein shells similar in structure to a viral capsid and enclose two or more enzymes in a particular reaction pathway. Carboxysomes from cyanobacteria and chemoauxotrophs are the most well known bacterial microcompartments. Within the last few years, the crystal structures of several shell components have been solved and have led to remarkable progress in the knowledge of subunit assembly and function of individual components. So far, there has been no report of heterologous assemblies of bacterial microcompartments in eukaryotic organisms. This project will target multiple carboxysome proteins into chloroplasts. This study will employ transient expression of foreign proteins fused with fluorescent tags by agroinfiltration technique. Any molecular complex assembled from the expressed proteins will be monitored with confocal microscopy, and the structures formed will be characterized by electron microscopy. In addition, this work will generate stable transgenic lines, in which carboxysome proteins are expressed from either the nucleus or the chloroplast genome. By expressing all necessary carboxysome structural components, this project will attempt to assemble the carboxysome microcompartment shells in eukaryotes. In addition, this study will determine whether it is possible to target foreign proteins to the carboxysome shells. The proposed study is an important step for engineering novel microcompartments and will have potential future applications in areas such as drug delivery, metabolic engineering and biotechnology.