Assembly of Cyanobacterial Carboxysomes

描述（由申请人提供）：许多细菌具有复杂的代谢调节机制，涉及微区室。这些细菌微区室由独特的蛋白质外壳组成，其结构与病毒衣壳相似，并在特定的反应途径中包含两种或更多种酶。来自蓝藻和化学营养缺陷型的羧基体是最知名的细菌微区室。在过去的几年里，几个壳组件的晶体结构已经解决，并导致了显着的进步，在知识的亚基组装和功能的个别组件。到目前为止，还没有关于细菌微区室在真核生物中异源组装的报道。该项目将靶向多个羧基体蛋白进入叶绿体。本研究将利用农杆菌渗入技术瞬时表达与荧光标记融合的外源蛋白。将用共聚焦显微镜监测从表达的蛋白质组装的任何分子复合物，并且将通过电子显微镜表征形成的结构。此外，这项工作将产生稳定的转基因株系，其中羧基体蛋白从细胞核或叶绿体基因组表达。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区室外壳。此外，本研究将确定是否有可能将外源蛋白靶向到羧基体壳。这项研究是设计新型微区室的重要一步，并将在药物输送，代谢工程和生物技术等领域具有潜在的未来应用。

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.