Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins
Production and Characterization of Synthetic Carboxysome Shells with Incorporated Luminal Proteins
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DOI：
10.1104/pp.15.01822
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发表时间：
2016-03-01
期刊：
影响因子：
7.4
通讯作者：
Kerfeld, Cheryl A.
中科院分区：
文献类型：
--
作者：
Cai, Fei;Bernstein, Susan L.;Kerfeld, Cheryl A.
代谢的空间分离，如C4植物或蓝藻羧基体中的细胞定位的CO2固定，通过选择性地将它们与其底物一起浓缩来增强低效酶的活性。碳氧体和其他细菌微区室（BMC）引起了纳米反应器生物工程的特别关注，因为它们是自组装的蛋白质细胞器。所有的BMC都有一个结构相似的、可选择性渗透的外壳，包裹着酶。工程carboxysomes和其他BMC在植物合成生物学和代谢工程中的应用的基础是理解货物包装和壳渗透性的结构决定因素。在这里，我们描述了产生羧基体壳的合成操纵子在大肠杆菌中的表达。使用羧基体核心天然的蛋白质结构域将外来货物包封到合成壳中。这些合成壳可以在有或没有腔蛋白的情况下纯化至同质。我们的研究结果不仅进一步了解蛋白质-蛋白质相互作用的羧基体组装，但也建立了一个平台，研究壳渗透性和完整的BMC壳功能的结构基础在体内和体外。该系统将特别适用于开发用于植物工程的合成羧基体。
Spatial segregation of metabolism, such as cellular-localized CO2 fixation in C4 plants or in the cyanobacterial carboxysome, enhances the activity of inefficient enzymes by selectively concentrating them with their substrates. The carboxysome and other bacterial microcompartments (BMCs) have drawn particular attention for bioengineering of nanoreactors because they are selfassembling proteinaceous organelles. All BMCs share an architecturally similar, selectively permeable shell that encapsulates enzymes. Fundamental to engineering carboxysomes and other BMCs for applications in plant synthetic biology and metabolic engineering is understanding the structural determinants of cargo packaging and shell permeability. Here we describe the expression of a synthetic operon in Escherichia coli that produces carboxysome shells. Protein domains native to the carboxysome core were used to encapsulate foreign cargo into the synthetic shells. These synthetic shells can be purified to homogeneity with or without luminal proteins. Our results not only further the understanding of protein-protein interactions governing carboxysome assembly, but also establish a platform to study shell permeability and the structural basis of the function of intact BMC shells both in vivo and in vitro. This system will be especially useful for developing synthetic carboxysomes for plant engineering.