Deciphering correct strategies for multiprotein complex assembly by co-expression: Application to complexes as large as the histone octamer
Deciphering correct strategies for multiprotein complex assembly by co-expression: Application to complexes as large as the histone octamer
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DOI：
10.1016/j.jsb.2011.02.001
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发表时间：
2011-08-01
影响因子：
3
通讯作者：
Romier, Christophe
中科院分区：
文献类型：
--
作者：
Diebold, Marie-Laure;Fribourg, Sebastien;Romier, Christophe
大分子复合体负责细胞内的大多数基本机制，导致了人们对它们的纯化和表征的广泛兴趣。共表达是目前公认的组装多蛋白复合体的主要技术，目前有许多共表达系统可用于在不同宿主中进行共表达实验。然而，关于共表达策略的比较知识仍然非常缺乏。以大肠杆菌的多功能共表达系统、pET-MCN和pET-MCP系列以及三元蛋白质复合体为例，我们演示了如何成功地描述正确的共表达策略。具体地说，一种适当的、依赖于络合物的方法可以缓解化学计量比不平衡和产量问题，甚至可以减少生产络合物的失败。重要的是，影响共表达策略的一些参数似乎与表达宿主无关，因此对真核宿主的共表达具有一定的意义。通过进一步使用这些策略，我们证明在大肠杆菌中的共表达能够重建与SAGA转录因子和组蛋白八聚体的去泛素化模块一样大的蛋白质复合体。(C)2011 Elsevier Inc.保留所有权利。
Macromolecular complexes are responsible for most of the essential mechanisms in cells, leading to a broad interest in their purification and characterization. Co-expression is now widely recognized as a major technique for assembling multiprotein complexes and many co-expression systems are currently available for performing co-expression experiments in different hosts. However, comparative knowledge on co-expression strategies is still crucially lacking. Using versatile co-expression systems for Escherichia coli, the pET-MCN and pET-MCP series, and ternary protein complexes as examples, we demonstrate how to successfully delineate correct co-expression strategies. Specifically, an appropriate, complex-dependent approach alleviates stoichiometry imbalance and yield problems, and even failure in producing complexes. Importantly, some of the parameters influencing co-expression strategies appear independent of the expression host, thus having implications for co-expression in eukaryotic hosts. By further using these strategies, we show that co-expression in E. coli enables reconstitution of protein complexes as large as the deubiquitination module of the SAGA transcription factor and the histone octamer. (C) 2011 Elsevier Inc. All rights reserved.