Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E-coli
Dissecting the interaction network of multiprotein complexes by pairwise coexpression of subunits in E-coli
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DOI：
10.1006/jmbi.2000.4376
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发表时间：
2001-02-16
影响因子：
5.6
通讯作者：
Moras, D
中科院分区：
文献类型：
--
作者：
Fribourg, S;Romier, C;Moras, D
以人类基础转录因子 TFIID 和 TFIIH 为例，我们表明多肽在大肠杆菌中的成对共表达可以用作鉴定多蛋白复合物中特异性相互作用的亚基的工具。我们发现适当组合的共表达通常会导致所涉及的多肽的溶解度和稳定性增加，这意味着可以立即获得大量所得复合物用于后续的生化和结构分析。此外，我们证明蛋白质的天然伴侣的溶解和/或正确折叠可以用作缺失图谱的监测器，以确定精确的相互作用域。共表达可用作相互作用研究（例如酵母双杂交分析、GST Pulldown 和免疫沉淀）的常规技术的替代或补充方法。 (C) 2001 年学术出版社。
Using the human basal transcription factors TFIID and TFIIH as examples, we show that pairwise coexpression of polypeptides in Escherichia coli can be used as a tool for the identification of specifically interacting subunits within multiprotein complexes. We find that coexpression of appropriate combinations generally leads to an increase in the solubility and stability of the polypeptides involved, which means that large amounts of the resulting complexes can immediately be obtained for subsequent biochemical and structural analysis. Furthermore, we demonstrate that the solubilization and/or the proper folding of a protein by its natural partner can be used as a monitor for deletion mapping to determine precise interaction domains. Coexpression can be used as an alternative or complementary approach to conventional techniques for interaction studies such as yeast two-hybrid analysis, GST pulldown and immunoprecipitation. (C) 2001 Academic Press.