Borloo Jimmy, De Smet Lina, Vergauwen Bjorn, Van Beeumen Jozef J, Devreese Bart
Department of Biochemistry, Physiology and Microbiology, Ghent University, Laboratory for Protein Biochemistry and Protein Engineering, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium.
J Proteome Res. 2007 Jul;6(7):2587-95. doi: 10.1021/pr070037j. Epub 2007 Jun 1.
The vast majority of proteins functions in complex with one or more of the same or other proteins, indicating that protein-protein interactions play crucial roles in biology. Here, we present a beta-galactosidase reconstitution-based bacterial two-hybrid system in which two proteins of interest are fused to two non-functional but complementing beta-galactosidase truncations (Delta alpha and Delta omega). The level of complemented beta-galactosidase activity, driven by the protein-protein recognition between both non-beta-galactosidase parts of the chimeras, reflects whether or not the proteins of interest interact. Our approach was validated by reconfirming some well-established Escherichia coli cytoplasmic and membranous interactions, including well-chosen mutants, and providing the first in vivo evidence for the transient periplasmic interaction between Rhodobacter capsulatus cytochrome c2 and cytochrome c peroxidase. We demonstrated the major advantages of this in vivo two-hybrid technique: i) analyses of interactions are not limited to particular cellular compartments, ii) the potential of using the system in mutation-driven structure-function studies, and iii) the possibility of its application to transiently interacting proteins. These benefits demonstrate the relevance of the method as a powerful new tool in the broad spectrum of interaction assessment methods.
绝大多数蛋白质与一种或多种相同或其他蛋白质形成复合物发挥功能，这表明蛋白质 - 蛋白质相互作用在生物学中起着关键作用。在此，我们提出了一种基于β - 半乳糖苷酶重建的细菌双杂交系统，其中将两个感兴趣的蛋白质分别与两个无功能但可互补的β - 半乳糖苷酶截短片段（Δα和Δω）融合。由嵌合体的两个非β - 半乳糖苷酶部分之间的蛋白质 - 蛋白质识别驱动的互补β - 半乳糖苷酶活性水平，反映了感兴趣的蛋白质是否相互作用。我们通过再次确认一些已确立的大肠杆菌细胞质和膜相互作用（包括精心挑选的突变体），并为荚膜红细菌细胞色素c2和细胞色素c过氧化物酶之间的瞬时周质相互作用提供首个体内证据，验证了我们的方法。我们展示了这种体内双杂交技术的主要优点：i）相互作用分析不限于特定的细胞区室；ii）该系统在突变驱动的结构 - 功能研究中的潜力；iii）其应用于瞬时相互作用蛋白质的可能性。这些优点证明了该方法作为广泛的相互作用评估方法中的一种强大新工具的相关性。
