Ouellette Scot P, Gauliard Emilie, Antosová Zuzana, Ladant Daniel
Département de Biologie Structurale et Chimie, Institut Pasteur, Unité de Biochimie des Interactions Macromoléculaires, 28 rue du Dr. Roux, F-75015, Paris, France; CNRS, UMR 3528, 28 rue du Dr. Roux, F-75015, Paris, France.
Environ Microbiol Rep. 2014 Jun;6(3):259-67. doi: 10.1111/1758-2229.12123. Epub 2013 Nov 25.
The bacterial adenylate cyclase two-hybrid (BACTH) system has been widely used to characterize protein-protein interactions in the prokaryotic world. This system relies on the interaction-mediated reconstitution of adenylate cyclase activity in Escherichia coli by bringing together two complementary fragments of the catalytic domain of the adenylate cyclase toxin of Bordetella pertussis. A limiting factor in performing large-scale two-hybrid interaction screens with full-length open reading frames (ORFs) is the need to clone each ORF individually into the plasmids used to express the hybrid proteins. The Gateway(®) (GW) cloning system (Life Technologies, Grand Island, NY, USA) partially circumvents this limitation, and we describe here modifications to the BACTH system for compatibility with this recombineering technology. We validated and tested the functionality of the BACTH Gateway (BACTHGW ) system using several models of protein-protein interactions, focusing particularly on those involved in bacterial cell division. We further modified the BACTH plasmids to incorporate a transmembrane (TM) segment downstream of the cyclase fragments to permit analysis of extracytoplasmic protein interactions. This approach was also useful to identify putative TM segments and to experimentally validate bioinformatically identified TM domains. The BACTHGW system will prove a useful addition to the study of protein-protein interactions.
细菌腺苷酸环化酶双杂交（BACTH）系统已被广泛用于表征原核生物中的蛋白质-蛋白质相互作用。该系统通过将百日咳博德特氏菌腺苷酸环化酶毒素催化结构域的两个互补片段聚集在一起，依赖于大肠杆菌中相互作用介导的腺苷酸环化酶活性的重建。使用全长开放阅读框（ORF）进行大规模双杂交相互作用筛选的一个限制因素是需要将每个ORF单独克隆到用于表达杂交蛋白的质粒中。Gateway®（GW）克隆系统（美国纽约州大岛市生命技术公司）部分克服了这一限制，我们在此描述了对BACTH系统的修改，使其与这种重组技术兼容。我们使用几种蛋白质-蛋白质相互作用模型验证并测试了BACTH Gateway（BACTHGW）系统的功能，特别关注那些参与细菌细胞分裂的相互作用。我们进一步修改了BACTH质粒，以便在环化酶片段下游整合一个跨膜（TM）区段，从而能够分析胞外蛋白质相互作用。这种方法对于识别假定的TM区段以及通过实验验证生物信息学鉴定的TM结构域也很有用。BACTHGW系统将被证明是蛋白质-蛋白质相互作用研究的一个有用补充。
