A Gateway® -compatible bacterial adenylate cyclase-based two-hybrid system
A Gateway® -compatible bacterial adenylate cyclase-based two-hybrid system
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DOI：
10.1111/1758-2229.12123
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发表时间：
2014-06-01
影响因子：
3.3
通讯作者：
Ladant, Daniel
中科院分区：
文献类型：
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作者：
Ouellette, Scot P.;Gauliard, Emilie;Ladant, Daniel
细菌腺苷酸环化酶双杂交（BACTH）系统已被广泛用于表征原核世界中的蛋白质-蛋白质相互作用。该系统依赖于相互作用介导的重组大肠杆菌中的腺苷酸环化酶活性，将百日咳杆菌腺苷酸环化酶毒素的催化结构域的两个互补片段放在一起。用全长开放阅读框（ORF）进行大规模双杂交相互作用筛选的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（Life Technologies，Grand Island，NY，USA）部分地规避了这种限制，并且我们在此描述了对BACTH系统的修改以与这种重组工程技术兼容。我们使用几种蛋白质-蛋白质相互作用模型验证和测试了BACTH Gateway（BACTH（GW））系统的功能，特别关注那些参与细菌细胞分裂的蛋白质-蛋白质相互作用。我们进一步修改了BACTH质粒，在环化酶片段的下游加入一个跨膜（TM）片段，以分析胞质外蛋白质的相互作用。这种方法也是有用的，以确定假定的TM段和实验验证生物信息学确定的TM域。BACTH（GW）系统将被证明是对蛋白质-蛋白质相互作用研究的有益补充。
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((R)) (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 (BACTH(GW)) 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 BACTH(GW) system will prove a useful addition to the study of protein-protein interactions.