Novel mobilizable prokaryotic two-hybrid system vectors for high-throughput protein interaction mapping in Escherichia coli by bacterial conjugation.
Novel mobilizable prokaryotic two-hybrid system vectors for high-throughput protein interaction mapping in Escherichia coli by bacterial conjugation.
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DOI：
10.1093/nar/gni011
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发表时间：
2005-02-01
影响因子：
14.9
通讯作者：
O'Connell M
中科院分区：
文献类型：
--
作者：
Clarke P;Cuív PO;O'Connell M
酵母双杂交（Y2H）系统自问世以来，已被广泛应用于蛋白质间相互作用的检测和分析。基于交配的策略已经开发，允许其应用程序的自动化蛋白质组相互作用映射项目使用详尽和高通量的策略。最近，一些原核双杂交（P2H）系统已被开发，但尽管有许多优势，如大肠杆菌为基础的系统比Y2H系统，他们还没有被广泛实施的蛋白质组相互作用映射。这可能在很大程度上是由于这样一个事实，即采用细菌转化的高通量策略不像基于Y2H交配的策略那样易于自动化。在这里，我们描述了新的共轭P2H系统载体的建设。这些载体携带IncPα组质粒RP4的动员元件，因此可以从编码所有所需转运功能的大肠杆菌供体菌株中高效动员。我们展示了这些载体如何允许利用细菌接合技术简化和自动化大肠杆菌中的蛋白质组相互作用作图策略，类似于为Y2H系统开发的基于交配的策略。
Since its initial description, the yeast two-hybrid (Y2H) system has been widely used for the detection and analysis of protein–protein interactions. Mating-based strategies have been developed permitting its application for automated proteomic interaction mapping projects using both exhaustive and high-throughput strategies. More recently, a number of prokaryotic two-hybrid (P2H) systems have been developed but, despite the many advantages such Escherichia coli-based systems have over the Y2H system, they have not yet been widely implemented for proteomic interaction mapping. This may be largely due to the fact that high-throughput strategies employing bacterial transformation are not as amenable to automation as Y2H mating-based strategies. Here, we describe the construction of novel conjugative P2H system vectors. These vectors carry a mobilization element of the IncPα group plasmid RP4 and can therefore be mobilized with high efficiency from an E.coli donor strain encoding all of the required transport functions in trans. We demonstrate how these vectors permit the exploitation of bacterial conjugation for technically simplified and automated proteomic interaction mapping strategies in E.coli, analogous to the mating-based strategies developed for the Y2H system.
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影响因子：
4.8
作者：
Hu, JC;Kornacker, MG;Hochschild, A
通讯作者：
Hochschild, A
影响因子：
30.8
作者：
Bartel, PL;Roecklein, JA;Fields, S
通讯作者：
Fields, S
影响因子：
56.9
作者：
HU, JC;OSHEA, EK;SAUER, RT
通讯作者：
SAUER, RT
影响因子：
3.5
作者：
INOUE, H;NOJIMA, H;OKAYAMA, H
通讯作者：
OKAYAMA, H
DOI：
10.1073/pnas.88.21.9578
发表时间：
1991-11-01
影响因子：
11.1
作者：
CHIEN, CT;BARTEL, PL;FIELDS, S
通讯作者：
FIELDS, S