New GATEWAY vectors for High Throughput Analyses of Protein-Protein Interactions by Bimolecular Fluorescence Complementation

作     者：Christian Gehl Rainer Waadt Jorg Kudla Ralf-R. Mendel Robert Hansch 

作者机构：Institut for Pflanzenbiologie Technische Universitat Braunschweig Humboldtstrasse 1 D-38106 Braunschweig Germany Molekulare Entwicklungsbiologie der Pflanzen Institut for Botanik und Botanischer Garten Universitat MOnster Schlossplatz 4 D-48149 MOnster Germany 

出 版 物：《Molecular Plant》 (分子植物（英文版）)

年 卷 期：2009年第2卷第5期

页      面：1051-1058页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 070207[理学-光学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 0901[农学-作物学] 0902[农学-园艺学] 0803[工学-光学工程] 0702[理学-物理学] 

基　　金：the Deutsche Forschungs-gemeinschaft to R.R.M 

主　　题：BiFC GATEWAY molybdenum cofactor nitrate reductase. 

摘      要：Complex protein interaction networks constitute plant metabolic and signaling systems. Bimolecular fluorescence complementation （BiFC） is a suitable technique to investigate the formation of protein complexes and the localization of protein-protein interactions in planta. However, the generation of large plasmid collections to facilitate the exploration of complex interaction networks is often limited by the need for conventional cloning techniques. Here, we report the implementation of a GATEWAY vector system enabling large-scale combination and investigation of candidate proteins in BiFC studies. We describe a set of 12 GATEWAY-compatible BiFC vectors that efficiently permit the combination of candidate protein pairs with every possible N-or C-terminal sub-fragment of S（CFP）3A or Venus, respectively, and enable the performance of multicolor BiFC （mcBiFC）. We used proteins of the plant molybdenum metabolism, in that more than 20 potentially interacting proteins are assumed to form the cellular molybdenum network, as a case study to establish the functionality of the new vectors. Using these vectors, we report the formation of the molybdopterin synthase complex by interaction of Arabidopsis proteins Cnx6 and Cnx7 detected by BiFC as well as the simultaneous formation of Cnx6/Cnx6 and Cnx6/Cnx7 complexes revealed by mcBiFC. Consequently, these GATEWAY-based BiFC vector systems should significantly facilitate the large-scale investigation of complex regulatory networks in plant cells.