An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease
An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease
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基于 PPV NIa 蛋白酶体内裂解的检测蛋白质-蛋白质相互作用的有效系统
DOI：
10.1007/s13238-012-2101-y
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发表时间：
2012-10
期刊：
影响因子：
--
通讯作者：
Xie, Qi
中科院分区：
文献类型：
--
作者：
Zheng, Nuoyan;Huang, Xiahe;Yin, Bojiao;Wang, Dan;Xie, Qi
蛋白质相互作用的检测可以为研究蛋白质的生物学功能提供有价值的信息。目前应用于蛋白质与蛋白质相互作用的方法，如免疫共沉淀、下拉等，由于繁重的克隆和纯化程序，经常导致大量的工作时间。我们建立了一个系统，通过在大肠杆菌中共表达和简单纯化来自一个表达盒的目的蛋白来完成蛋白质-蛋白质相互作用的表征。coli系统。我们将pET载体改造成共表达载体pInvivo，该共表达载体编码PPV NIa蛋白酶、两个切割位点F和位于切割位点两侧的两个多克隆位点。在多克隆位点插入靶蛋白（例如：蛋白A和蛋白B），并按照MBP标签-蛋白A-位点F-PPV NIa蛋白酶-位点F-蛋白B-His（6）标签的顺序翻译成多聚蛋白。PPV NIa蛋白酶沿着表达进行胞内切割，然后导致多聚蛋白组分分离，因此可以通过一步纯化和分析来检测蛋白A-蛋白B之间的相互作用。将蛋白质B的阴性对照引入该系统中以监测相互作用特异性。我们成功地利用该系统证明了两个已报道的蛋白质-蛋白质相互作用的案例：RHA 2a/ANAC和FTA/FTB。综上所述，本研究成功地建立了一种简便、高效的蛋白质相互作用检测系统。
Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.
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影响因子：
5
作者：
S. Urcuqui-Inchima;A. Haenni;F. Bernardi
通讯作者：
S. Urcuqui-Inchima;A. Haenni;F. Bernardi
重组李痘病毒NIa蛋白酶特异性高效切割融合蛋白
影响因子：
1.6
作者：
Zheng N;Pérez Jde J;Zhang Z;Domínguez E;Garcia JA;Xie Q
通讯作者：
Xie Q
影响因子：
5.5
作者：
S. Patial;V. Chaturvedi;A. Rai;M. Saini;R. Chandra;Y. Saini;P. Gupta
通讯作者：
S. Patial;V. Chaturvedi;A. Rai;M. Saini;R. Chandra;Y. Saini;P. Gupta
影响因子：
1.6
作者：
Dzivenu, OK;Park, HH;Hao, W
通讯作者：
Hao, W
影响因子：
8
作者：
Chen, Xi;Pham, Elizabeth;Truong, Kevin
通讯作者：
Truong, Kevin