An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease  

作　　者：Nuoyan zheng Xiahe Huang Bojiao Yin Dan Wang Qi Xie 

机构地区：[1]Department of Nephrology,the First Affiliated Hospital,Sun Yat-Sen University,Guangzhou 510000,China [2]Research Center of Translational Medicine,the First Affiliated Hospital,Sun Yat-Sen University,Guangzhou 510000,China [3]State Key Laboratory of Plant Genomics,National Centre for Plant Gene Research,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China [4]Animal Center,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,Beijing 100101,China

出　　处：《Protein & Cell》2012年第12期921-928,共8页蛋白质与细胞（英文版）

基　　金：supported by National Natural Science Foundation of China(Grant No.31030047);the National Basic Research Program(973 Program)(No.2011CB915402).

摘　　要：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 puri-fication 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-His6 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.

关 键 词：PPV NIa protease protein-protein interaction in vivo cleavage fusion protein 

分 类 号：Q78[生物学—分子生物学]