Volume 3 Issue 12
Dec.  2012
Turn off MathJax
Article Contents
Nuoyan zheng, Xiahe Huang, Bojiao Yin, Dan Wang, Qi Xie. An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease[J]. Protein&Cell, 2012, 3(12): 921-928. doi: 10.1007/s13238-012-2101-y
Citation: Nuoyan zheng, Xiahe Huang, Bojiao Yin, Dan Wang, Qi Xie. An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease[J]. Protein&Cell, 2012, 3(12): 921-928. doi: 10.1007/s13238-012-2101-y

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

doi: 10.1007/s13238-012-2101-y
Funds:

We thank Professor Juan Antonio Garcia for providing plasmid pGGNIa which contain the cDNA region of PPV NIa protease. This research was supported by National Natural Science Foundation of China (Grant No. 31030047) and the National Basic Research Program (973 Program) (No. 2011CB915402).

  • Received Date: 2011-12-19
  • Rev Recd Date: 2012-09-25
  • 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-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.
  • loading
  • 加载中

Catalog

    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Article Metrics

    Article views (153) PDF downloads(154) Cited by()
    Proportional views
    Related

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return