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

doi:10.1007/s13238-012-2101-y

Prot Cell

2012, Vol. 3

Issue (12) : 921-928 https://doi.org/10.1007/s13238-012-2101-y PMID: 23096592

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

Nuoyan zheng^1,2,3, Xiahe Huang³, Bojiao Yin³, Dan Wang⁴, Qi Xie³(

)

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

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Abstract

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.

Keywords PPV NIa protease protein-protein interaction in vivo cleavage fusion protein

Corresponding Author(s): Xie Qi,Email:qxie@genetics.ac.cn

Issue Date: 01 December 2012

Cite this article:

Xiahe Huang,Bojiao Yin,Dan Wang, et al. An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease[J]. Prot Cell, 2012, 3(12): 921-928.

URL:

https://academic.hep.com.cn/pac/EN/10.1007/s13238-012-2101-y
https://academic.hep.com.cn/pac/EN/Y2012/V3/I12/921

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