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. 2012 Oct 24;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

Nuoyan Zheng 1,2,3, Xiahe Huang 3, Bojiao Yin 3, Dan Wang 4, Qi Xie 3,✉
PMCID: PMC4875385  PMID: 23096592

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

Footnotes

These authors contributed equally to the work.

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