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An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.
- Source :
- Protein & Cell; Dec2012, Vol. 3 Issue 12, p921-928, 8p
- Publication Year :
- 2012
-
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-His 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 1674800X
- Volume :
- 3
- Issue :
- 12
- Database :
- Complementary Index
- Journal :
- Protein & Cell
- Publication Type :
- Academic Journal
- Accession number :
- 84088107
- Full Text :
- https://doi.org/10.1007/s13238-012-2101-y