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

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 burdensom...

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Bibliographic Details
Published in:Protein & cell 2012-12, Vol.3 (12), p.921-928
Main Authors: Zheng, Nuoyan, Huang, Xiahe, Yin, Bojiao, Wang, Dan, Xie, Qi
Format: Article
Language:English
Subjects:
Biochemistry
Biomedical and Life Sciences
Cell Biology
Communication
Developmental Biology
Endopeptidases - genetics
Endopeptidases - metabolism
Escherichia coli - genetics
Human Genetics
Life Sciences
Plum Pox Virus - enzymology
Plum Pox Virus - genetics
Protein Interaction Mapping - methods
Protein Science
Proteolysis
Stem Cells
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Summary: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 6 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.
ISSN:1674-800X
1674-8018
DOI:10.1007/s13238-012-2101-y