An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.
10.1007/s13238-012-2101-y
- Author:
Nuoyan ZHENG
1
;
Xiahe HUANG
;
Bojiao YIN
;
Dan WANG
;
Qi XIE
Author Information
1. Department of Nephrology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510000, China.
- Publication Type:Journal Article
- MeSH:
Endopeptidases;
genetics;
metabolism;
Escherichia coli;
genetics;
Plum Pox Virus;
enzymology;
genetics;
Protein Interaction Mapping;
methods;
Proteolysis
- From:
Protein & Cell
2012;3(12):921-928
- CountryChina
- Language:English
-
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(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.
