title: An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.
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.
authors.affiliation: Department of Nephrology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510000, China.
authors.forename: Nuoyan
authors.initials: N
authors.lastname: Zheng
authors.forename: Xiahe
authors.initials: X
authors.lastname: Huang
authors.forename: Bojiao
authors.initials: B
authors.lastname: Yin
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Xie
mesh_terms: D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D017801:Plum Pox Virus / Q000201:enzymology* / Q000235:genetics; D025941:Protein Interaction Mapping / Q000379:methods*; D059748:Proteolysis
chemical_list: D010450:Endopeptidases