Bimolecular Fluorescent Complementary (BiFC) Assay For Protein-protein Interactions In KRAS Signal Pathway And The Optimization Of BiFC System

Bimolecular fluorescence complementation (BiFC) assay is a powerful tool forresearches of protein-protein interactions in vivo. With high sensitivity, BiFC is a simplemeaning to directly observe protein-protein interactions in living cells. It is based on theassociation of fluorescent protein fragments that are attached to components of the samemacromolecular complex. Proteins that are postulated to interact are fused to unfoldedcomplementary fragments of a fluorescent reporter protein and expressed in live cells.Interaction of these proteins will bring the fluorescent fragments within proximity,allowing the reporter protein to reform in its native three-dimensional structure and emitits fluorescent signal. In this paper, the efficiency of BiFC systems with differentcharacteristics was compared; while dimerization of EGFR was observed and analyzedused the Vn173/Vc155BiFC system based on yellow fluorescent protein variant, Venus ina variety of cells.On this basis, a BiFC system in a dual-expression eukaryotic vector based onVn173/Vc155was built, which simplified the two-vector system into single-vector; theninteractions between KRAS (Kirsten rat sarcoma viral oncogene homolog) and RAF1(RAF proto-oncogene serine/threonine-protein kinase), KRAS and RASSF2(Ras-association domain family2), KRAS and GRB2(Growth factor receptor-boundprotein2) were observed respectively with this system. Through observation ofsubcellular localization of fluorescent complex formed in the assay, the interactionsbetween these proteins as well as the activation of cellular signal transduction mechanismsare discussed. However, BiFC fluorescent intensity and protein interaction strength does not have obvious proportional relationship in these systems, because of spontaneouscombination between Vn173and Vc155, which limits its use in quantitative analysis ofprotein interactions. To this end, we use a new pair of BiFC fluorescent fragments,mLumin1-151(Ln) and mLumin152-231(Lc), to construct BiFC system in a dualexpression vector, which significantly reduced false positive produced by spontaneouscombination of fluorescent protein fragments. With this system, interaction betweenKRAS and GRB2was quantified. Comparison between these BiFC systems provides usprinciples for the choice in the studies of protein-protein interaction with differentpurposes: Venus-based BiFC system with higher sensitivity is more suitable forsubcellular localization studies of proteins with weak interaction; whereas the GFP-basedBiFC system will be the better choice in studies on cells or organisms which grow incondition below30oC, such as plants, nematodes; and for quantitative analysis ofinteractions as well as studies requires a lower false-positive, mLumin-based BiFC systemin dual expression vector has greater advantage.In short, this paper establishes a mLumin-based BiFC system in dual expressionvector that largely reduces the BiFC false-positive rate; use of different BiFC systems,several protein-protein interactions in the KRAS signaling pathway were observed.Results show that our BiFC system is suitable for quantitative analysis of proteininteraction and lower false-positive-required researches, such as library screening of BiFCanalysis.