title: Low false-positives in an mLumin-based bimolecular fluorescence complementation system with a bicistronic expression vector.
abstract: The simplicity and sensitivity of the bimolecular fluorescence complementation (BiFC) assay make it a powerful tool to investigate protein-protein interactions (PPIs) in living cells. However, non-specific association of the fluorescent protein fragments in a BiFC system can complicate evaluation of PPIs. Here, we introduced a bicistronic expression vector, pBudCE4.1, into an mLumin-based BiFC system, denoted as the BEVL-BiFC system. The BEVL-BiFC system achieved a 25-fold contrast in BiFC efficiency between positive (Fos/Jun) and negative (ΔFos/Jun) PPIs. The high BiFC efficiency was due to a low false-positive rate, where less than 2% of cells displayed BiFC in the negative control. K-Ras and its interactive proteins, Ras binding domain (RBD) of Raf-1 and Grb2 were used to confirm the accuracy of the BEVL-BiFC system. The results also provide direct evidence in individual cells that post-translational modification of K-Ras and its localization at the plasma membrane (PM) were not essential for the interaction of K-Ras and Raf-1, whereas the interaction of Grb2 and K-Ras did depend on the PM localization of K-Ras. Taken together, the BEVL-BiFC system was developed to reduce the false-positive phenomenon in BiFC assays, resulting in more robust and accurate measurement of PPIs in living cells.
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. liushun@ibp.hust.edu.cn.
authors.forename: Shun
authors.initials: S
authors.lastname: Liu
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. xlxyongli@gmail.com.
authors.forename: Xiangyong
authors.initials: X
authors.lastname: Li
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. yangjie@mail.hust.edu.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.affiliation: Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and Technology, Wuhan 430074, China. czyzzh@mail.hust.edu.cn.
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D051380:GRB2 Adaptor Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000378:metabolism; D008856:Microscopy, Fluorescence; D060066:Protein Interaction Maps; D017434:Protein Structure, Tertiary; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D051380:GRB2 Adaptor Protein; D018631:ras Proteins