title: LEC-BiFC: a new method for rapid assay of protein interaction.
abstract: Abstract Protein-protein interactions play fundamental roles in most biological processes. Bimolecular fluorescence complementation (BiFC) is a promising method for its simplicity and direct visualization of protein-protein interactions in cells. This method, however, is limited by background fluorescence that appears without specific interaction between the proteins. We report here a point mutation (V150L) in one Venus BiFC fragment that efficiently decreases background fluorescence of BiFC assay. Furthermore, by combining this modified BiFC and linear expression cassette (LEC), we develop a simple and rapid method (LEC-BiFC) for protein interaction analysis that is demonstrated by a case study of the interaction between Bcl-X(L) and Bak BH3 peptide. The total analysis procedure can be completed in two days for screening tens of mutants. LEC-BiFC can be applied easily in any lab equipped with a fluorescence microscope.
authors.affiliation: Beijing Nuclear Magnetic Resonance Center, Peking University, China.
authors.forename: J
authors.initials: J
authors.lastname: Lin
authors.forename: N
authors.initials: N
authors.lastname: Wang
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Tian
authors.forename: L
authors.initials: L
authors.lastname: Zhao
authors.forename: Y
authors.initials: Y
authors.lastname: Zheng
authors.forename: S
authors.initials: S
authors.lastname: Liu
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: C
authors.initials: C
authors.lastname: Jin
authors.forename: B
authors.initials: B
authors.lastname: Xia
mesh_terms: D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics*; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D014162:Transfection; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000032:analysis* / Q000737:chemistry; D051020:bcl-X Protein / Q000032:analysis* / Q000737:chemistry
chemical_list: D008164:Luminescent Proteins; D010446:Peptide Fragments; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein