LEC-BiFC: a new method for rapid assay of protein interaction.
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.
Lin
J
J
Beijing Nuclear Magnetic Resonance Center, Peking University, China.
Wang
N
N
Li
Y
Y
Liu
Z
Z
Tian
S
S
Zhao
L
L
Zheng
Y
Y
Liu
S
S
Li
S
S
Jin
C
C
Xia
B
B
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
D008164:Luminescent Proteins; D010446:Peptide Fragments; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein