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–XL 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.

[1]  S. Johnston,et al.  Linear expression elements: a rapid, in vivo, method to screen for gene functions , 1999, Nature Biotechnology.

[2]  S. F. Konieczny,et al.  Inhibition of Mist1 Homodimer Formation Induces Pancreatic Acinar-to-Ductal Metaplasia , 2004, Molecular and Cellular Biology.

[3]  J. Lamerdin,et al.  Identifying off-target effects and hidden phenotypes of drugs in human cells , 2006, Nature chemical biology.

[4]  S. Michnick,et al.  Capturing protein interactions in the secretory pathway of living cells. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Chang‐Deng Hu,et al.  Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. , 2002, Molecular cell.

[6]  W. Thilly,et al.  Fidelity and mutational spectrum of Pfu DNA polymerase on a human mitochondrial DNA sequence. , 1997, Genome research.

[7]  L. Regan,et al.  Antiparallel Leucine Zipper-Directed Protein Reassembly: Application to the Green Fluorescent Protein , 2000 .

[8]  D. Kaslow,et al.  Vaccination with polymerase chain reaction-generated linear expression cassettes protects mice against lethal influenza A challenge. , 2007, Human gene therapy.

[9]  R. Meadows,et al.  Structure of Bcl-xL-Bak Peptide Complex: Recognition Between Regulators of Apoptosis , 1997, Science.

[10]  Han Liu,et al.  Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. , 2006, BioTechniques.

[11]  F. Avilés,et al.  Detection of transient protein–protein interactions by bimolecular fluorescence complementation: The Abl‐SH3 case , 2007, Proteomics.

[12]  CUPSAT : Predicting Protein Stability Upon Point Mutations , 2006 .

[13]  L. Huang,et al.  Delivery of a PCR amplified DNA fragment into cells: a model for using synthetic genes for gene therapy , 1997, Gene Therapy.

[14]  T. Kerppola,et al.  Visualization of molecular interactions by fluorescence complementation , 2006, Nature Reviews Molecular Cell Biology.

[15]  Thomas J Magliery,et al.  Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism. , 2005, Journal of the American Chemical Society.

[16]  L. Pease,et al.  Gene splicing and mutagenesis by PCR-driven overlap extension , 2007, Nature Protocols.

[17]  Z. Li,et al.  Efficient in vivo gene transfer by PCR amplified fragment with reduced inflammatory activity , 2001, Gene Therapy.

[18]  F. Avilés,et al.  Study and selection of in vivo protein interactions by coupling bimolecular fluorescence complementation and flow cytometry , 2007, Nature Protocols.