A simple method to generate stable cell lines for the analysis of transient protein-protein interactions
| Autor: | Patrick M. Sexton, Denise Wootten, Arthur Christopoulos, Sebastian G.B. Furness, Emilia Elizabeth Savage |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
Arrestins
Recombinant Fusion Proteins Genetic Vectors Molecular Sequence Data Gene Expression Computational biology Ligands Transfection General Biochemistry Genetics and Molecular Biology Cell Line Receptors G-Protein-Coupled Protein–protein interaction Protein Interaction Mapping Gene expression Arrestin Animals Humans Amino Acid Sequence Receptor G protein-coupled receptor Chemistry Isogenic human disease models Luminescent Proteins Cell culture Luminescent Measurements Signal transduction Biotechnology |
| Zdroj: | BioTechniques. 54:217-221 |
| ISSN: | 1940-9818 0736-6205 |
| Popis: | Transient protein-protein interactions form the basis of signal transduction pathways in addition to many other biological processes. One tool for studying these interactions is bioluminescence resonance energ y transfer (BRET). This technique has been widely applied to study signaling pathways, in particular those initiated by G protein-coupled receptors (GPCRs). These assays are routinely performed using transient transfection, a technique that has limitations in terms of assay cost and variability, overexpression of interacting proteins, vector uptake limited to cycling cells, and non-homogenous expression across cells within the assay. To address these issues, we developed bicistronic vectors for use with Life Technology's Gateway and flpIN systems. These vectors provide a means to generate isogenic cell lines for comparison of interacting proteins. They have the advantage of stable, single copy, isogenic, homogeneous expression with low inter-assay variation. We demonstrate their utility by assessing ligand-induced interactions between GPCRs and arrestin proteins. |
| Databáze: | OpenAIRE |
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