A Split-Luciferase Reporter Recognizing GFP and mCherry Tags to Facilitate Studies of Protein–Protein Interactions
| Autor: | Yann Gambin, Emma Sierecki, Mehdi Moustaqil, Laura Gonzalez, Dominic J. B. Hunter, Lisa Raoul, Pascal Carrive, Akshay Bhumkar |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Proteomics Protein combining Green Fluorescent Proteins Catalysis Article Protein–protein interaction Green fluorescent protein lcsh:Chemistry Inorganic Chemistry 03 medical and health sciences Genes Reporter Protein Interaction Maps Physical and Theoretical Chemistry Luciferases lcsh:QH301-705.5 Molecular Biology Spectroscopy universal reporter Luciferase reporter Cell-Free System Chemistry Organic Chemistry General Medicine 3. Good health Computer Science Applications Cell biology Complementation Luminescent Proteins 030104 developmental biology protein–protein interaction lcsh:Biology (General) lcsh:QD1-999 Microscopy Fluorescence Leishmania tarentolae cell-free split-luciferase mCherry Genetic Engineering |
| Zdroj: | International Journal of Molecular Sciences International Journal of Molecular Sciences; Volume 18; Issue 12; Pages: 2681 International Journal of Molecular Sciences, Vol 18, Iss 12, p 2681 (2017) |
| ISSN: | 1422-0067 |
| Popis: | The use of fluorescently-tagged proteins in microscopy has become routine, and anti-GFP (Green fluorescent protein) affinity matrices are increasingly used in proteomics protocols. However, some protein–protein interactions assays, such as protein complementation assays (PCA), require recloning of each protein as a fusion with the different parts of the complementation system. Here we describe a generic system where the complementation is separated from the proteins and can be directly used with fluorescently-tagged proteins. By using nanobodies and performing tests in cell-free expression systems, we accelerated the development of multiple reporters, detecting heterodimers and homodimers or oligomers tagged with GFP or mCherry. We demonstrate that the system can detect interactions at a broad range of concentrations, from low nanomolar up to micromolar. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|