Production, Purification and Characterization of Recombinant Biotinylated Phytochrome B for Extracellular Optogenetics.
| Autor: | Hörner M; Faculty of Biology, University of Freiburg, Freiburg, Germany.; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany., Yousefi OS; Faculty of Biology, University of Freiburg, Freiburg, Germany.; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany., Schamel WWA; Faculty of Biology, University of Freiburg, Freiburg, Germany.; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany.; Center for Chronic Immunodeficiency (CCI), Medical Center Freiburg and Faculty of Medicine, University of Freiburg, Freiburg, Germany., Weber W; Faculty of Biology, University of Freiburg, Freiburg, Germany.; Signalling Research Centres BIOSS and CIBSS, University of Freiburg, Freiburg, Germany. |
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| Jazyk: | angličtina |
| Zdroj: | Bio-protocol [Bio Protoc] 2020 Mar 05; Vol. 10 (5), pp. e3541. Date of Electronic Publication: 2020 Mar 05 (Print Publication: 2020). |
| DOI: | 10.21769/BioProtoc.3541 |
| Abstrakt: | In the field of extracellular optogenetics, photoreceptors are applied outside of cells to obtain systems with a desired functionality. Among the diverse applied photoreceptors, phytochromes are the only ones that can be actively and reversibly switched between the active and inactive photostate by the illumination with cell-compatible red and far-red light. In this protocol, we describe the production of a biotinylated variant of the photosensory domain of A. thaliana phytochrome B (PhyB-AviTag) in E. coli with a single, optimized expression plasmid. We give detailed instructions for the purification of the protein by immobilized metal affinity chromatography and the characterization of the protein in terms of purity, biotinylation, spectral photoswitching and the light-dependent interaction with its interaction partner PIF6. In comparison to previous studies applying PhyB-AviTag, the optimized expression plasmid used in this protocol simplifies the production process and shows an increased yield and purity. Competing Interests: Competing interestsThe authors do not declare any competing interests. (©Copyright Hörner et al.) |
| Databáze: | MEDLINE |
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