Dokument: Overexpression and isolation of the intermediate state of serotonin transporter from Echinococcus multilocularis –‒ the ER localized HSP complexes of the folding trajectory
| Titel: | Overexpression and isolation of the intermediate state of serotonin transporter from Echinococcus multilocularis –‒ the ER localized HSP complexes of the folding trajectory | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=56576 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20210616-103017-4 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Guo, Weihou [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof.Dr. Labahn, Jörg [Gutachter] Prof. Dr. Heise, Henrike [Gutachter] | |||||||
| Stichwörter: | Serotonin transporter, Echinococcus multilocularis | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibung: | This study set out to achieve the overexpression and isolation of serotonin transporter from E. multilocularis. Being the first membrane protein of E. multilocolaris that was heterologously expressed, the overexpression of emST was carried out in three different expression systems i.e. bacteria, insect and mammalian systems.
In the bacterial system, a 50kDa degradation product was observed. By CD spectroscopy the full-length protein was shown to be significantly different from the prediction, probably because of missing glycosylation in the bacterial system. Indeed, folded full-length protein was obtained in the mammalian expression system. Even for the in vitro expressed emST-MSP1D1 complex the CD spectra showed a random coil structure, which indicated unfolded state. In this case, the nanodisc environment was not sufficient to maintain a folded state of the protein. In mammalian cells, the emST was isolated as emST-HSPs complexes. This provided the insight that the isolated protein was in an intermediary state in ER. The ER localization was confirmed by both confocal microscopy-based localization analysis using a mCherry-construct and subcellular fractionation analysis. The investigation of glycosylation has shown that the glycan-moieties present on emST and its variants were obviously of different accessibility for processing, the mCherry construct being the least processed. The CD analysis of both complexes showed secondary structure close to the predicted values, which indicated that the HSP, as a co-chaperone, was able to keep the emST in a properly folded state. The helical content was significantly increased when CHS was added to detergent purified emST, which had a low helical content after detergent solubilization. The hypothesized relevance of CHS for maintaining folding was thereby shown. But the reconstitution of the secondary structure of emST was found to be incomplete which indicated that deviation from the proper intracellular processing of emST caused irreversible changes in the protein structure. In this work, for the first time the HSP complexes of a target protein were isolated. The isolated complexes have not been described previously and provide an extension to the COPII/exchange model of membrane protein transport. The yield of about 5-7 μg / g cell pellet allowed characterizing the biochemical and biophysical properties, especially wrt the folding trajectory of the protein. These complexes will allow further studies relevant to the transport and folding process and present a suitable target for pharmaceutical intervention in AHD. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie | |||||||
| Dokument erstellt am: | 16.06.2021 | |||||||
| Dateien geändert am: | 16.06.2021 | |||||||
| Promotionsantrag am: | 13.04.2021 | |||||||
| Datum der Promotion: | 08.06.2021 |
