| Titel: | Expression and purification of human connexin 26 |
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=40233 |
| URN (NBN): | urn:nbn:de:hbz:061-20161027-092816-0 |
| Kollektion: | Dissertationen |
| Sprache: | Englisch |
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation |
| Medientyp: | Text |
| Autor: | Balandin, Taras [Autor]
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| Dateien: | |
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie |
| Beschreibung: | Here, we explored the MstX tag from B.subtilis as an expression and membrane insertion driver to overproduce human connexin 26 in E.coli. The MstX tag, while being a very efficient Cx26 expression booster, showed a particular resistance to its removal what made us look for alternatives. Thus, we evaluated bacteriorhodopsin from Exiguobacterium sibiricum as a new tag enhancing membrane-targeting expression. However, the best results we have achieved with cell-free expression system based on E.coli S30 extract that yielded 1 mg of Cx 26 from 1 mL of reaction mixture. Moreover, cell-free expression system flexibility permitted us to develop an original purification approach for Cx 26 expressed with no any tags. The purified protein self-assembled into hexamers and after reconstitution into unilamellar liposomes demonstrated to be functional by responding to free Ca++ level by switching between closed and opened states. The yield achieved is sufficient for the modern high-throughput crystallization techniques.
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| Lizenz: | 
Urheberrechtsschutz
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| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Physikalische Biologie |
| Dokument erstellt am: | 27.10.2016 |
| Dateien geändert am: | 27.10.2016 |
| Promotionsantrag am: | 23.10.2014 |
| Datum der Promotion: | 25.11.2014 |
