Publikationsserver der RWTH Aachen University
Charakterisierung fluoreszierender Varianten des Zytokinrezeptors gp130 hinsichtlich biologischer Aktivität und Dimerisierung mit molekularbiologischen und fluoreszenzspektroskopischen Methoden
Abstract
dc:descriptionThe glycoprotein gp130 is expressed as a trans-membrane receptor on the cell surface of many cells. Because of its function as a central signal transducer component of the cytokines IL-6, IL-11, LIF, CNTF, OSM and CT-1 the behaviour of gp130 towards its ligands is of major interest. For IL-6 the cell surface complex, which is necessary to start the intracellular signal cascade, is composed out of the ligand, the specific IL-6-Receptor and at least two molecules of gp130. LIF induces a heterodimerisation of gp130 and LIF-Receptor. The fluorescent proteins Cyan fluorescent protein (CFP) and Yellow fluorescent protein (YFP) derived from jelly fish are important tools in protein biochemistry. Fluorescence resonance energy transfer (FRET) offers a possibility to visualize protein-protein-interactions. The aim of this work was the characterization of the fluorescent variants of the cytokine receptor gp 130 including biological activity and dimerization with the use of molecular biological methods and Fluorescence Spectroscopy. · The biological activity of the fluorescent variants of gp130 was analysed. The full length construct gp130-ETC-YFP has been shown to perform signal transduction, however, this could be excluded for the shortened gp130-ET-YFP. · With molecular biological methods and Fluorescence Spectroscopy it has been proven that gp 130 forms homodimers before the interaction with its ligands. · A method to measure Fluorescence Resonance Energy Transfer (FRET) in living cells, as well as in cell-lysates has been set up. This analysis was performed both for the soluble and the receptor-bound fluorescent proteins. · By using Fluorescence Spectroscopy the soluble fluorescent proteins could be characterized in more details concerning their reaction towards different environmental conditions e.g. Excitation-wavelength, temperature and pH. The fusion protein composed out of CFP and YFP, CYFP, could be used as a cytosolic FRET-positive control. Based on this the receptor-bound positive control gp130-ET-CYFP has been constructed. After expressing in COS7-cells its behaviour has been analysed during Laser-scanning Microscopy and Fluorescence Spectroscopy.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2004
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Wortmann, Saskia Brigitte
- Contributors dc:contributor
-
- Heinrich, Peter C.
Subjects
dc:subject × 8Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger
Identifiers
dc:identifier.*- OAI identifier oai:identifier
- oai:publications.rwth-aachen.de:59653