Expression and purification of membrane proteins: Focus on the G-protein coupled receptor MC4r
Abstract: Membrane proteins are crucial components of the cell and are involved in many biological processes. Recombinant overexpression systems together with different purification methods are necessary to obtain large amounts of purified receptor for biophysical and functional studies. More knowledge about membrane proteins, and especially G-protein coupled receptors, would facilitate the development of imporatant future drug candidates. Intrinsic membrane proteins are embedded in the membrane and detergents are used to extract them from the membrane prior to purification. It is important to perform solubilisation with suitable detergents in order to prevent aggregation and denaturation. This thesis presents results from the study of two membrane proteins; the human melanocortin 4 receptor and the human membrane bound enzyme 11beta hydroxysteroid dehydrogenase type 1 (11beta-HSD 1). The MC4r is a G-protein coupled receptor with seven transmembrane regions, which mediates signalling to a G-protein upon receptor activation. The G-protein, activated by MC4r, is able to stimulate adenylate cyclase, and thus stimulate the formation of cAMP. The 11beta-HSD 1 is a membrane bound enzyme in the endoplasmatic reticulum. The enzyme contains one transmembrane region and the protein is a NADP(H) dependent enzyme and is responsible for the reversible interconversion of active cortisol to inactive cortisone. The results present overexpression of MC4r in mammalian CHO cells and His-tagged MC4r in insect cells using the baculovirus infection system. The enzyme 11 beta-HSD 1 was succesfully overexpressed in yeast cells. Purification strategies were developed for the target proteins in order to obtain enriched and pure fractions of the proteins. Both MC4r and the 11 beta-HSD1 were expressed with histidine tags to enhance purification. The tagged MC4r was successfully purified using two-affinity steps (Ni-NTA and Heparin) together with an ion-exchange chromatography step. The enzyme 11 beta-HSD 1 was efficiently purified in a detergent/polymer system (Dextran 500/Tween 20) in combination with affinity resin. Most of the work was performed on the MC4 receptor, and is therefore the main focus of the thesis.
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