Expression and purification of membrane proteins: Focus on the G-protein coupled receptor MC4r

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

Overexpression and functional characterisation of the human melanocortin 4 receptor in Sf9 cells

The human melanocortin 4 receptor (MC4r) was successfully expressed in Sf9 cells using the baculovirus infection system. N- and C-terminally His-tagged receptors generated B-max values of 14 and 23 pmol receptor/mg membrane protein, respectively. The highest expression level obtained with the C-terminally His-tagged MC4r corresponded to 0.25 mg active receptor/litre culture volume. Addition of a viral signal peptide at the N-terminus of the His-tagged MC4r did not improve the expression level. Confocal laser microscopy studies revealed that both the N- and C-terminally tagged MC4r did not accumulate intracellularly and were mainly located in the plasma membrane. The recombinant receptors showed similar affinity for the agonist NDP-MSH (K-d = 11 nM) as to MC4r expressed in mammalian cells. Functional coupling of the highest expressed C-terminal tagged receptor to endogenous Galpha protein was demonstrated through 6TPgammaS binding upon agonist stimulation of the receptor. K-i values for the ligands MTII, HS014, alpha-, beta-, and gamma-MSH are comparable to the values obtained for MC4r expressed in mammalian cells. (C) 2004 Elsevier Inc. All rights reserved

Effects of pH, salt and time on ligand binding properties of overexpressed melanocortin 4 receptor.

The G-protein coupled melanocortin 4 receptor (MC4r) plays an important role in the energy metabolism. We overexpressed the MC4r in CHO cells and performed characterisation studies on the cell membranes to determine functional stability and ligand binding properties of the receptor. The affinity for the ligands [Nle4, Image-Phe7]-αMSH and MTII was lost below pH 6 but could be restored by returning to physiological pH. Increasing NaCl concentration up to 1 M had little influence on the binding of either ligand. At neutral pH, physiological salt concentration and 4 °C the ligand affinity of the receptor was stable for up to 6 days. These findings will facilitate design of purification methods for the receptor

Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems - Purification of the human type 1 11 beta-hydroxysteroid dehydrogenase

Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound I I p-hydroxysteroid dehydrogenase type I (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved. (C) 2004 Elsevier B.V. All rights reserved