Selective extraction systems for detection and purification of membrane proteins
Abstract: Increased understanding of membrane proteins is important for characterization of biochemical processes. Improved membrane protein isolation methods is a key issue for effective functional and structural determination of the large amount of unknown membrane proteins. In this thesis, a novel approach towards selective membrane protein purification is introduced. Polymer induced micelle extraction (PIME) systems are formed in mixtures between nonionic detergents and water-soluble polymers. Over a critical concentration the detergent/polymer/water mixture separates into two phases and forms a detergent-rich and a polymer-rich phase. Separation of membrane proteins from cytosolic proteins and insoluble materials is achieved by the large difference in distribution between the phases. In general, membrane proteins are extracted into the micelle-enriched phase, while water-soluble proteins partitions in the polymer-rich phase. Membranes can be solubilized directly into the PIME system at low temperature with many commonly used nonionic detergents. The developed micelle-based extraction system is suggested to replace the traditional solubilization step in membrane protein purification. A combined solubilization and primary purification of the target proteins can be obtained. Such smart solubilization in PIME systems was utilized to pre-fractionate membrane proteins prior to detection of a putative membrane bound receptor. The partitioning and phase separation mechanism in the PIME systems was examined in detail and explained in a thermodynamic context. An understanding how to optimize the system for membrane protein extraction was gained. Several approaches to further increase the separation between hydrophobic and hydrophilic proteins was studied such as addition of affinity ligands, salts and ionic detergents. A novel high-resolution method was developed, called affinity PIME systems. A selective membrane protein extraction system was obtained by affinity partitioning of the target membrane protein into the polymer-rich phase, while contaminating membrane proteins remained in the opposite micelle-rich phase. The concept was successfully demonstrated by metal affinity purification of the His-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase from Escherichia coli membranes. Affinity PIME systems should be of great importance for a fast purification of structural intact membrane proteins due the mild and selective properties of the system. PIME systems can be applied for purification of amphiphilic fusion proteins. An effective purification of the amphiphilic fusion protein endoglucanase I-hydrophobin I was obtained from a Trichoderma reesei fermentation filtrate. A method was developed for removal of detergent from the purified fusion protein.
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