Reconstitution of Membrane Proteins into Liposomes

Abstract

The reconstitution of membrane proteins into liposomes is a powerful tool that can be used to identify the mechanism of the action of membrane proteins. The prospects of achieving optimal proteoliposome reconstitution are good when reliable methods and systematic experimental analysis are used. This chapter deals with the various strategies commonly used to reconstitute proteoliposomes and focuses on approaches that have led to the production of highly functional proteoliposomes. Four basic strategies are outlined—mechanical means, freeze-thawing, organic solvents, and detergents. The chapter also introduces a new method for membrane protein reconstitution. The new reconstitution strategy proceeds in four stages: (1) preparation of large, homogeneous, and unilamellar liposomes, (2) addition of detergent to the preformed liposomes, through all the range of the solubilization process, (3) addition of solubilized protein at each well-defined step of the solubilization process, and (4) detergent removal and characterization of the reconstituted products. Besides the need for measuring the activity of the protein, any method of membrane-protein reconstitution should fulfill a number of important criteria that must be analyzed to characterize unequivocally the efficiency of the reconstitution. The first parameter to analyze to check the efficiency of a reconstitution trial is the activity of the protein after reconstitution. The most accurate method to analyze the efficiency of membrane-protein incorporation is a density gradient.