Abstract
A novel generic approach is described for the selective extraction of detergents from mixed detergent/lipid/protein micelles for the preparation of proteoliposomes of defined lipid-protein ratio. The approach is based on the much higher affinity of inclusion compounds of the cyclodextrin type for detergents in comparison with bilayer-forming lipids. This approach has distinct advantages over other procedures currently in use. It produces good results with all detergents tested, independent of type and critical micelle concentration, and appears to be generally applicable. It yields nearly quantitative recovery of membrane protein in the proteoliposome fraction. Finally, no large excess of lipid is required; a molar ratio of lipid to protein of 100 to 1 already produces proteoliposomes with functional membrane protein, but higher ratios are well tolerated. The size of the vesicles thus obtained depends on the detergent used. Separation of the resulting proteoliposomes from the detergent-cyclodextrin complexes was most easily achieved by centrifugation through a discontinuous sucrose gradient. A variety of detergents was tested in this procedure on the bovine rod visual pigment rhodopsin in combination with retina lipids. In all cases good yields of proteoliposomes were obtained, which contained fully functional rhodopsin.