Research and Development for Purification of Membrane Proteins

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Crystallization of membrane proteins requires hard work, primarily because of difficulty in preparation of a stable, highly-purified and concentrated protein sample. In order to simplify the purification step of membrane proteins, we have tried to develop a novel procedure for selective isolation of a membrane protein by inducing self-association in a biological membrane and subsequent membrane vesicularization.Bacteriorhodopsin, a transmembrane protein found in halobacterium halobium, forms a two-dimensional crystal (called purple mebrane) under physiological condition. When purple membrane was incubated at high temperature with a small amount of detergent (octylthioglucoside) in the presence of a high concentration of precipitant, uniformly-sized spherical vesicles (polyhedral assembly) of bacteriorhodopsin was produced. The stability of the polyhedral assembly decreased at low temperature. By promoting fusion processes of the polyhedral assembly at low temperature, we obtained a new … More three-dimensional that diffracts X-ray diffract beyond 3.0 angstrom. This crystal belongs to the space group P622 with cell dimensions of a=b=104.7*, and c=114.1*, and it is shown to be made up of stacked planar membranes, in each of bacteriorhodopin trimers are arranged on a honey-comb lattice. The crystal contains native lipids (5 phospholipids per bR) and one phospholipid is bound firmly to a crevice between adjacent monomers in the trimeric unit. This lipid is suggested to act as a glue for formation of the trimeric structure.Light-harvesting chlorophyll-protein complex from pea is shown to form a polyhedral structure with a diameter of 27 nm under crystallization condition. It is assembled into an octahedral crystal that belongs to the space group of P2_13 with cell dimensions of a=b=c=390*.Another purification procedure was developed for bovine rhodopsin, a protein with a 7-fold transmembrane alpha-helices. The disk membrane of the photoreceptor cell was purified by a density-gradient centrifugation and the purified membrane was treated with detergent (alkylglucoside) in the presence of a high concentration of divalent cation. A single step of centrifugation of the mixture yielded a highly-purified sample of rhodopsin. Using this purified sample, we obtained 3D crystals of rhodopsin by the vapor diffusion hanging drop method. Less