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The Improved Method for Isolation of Photochrome Trans-membrane Protein Bacteriorhodopsin from Purple Membranes of Halobacterium Halobacterium Halobium ET 1001
Authors: Oleg Mosin, Ignat Ignatov
Number of views: 552
It was developed the improved method for isolation of photochrome trans-membraine protein bacteriorhodopsin (output – 5 mg from 100 g of wet biomass) capable to transform light energy to electrochemical energy of generated protons H+ and АТP. The protein was isolated from purple membranes of photo-organotrophic halobacterium Halobacterium halobium ET 1001 by cellular autolysis by distilled water, processing of bacterial biomass by ultrasound at 22 KHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, solubilization with 0.5% (w/v) SDS-Na, fractionation by MeOH and column gel permeation chromatography (GPC) of the final protein on Sephadex G-200 with 0.1% (w/v) SDS-Na and 2.5 mM ETDA. The homogeneity of the isolated bacteriorhodopsin was proved by combination of preparative and analytical methods, including elecrtophoresis in 12.5% (w/v) PAAG with 0.1% (w/v) SDS-Na and regeneration of apomembranes with 13-trans-retinal.