Preparative purification of functional bacteriorhodopsin by high-performance size-exclusion chromatography.
High-performance size-exclusion chromatography (HPSEC) was used to produce stable bacteriorhodopsin essentially free of native lipids. The purified bacteriorhodopsin was shown to be highly functional when reconstituted into phospholipid vesicles. Purple membrane was washed in the detergent 3-[( 3-cholamidopropyl)dimethylammonio]-2,2-hydroxy-1-propanesulfonate (CHAPSO) to remove a large fraction (65%) of the membrane lipids, solubilized in Triton X-100 and purified on a Bio-Sil TSK G3000SW column using a CHAPSO mobile phase. Pooled column fractions of bacteriorhodopsin from 25-mg sample loads show a 280/548 nm absorbance ratio of 1.5-1.6 and contain less than 4% endogenous lipids. This HPSEC method requires much less expensive synthetic detergent and is much faster than open column methods [cf. L.J.W. Miercke, P.E. Ross, R.M. Stroud and E.A. Dratz, J. Biol. Chem., 264 (1989) 7531-7535].
Miercke
L J
LJ
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448.
Stroud
R M
RM
Dratz
E A
EA
D001436:Bacteriorhodopsins / Q000302:isolation & purification* / Q000378:metabolism; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D011522:Protons
D002793:Cholic Acids; D003902:Detergents; D011522:Protons; D001436:Bacteriorhodopsins; C048531:chapso