Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo(3) ubiquinol oxidase, in a detergent-polymer aqueous two-phase system containing metal-chelating polymer

A system has been developed for selective partitioning of membrane proteins . For the first time, an integral membrane protein, cytochrome bo, ubiquino l oxidase from Escherichia coli, has been affinity partitioned in an aqueou s two-phase system. The systems used were different detergent/polymer aqueo us two-phase systems containing a metal-chelating polymer, such as poly(eth yleneglycol)-iminodiacetic acid-Cu(II) as well as dextran-iminodiacetic aci d-Cu(II). Many non-ionic detergents, such as alkyl(polyethyleneoxide) (CmEO n), Triton, Tween and alkylglucosides, form two-phase systems in mixture wi th polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enri ched phase in equilibrium with a polymer-enriched phase are formed. In gene ral, membrane proteins partition strongly to the micelle phase. We show tha t it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, wi th a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold). Th e affinity partitioning was characterized and the effects of ligand concent ration, pH, time, salts, buffer type, imidazole and charged detergent are d iscussed. Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer a queous two-phase systems, and the method is especially suitable for the pur ification of labile integral membrane proteins, such as receptors. (C) 2000 Elsevier Science B.V. All rights reserved.