Affinity partitioning of a poly(histidine)-tagged integral membrane protein, cytochrome bo3 ubiquinol oxidase, in a detergent–polymer aqueous two-phase system containing metal-chelating polymer

Abstract

A system has been developed for selective partitioning of membrane proteins. For the first time, an integral membrane protein, cytochrome bo 3 ubiquinol oxidase from Escherichia coli, has been affinity partitioned in an aqueous two-phase system. The systems used were different detergent/polymer aqueous two-phase systems containing a metal-chelating polymer, such as poly(ethyleneglycol)–iminodiacetic acid–Cu(II) as well as dextran–iminodiacetic acid–Cu(II). Many non-ionic detergents, such as alkyl(polyethyleneoxide) (C mEO n), Triton, Tween and alkylglucosides, form two-phase systems in mixture with polymers, such as dextran and poly(ethyleneglycol), i.e., a micelle-enriched phase in equilibrium with a polymer-enriched phase are formed. In general, membrane proteins partition strongly to the micelle phase. We show that it is possible to selectively partition a poly(histidine)-tagged integral membrane protein into the polymer phase by metal affinity partitioning, with a shift in the partitioning coefficient from 0.015 to 4.8 (300-fold). The affinity partitioning was characterized and the effects of ligand concentration, pH, time, salts, buffer type, imidazole and charged detergent are discussed. Thus, a fast and mild affinity procedure for the purification of integral membrane proteins can be developed in affinity detergent/polymer aqueous two-phase systems, and the method is especially suitable for the purification of labile integral membrane proteins, such as receptors.