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.
A system has been developed for selective partitioning of membrane proteins. For the first time, an integral membrane protein, cytochrome bo3 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) (CmEOn), 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.
Sivars
U
U
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
Abramson
J
J
Iwata
S
S
Tjerneld
F
F
D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003902:Detergents / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000302:isolation & purification*; D006639:Histidine; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008670:Metals / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry
D002614:Chelating Agents; D003902:Detergents; D008565:Membrane Proteins; D008670:Metals; D010455:Peptides; D011108:Polymers; C033223:polyhistidine; D006639:Histidine; C038862:cytochrome o oxidase; D003576:Electron Transport Complex IV