Aqueous multiple-phase isolation of polypeptide

A method for recovering from cells a biologically active exogenous polypeptide of interest comprising contacting the cells with a chaotropic agent in an amount sufficient to extract from the cells the polypeptide, which is in a non-native conformation, and maintain its solubility and with an effective amount of phase-forming species consisting essentially of salt and polymer to form two aqueous phases, the upper one of which is enriched in the polypeptide and depleted in biomass solids and nucleic acids originating from the cells, recovering the polypeptide by separating the phases, and incubating said recovered polypeptide in a buffer of pH 7-12 comprising about 5-40% (v/v) of an alcoholic or polar aprotic solvent, about 0.2 to 3M of an alkaline earth, alkali metal, or ammonium salt, about 0.1 to 9M of a chaotropic agent, and about 0.01 to 15 .mu.M of a copper or manganese salt, wherein an oxygen source is introduced, so that refolding of the polypeptide occurs during the incubation.

A two-phase aqueous solution comprising an upper phase enriched in an exogenous polypeptide of interest in a non-native conformation and depleted in biomass solids and nucleic acids from cells in which the polypeptide was produced, wherein the aqueous solution also comprises phase-forming species, which species consist essentially of salt and polymer, and a chaotropic agent in an amount sufficient to maintain the solubility of the polypeptide, whereby said polypeptide is further characterized as refolding to active confirmation under conditions whereby the polypeptide recovered from the upper phase is incubated in a buffer of pH 7-12 into which an oxygen source is introduced comprising about 5-40% (v/v) of an alcoholic pr polar aprotic solvent, about 0.2 to 3M of an alkaline earth, alkali metal, or ammonium salt, about 0.1 to 9M of chaotropic agent, and about 0.01 to 15 .mu.M of a copper or manganese salt.