PURIFICATION OF RECOMBINANT APOLIPOPROTEIN A-1(MILANO) EXPRESSED IN ESCHERICHIA-COLI USING AQUEOUS 2-PHASE EXTRACTION FOLLOWED BY TEMPERATURE-INDUCED PHASE-SEPARATION

A method for purification of recombinant apolipoprotein Al in aqueous two-phase systems has been studied. A mutant of apolipoprotein A-I, th e Milano variant, was expressed in E, coli. Phase systems containing e thylene oxide (EO)-propylene oxide (PO) random copolymers have been us ed. These polymers are thermoseparating and have the ability to separa te into one water-rich and one polymer-rich phase when heated above a critical temperature i.e. the cloud point. The filtrate from an E. col i fermentation was added to a primary aqueous two-phase system compose d of an EO-PO copolymer and Reppal, which is an inexpensive hydroxypro pyl starch. Apolipoprotein A-I was partitioned to the top EO-PO copoly mer phase and contaminating proteins to the bottom starch phase. The p hase diagrams for Reppal PES 100-EO50PO50 (Ucon) and Reppal PES 100-EO 30PO70 were determined. The effect on partitioning, when changing para meters such as polymer concentration, type of polymer, protein concent ration, pH, salt concentration and volume ratio. were studied. Studies on E. coli DNA partitioning showed that DNA could be partitioned stro ngly to the bottom phase. An optimal system was scaled up from 5 g to 5 kg with similar degrees of purification, i.e. 2.5 and 2.7 and yields of 79% and 82% respectively. Furthermore temperature-induced phase fo rmation was used for separation of apolipoprotein A-I from the copolym er by raising the temperature above the copolymer cloud point. thus, r ecovering protein in a 'clean' water phase. (C) 1998 Elsevier Science BN. All rights reserved.