Purification of recombinant apolipoprotein A-1Milano expressed in Escherichia coli using aqueous two-phase extraction followed by temperature-induced phase separation.
A method for purification of recombinant apolipoprotein A1 in aqueous two-phase systems has been studied. A mutant of apolipoprotein A-1, the Milano variant, was expressed in E. coli. Phase systems containing ethylene oxide (EO)-propylene oxide (PO) random copolymers have been used. These polymers are thermoseparating and have the ability to separate 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. coli fermentation was added to a primary aqueous two-phase system composed of an EO-PO copolymer and Reppal, which is an inexpensive hydroxypropyl starch. Apolipoprotein A-1 was partitioned to the top EO-PO copolymer phase and contaminating proteins to the bottom starch phase. The phase diagrams for Reppal PES 100-EO50PO50 (Ucon) and Reppal PES 100-EO30PO70 were determined. The effect on partitioning, when changing parameters such as polymer concentration, type of polymer, protein concentration, pH, salt concentration and volume ratio, were studied. Studies on E. coli DNA partitioning showed that DNA could be partitioned strongly 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 formation was used for separation of apolipoprotein A-1 from the copolymer by raising the temperature above the copolymer cloud point; thus, recovering protein in a 'clean' water phase.
Department of Biochemistry, Lund University, Sweden.
J
J
Persson
L
L
Nyström
H
H
Ageland
F
F
Tjerneld
D016632:Apolipoprotein A-I / Q000235:genetics / Q000302:isolation & purification*; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000302:isolation & purification; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007477:Ions; D011095:Polyethylenes / Q000737:chemistry; D011126:Polypropylenes / Q000737:chemistry; D011994:Recombinant Proteins / Q000302:isolation & purification; D012492:Salts; D013696:Temperature; D014508:Urea
D016632:Apolipoprotein A-I; D004274:DNA, Recombinant; D007477:Ions; D011095:Polyethylenes; D011126:Polypropylenes; D011994:Recombinant Proteins; D012492:Salts; C080631:apolipoprotein A-I Milano; D014508:Urea; C078661:UCON 50-HB-5100