Purification of fusion proteins using affinity microspheres in aqueous two-phase systems

Abstract

Affinity microspheres were prepared by immobilizing human γ-globulin (HγGb) onto carboxylated poly (styrene/acrylamide) latex particles [P(St/AAm)-H; average diameter 0.33 μm], which were prepared by emulsifier-free emulsion polymerization. HγGB was covalently immobilized onto the latex particles with high efficiency by the carbodiimide method. A fusion protein (ZZB1B2) of immunoglobulin G and albumin-binding domains (ZZ and B1B2, respectively) was expressed intracellularly and extracellularly in Escherichia coli and was purified by the affinity microspheres. In poly (ethylene glycol) (PEG)/potassium phosphate aqueous two-phase system, the affinity microspheres were partitioned into the PEG-rich top phase, while cells and cell debris of E. coli were displaced into the salt-rich bottom phase. Therefore, ZZB1B2 was directly purified from cell disintegrate or culture broth by combining the affinity microspheres with the aqueous two-phase partitioning, and its purity was almost the same as that purified by conventional affinity chromatography. Therefore, by this purification method, the primary purification process and the subsequent high resolution purification process are combined, and the number of purification steps can be reduced.