AFFINITY PARTITIONING OF PROTEIN-A USING A MAGNETIC AQUEOUS 2-PHASE SYSTEM

An affinity extraction method combining an aqueous two-phase system an d affinity ligand-bound fine magnetic particles is proposed as a novel protein separation technique. Polymer-coated magnetic particles, Eudr agit-Mag, partitioned to the upper phase stably in three types of PEG- based aqueous two-phase systems. The phase separation time was shorten ed by the addition of the magnetic particles, and further reduced by a pplying a magnetic field. Human IgG was immobilized to Eudragit-Mag fo r the separation of staphylococcal protein A. The partition coefficien t of protein A increased up to 35-fold when IgG-bound Eudragit-Mag was added to a PEG-phosphate aqueous two-phase system. The IgG-bound Eudr agit-Mag was applied to the purification of protein A produced by reco mbinant Escherichia coil. In the separation, 90% of protein A added wa s selectively adsorbed to the affinity ligand, where the content of pr otein A was 89%. By using 3.5 M KSCN solution as an eluant, 39% of pro tein A was recovered with 49% of purity from a crude extract. In addit ion to the intrinsic advantages of aqueous two-phase extraction, the n ewly developed separation technique has advantages in the selective pa rtitioning of the target protein, a shortening of phase separation tim e, and easy recovery and reuse of the ligand.