Use of aqueous two-phase systems in the purification of human interferon-alpha 1 from recombinant Escherichia coli

Intracellular human recombinant interferon alpha 1 (rhIFN-alpha 1) has been purified using two consecutive aqueous two-phase extractions: selective extraction followed by back extraction. In the selective extraction step using polyethylene glycol (PEG)-potassium phosphate aqueous two-phase system, optimization of the systems was achieved by varying the concentrations of PEG-phosphate ester, phosphate, added inorganic salts, pH, and biomass load. The optimum condition was found to be 22% (w/w) synthesized PEG-phosphate ester, 16% (w/w) potassium phosphate and 3% (w/w) NaCl at pH 6.9 with 10% (wet w/w) biomass load. The corresponding top phase yield of rhIFN-alpha 1 was 99.6% with a purification factor of 25. It was observed that most of the modified PEG is partitioned to the phosphate-enriched bottom phase, so the mechanism for enhancement to the extraction is likely to be an electrostatic expulsion effect. In the back extraction step where the partially purified IFN is transferred from the PEG-rich top phase to a phosphate buffer, the concentrations of PEG-phosphate ester and phosphate, pH, and other parameters have been adjusted. The pH was found to be the most sensitive. A back extraction system using 20% (w/w) PEG-phosphate ester and 10% (w/w) potassium phosphate system at pH 6.0 gives the yield of rhIFN-alpha 1 in the bottom phase (approximately 76%). This newly established back extraction procedure avoids the use of a cumbersome gel chromatography while the yield achieved is fairly high.