Recyclable aqueous two-phase system based on two pH-responsive copolymers and its application to porcine circovirus type 2 Cap protein purification

Aqueous two-phase system (ATPS) has great potential in industrial applications of bio-separations and bio-reactions. However, its large-scale application is limited by recovery difficulty of phase systems. In this paper, a recyclable ATPS was prepared by two pH-responsive copolymers (PADB4.99 and PMDM7.08) and applied for purification of porcine circovirus type 2 Cap protein fermentation broth (PCV2 Cap protein). Phase separation mechanism was studied by using low-field nuclear magnetic resonance (LF-NMR). The results showed that relatively desirable ATPS were formed successfully when two copolymer concentrations were 4%-6% (w/w) with pH 7.5-8.6. The main parameters, such as copolymer concentration, temperature, pH, type and concentration of salts, were investigated. The results demonstrated that the best ATPS consisted of 4% (w/w) PADB4.99 and 6% (w/w) PMDM7.08, the optimal partition coefficient (K) and extraction recovery (ER) of PCV2 cap protein were 0.25 and 94.2% in the presence of 50 mM Li2SO4, 3.53 and 93.4% in the presence of 40 mM KCl, respectively, at 30 °C, pH 8.1. The maximum purity of PCV2 Cap protein was 88.4%. In addition, the two phase-forming copolymers could be recycled by adjusting solution pH to their isoelectric points (pI), with recoveries of over 97.5%.