Journal of Chromatography B, 743 (2000) 13–19 www.elsevier.com / locate / chromb Modelprocess for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems a b b b Hiroshi Umakoshi , Josefine Persson , MajKroon ,Hans-Olof Johansson , b a b , * DanielE. Otzen ,RyoichiKuboi, Folke Tjerneld a Department of Chemical Science and Engineering , Graduate School of Engineering Science , Osaka University , 1 - 3 Machikaneyama-cho , Toyonaka , Osaka560 - 8531, Japan b Department of Biochemistry , Center for Chemistry and Chemical Engineering , P. O . Box 124 Lund University , S- 221 00 Lund , Sweden Abstract For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide and propylene oxide (Breox) and dextran T-500. Between 25 and 508C, the partition coefficients of CI2 in Breox–dextran T-500 systems remain constant at neutral pH. However, a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 508C, respectively. The partitioning behavior of also investigated in thermoseparating water–Breox systems at 55–608C, where CI2 was partitioned to the polymer-ri at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference betw folded and the unfolded states of the protein, where the unfolded CI2 with a more hydrophobic surface is partitioned to the relatively hydrophobic Breox phase in both systems. A separation process is presented based on the partitioning beha unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The target protein can be recovered through (i) selective separation in Breox–dextran systems, (ii) refolding in Breox phase, and (iii) thermoseparation of primary Breox phase.  2000 Elsevier Science B.V. All rights reserved. Keywords : Aqueous two-phase systems; Protein folding; Chymotrypsin inhibitor 2; Thermoseparating polymers 1. Introduction purification of biological products [1,2].Recently, the use of thermoseparating polymers in aqueous Aqueoustwo-phase systems (ATPS),which are two-phasesystemshas been introduced [3–10]. spontaneously formed in solutions of two non-com- When such polymers are heated abovea critical patiblepolymers(e.g. dextranand poly(ethylene temperature, i.e. the cloud point, the solubility of the glycol) (PEG)) or a polymer (e.g. PEG) and a high polymerwill decrease and a system composed of concentration of salt (e.g.phosphate), have wide- water and a polymer phase is formed. This makes it spread use in biochemistry and biotechnology for possible to perform temperature-induced phase sepa- ration whereby a target protein can be separated from the polymer and recovered in a clean water phase. *Corresponding author. Fax:146-46-222-4534. E-mailaddress : folke.tjerneld@biokem.lu.se (F. Tjerneld) Such smartpolymersystemsmay improvethe 0378-4347 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 3 7 8 - 4 3 4 7 ( 0 0 ) 0 0 1 9 0 - 0