Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems

For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermose parating polymer two-phase systems with varying pH and temperature. Chymotr ypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state man ner, was partitioned in an aqueous two-phase system (ATPS) composed of a ra ndom copolymer of ethylene oxide and propylene oxide (Breox) and dextran T- 500. Between 25 and 50 degrees C, the partition coefficients of CI2 in Breo x-dextran T-500 systems remain constant at neutral pH. However, there is a drastic increase at pH values below 1.7, 2.1, and 2.7 at 25, 40 and 50 degr ees C, respectively. Thge partitioning behavior of CI2 was also investigate d in thermoseparating water-Breox systems at 55-60 degrees C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These result s on the CI2 partitioning can be explained by the conformational difference between the folded and the unfolded states of the protein, where the unfol ded CI2 with a more hydrophobic surface is partitioned to the relatively hy drophobic Breox phase in both systems. A separation process is presented ba sed on the partitioning behavior of unfolded and refolded CI2 by control of pH and temperature in thermoseparating polymer two-phase systems. The targ et protein can be recovered through (i) selective separation in Breox-dextr an systems, (ii) refolding in Breox phase, and (iii) thermoseparation of pr imary Breox phase. (C) 2000 Elsevier Science B.V. All rights reserved.