TY - JOUR

T1 - Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems

AU - Umakoshi, Hiroshi

AU - Persson, Josefine

AU - Kroon, Maj

AU - Johansson, Hans Olof

AU - Otzen, Daniel E.

AU - Kuboi, Ryoichi

AU - Tjerneld, Folke

PY - 2000/6/23

Y1 - 2000/6/23

N2 - 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 50°C, the partition coefficients of CI2 in Breox-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°C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60°C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the 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 behavior of 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. (C) 2000 Elsevier Science B.V.

AB - 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 50°C, the partition coefficients of CI2 in Breox-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°C, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60°C, where CI2 was partitioned to the polymer-rich phase at pH values below 2.4. These results on the CI2 partitioning can be explained by the conformational difference between the 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 behavior of 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. (C) 2000 Elsevier Science B.V.

KW - Aqueous two-phase systems

KW - Chymotrypsin inhibitor 2

KW - Protein folding

KW - Thermoseparating polymers

UR - http://www.scopus.com/inward/record.url?scp=0034705827&partnerID=8YFLogxK

U2 - 10.1016/S0378-4347(00)00190-0

DO - 10.1016/S0378-4347(00)00190-0

M3 - Journal article

C2 - 10942268

AN - SCOPUS:0034705827

VL - 743

SP - 13

EP - 19

JO - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

JF - Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

SN - 1570-0232

IS - 1-2

ER -