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

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  • Hiroshi Umakoshi, Grad. Sch. Eng. Sci.
  • ,
  • Josefine Persson, Lund University
  • ,
  • Maj Kroon, Lund University
  • ,
  • Hans Olof Johansson, Lund University
  • ,
  • Daniel E. Otzen
  • Ryoichi Kuboi, Grad. Sch. Eng. Sci.
  • ,
  • Folke Tjerneld, Lund University

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.

Original languageEnglish
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Pages (from-to)13-19
Number of pages7
Publication statusPublished - 23 Jun 2000

    Research areas

  • Aqueous two-phase systems, Chymotrypsin inhibitor 2, Protein folding, Thermoseparating polymers

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