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

Umakoshi, Hiroshi; Persson, Josefine; Kroon, Maj; Johansson, Hans-Olof LU ; Otzen, Daniel E; Kuboi, Ryoichi and Tjerneld, Folke LU (2000) In Journal of Chromatography. B 743(1-2). p.13-19
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 50oC, 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 50oC, respectively. The partitioning behavior of CI2 was also investigated in... (More)
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 50oC, 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 50oC, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60oC, 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. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Chymotrypsin inhibitor 2, Thermoseparating polymers
in
Journal of Chromatography. B
volume
743
issue
1-2
pages
13 - 19
publisher
Elsevier
external identifiers
  • scopus:0034705827
ISSN
1873-376X
DOI
10.1016/S0378-4347(00)00190-0
language
English
LU publication?
yes
id
d63a5b91-18c4-4538-8109-0b23030d657c (old id 125214)
date added to LUP
2007-07-09 09:44:12
date last changed
2017-06-25 03:33:13
@article{d63a5b91-18c4-4538-8109-0b23030d657c,
  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 50oC, 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 50oC, respectively. The partitioning behavior of CI2 was also investigated in thermoseparating water-Breox systems at 55-60oC, 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.},
  author       = {Umakoshi, Hiroshi and Persson, Josefine and Kroon, Maj and Johansson, Hans-Olof and Otzen, Daniel E and Kuboi, Ryoichi and Tjerneld, Folke},
  issn         = {1873-376X},
  keyword      = {Chymotrypsin inhibitor 2,Thermoseparating polymers},
  language     = {eng},
  number       = {1-2},
  pages        = {13--19},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography. B},
  title        = {Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems},
  url          = {http://dx.doi.org/10.1016/S0378-4347(00)00190-0},
  volume       = {743},
  year         = {2000},
}