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Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems - Purification of the human type 1 11 beta-hydroxysteroid dehydrogenase

Roobol-Boza, M; Dolby, Viveka LU ; Doverskog, M; Barrefelt, A; Lindqvist, F; Oppermann, U C; Van Alstine, K K and Tjerneld, Folke LU (2004) In Journal of Chromatography A 1043(2). p.217-223
Abstract
Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound I I p-hydroxysteroid dehydrogenase type I (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity... (More)
Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound I I p-hydroxysteroid dehydrogenase type I (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved. (C) 2004 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Aqueous two-phase systems, Affinity adsorption, Pichia pastoris, Enzymes, Membrane proteins
in
Journal of Chromatography A
volume
1043
issue
2
pages
217 - 223
publisher
Elsevier
external identifiers
  • wos:000222807500011
  • pmid:15330095
  • scopus:3042717658
ISSN
0021-9673
DOI
10.1016/j.chroma.2004.05.061
language
English
LU publication?
yes
id
159be09a-8f7a-4fbc-a7d9-ce05b0f17b2f (old id 138731)
date added to LUP
2007-07-06 14:30:41
date last changed
2017-01-01 06:51:28
@article{159be09a-8f7a-4fbc-a7d9-ce05b0f17b2f,
  abstract     = {Recently developed aqueous two-phase systems based on non-ionic detergents and polymers are suitable for the separation of membrane proteins. Moreover, within this relatively membrane protein "friendly" environment, changes in temperature can be controlled and stabilizing agents may be added to ensure integrity of the target protein during isolation. Here, we use aqueous two-phase partitioning for the isolation of membrane bound I I p-hydroxysteroid dehydrogenase type I (11beta-HSD1). Different detergents were used to find optimal conditions regarding solubilization and retaining target protein activity. We explored in situ solubilization by adding detergent directly to the aqueous two-phase system, as well as a batch metal affinity capture step of 6xHis tagged 11beta-HSD1 in the two-phase system. The use of detergent/polymer two-phase systems resulted in a specific enzyme activity of 3840 nmol mg(-1) min(-1) of the target membrane protein compared to a conventional purification protocol where a specific enzyme activity of 1440 nmol mg(-1) min(-1) was achieved. (C) 2004 Elsevier B.V. All rights reserved.},
  author       = {Roobol-Boza, M and Dolby, Viveka and Doverskog, M and Barrefelt, A and Lindqvist, F and Oppermann, U C and Van Alstine, K K and Tjerneld, Folke},
  issn         = {0021-9673},
  keyword      = {Aqueous two-phase systems,Affinity adsorption,Pichia pastoris,Enzymes,Membrane proteins},
  language     = {eng},
  number       = {2},
  pages        = {217--223},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Membrane protein isolation by in situ solubilization, partitioning and affinity adsorption in aqueous two-phase systems - Purification of the human type 1 11 beta-hydroxysteroid dehydrogenase},
  url          = {http://dx.doi.org/10.1016/j.chroma.2004.05.061},
  volume       = {1043},
  year         = {2004},
}