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| 2. |
- Kepka, C., et al.
(författare)
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Pilot-scale extraction of an intracellular recombinant cutinase from E-coli cell homogenate using a thermoseparating aqueous two-phase system
- 2003
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 103:2, s. 165-181
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Tidskriftsartikel (refereegranskat)abstract
- A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 1 to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO50PO50 (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)4 tag was used for enhanced target protein partitioning to the EO50PO50 phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO50PO50 phase from the first step was supplemented with a non-ionic detergent (C12-18EO5) and heated to the cloud point (CP) temperature (45 degreesC). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.
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| 3. |
- Roobol-Boza, M, et al.
(författare)
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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
- 2004
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1043:2, s. 217-223
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Tidskriftsartikel (refereegranskat)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.
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