Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system
(2003) In Journal of Biotechnology 103(2). p.165-181- 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 l 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... (More)
- 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 l 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 (45oC). 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/124659
- author
- Kepka, Cecilia LU ; Collet, Eric ; Persson, Josefine ; Ståhl, Åke ; Lagerstedt, Torgny ; Tjerneld, Folke LU and Veide, Andres
- organization
- publishing date
- 2003
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Aqueous two-phase system, Disk-stack separator, Extraction, Thermoseparation, Cutinase, Escherichia coli
- in
- Journal of Biotechnology
- volume
- 103
- issue
- 2
- pages
- 165 - 181
- publisher
- Elsevier
- external identifiers
-
- pmid:12814875
- wos:000183825000008
- scopus:0038119852
- ISSN
- 1873-4863
- DOI
- 10.1016/S0168-1656(03)00104-4
- language
- English
- LU publication?
- yes
- id
- 7af0c267-85e3-4c6a-8d21-1e0a33ad348a (old id 124659)
- date added to LUP
- 2016-04-01 12:25:25
- date last changed
- 2022-01-27 03:32:58
@article{7af0c267-85e3-4c6a-8d21-1e0a33ad348a, 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 l 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 (45oC). 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.}}, author = {{Kepka, Cecilia and Collet, Eric and Persson, Josefine and Ståhl, Åke and Lagerstedt, Torgny and Tjerneld, Folke and Veide, Andres}}, issn = {{1873-4863}}, keywords = {{Aqueous two-phase system; Disk-stack separator; Extraction; Thermoseparation; Cutinase; Escherichia coli}}, language = {{eng}}, number = {{2}}, pages = {{165--181}}, publisher = {{Elsevier}}, series = {{Journal of Biotechnology}}, title = {{Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system}}, url = {{http://dx.doi.org/10.1016/S0168-1656(03)00104-4}}, doi = {{10.1016/S0168-1656(03)00104-4}}, volume = {{103}}, year = {{2003}}, }