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Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system

Kepka, Cecilia LU ; Collet, Eric ; Persson, Josefine ; Ståhl, Åke ; Lagerstedt, Torgny ; Tjerneld, Folke LU and Veide, Andres (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)
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author
; ; ; ; ; and
organization
publishing date
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}},
}