Advanced

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)
Please use this url to cite or link to this publication:
author
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
2007-07-05 16:59:07
date last changed
2018-10-03 10:38:59
@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},
  keyword      = {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},
  volume       = {103},
  year         = {2003},
}