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Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography

Kepka, Cecilia LU ; Collet, E; Roos, Fredrik LU ; Tjerneld, Folke LU and Veide, A (2005) In Journal of Chromatography A 1075(1-2). p.33-41
Abstract
In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was... (More)
In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to > 95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process. © 2005 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Chromatography A
volume
1075
issue
1-2
pages
33 - 41
publisher
Elsevier
external identifiers
  • pmid:15974115
  • wos:000229550800003
  • scopus:19444361831
ISSN
0021-9673
DOI
10.1016/j.chroma.2005.03.054
language
English
LU publication?
yes
id
7052180c-051c-4823-be57-bac41540a725 (old id 151441)
date added to LUP
2007-07-05 16:51:12
date last changed
2017-06-18 04:35:51
@article{7052180c-051c-4823-be57-bac41540a725,
  abstract     = {In this work, the interfacing of a poly(ethylene glycol) (PEG)-phosphate aqueous two-phase system with hydrophobic interaction chromatography (HIC) for primary recovery of an intracellular protein was evaluated. As a model protein, a recombinant cutinase furnished with a tryptophan-proline (WP) peptide tag was used and produced intracellularly in Escherichia coli (E. coli). E. coli cell homogenate was partitioned in a two-phase system and the top phase yield, concentration and purity of the tagged ZZ-cutinase-(WP)(4) was evaluated as function of polymer sizes, system pH and phase volume ratio. The partition behaviour of cell debris, total protein and endotoxin was also monitored. In the HIC part, the chromatographic yield and purity was investigated with respect to ligand hydrophobicity, dilution of loaded top phase and elution conditions. Based on the results, a recovery process was demonstrated where a PEG 1500-K-Na phosphate salt aqueous two-phase system was interfaced with a HIC column. The interfacing was facilitated by the Trp-tagged peptide. The tagged ZZ-cutinase-(WP)4 was obtained in a PEG-free phase and purified to > 95% purity according to silver stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels with a total yield of 83% during the two-step recovery process. © 2005 Elsevier B.V. All rights reserved.},
  author       = {Kepka, Cecilia and Collet, E and Roos, Fredrik and Tjerneld, Folke and Veide, A},
  issn         = {0021-9673},
  language     = {eng},
  number       = {1-2},
  pages        = {33--41},
  publisher    = {Elsevier},
  series       = {Journal of Chromatography A},
  title        = {Two-step recovery process for tryptophan tagged cutinase: Interfacing aqueous two-phase extraction and hydrophobic interaction chromatography},
  url          = {http://dx.doi.org/10.1016/j.chroma.2005.03.054},
  volume       = {1075},
  year         = {2005},
}