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Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer

Nilsson, Anna LU ; Johansson, Hans-Olof LU ; Mannesse, Maurice; Egmond, Maarten R. and Tjerneld, Folke LU (2002) In Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00 1601(2). p.138-148
Abstract
Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C14H29) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12oC in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few... (More)
Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C14H29) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12oC in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Protein-peptide fusion, HM-EOPO, Hydrophobically modified, Partitioning, Aqueous two-phase system, Thermoseparating polymer
in
Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00
volume
1601
issue
2
pages
138 - 148
publisher
Elsevier
external identifiers
  • wos:000179479300003
  • pmid:12445475
  • scopus:0037121457
ISSN
1570-9639
DOI
10.1016/S1570-9639(02)00462-4
language
English
LU publication?
yes
id
8ebdb465-9c09-4071-814a-35fa93592228 (old id 124868)
date added to LUP
2007-07-06 12:28:29
date last changed
2017-04-23 04:27:06
@article{8ebdb465-9c09-4071-814a-35fa93592228,
  abstract     = {Genetic engineering has been used to construct hydrophobically modified fusion proteins of cutinase from Fusarium solani pisi and tryptophan-containing peptides. The aim was to enhance the partitioning of the tagged protein in a novel aqueous two-phase system formed by only one water-soluble polymer. The system was based on a hydrophobically modified random copolymer of ethylene oxide (EO) and propylene oxide (PO) units, HM-EOPO, with myristyl groups (C14H29) at both ends. The HM-EOPO polymer is strongly self-associating and has a lower critical solution temperature (cloud point) at 12oC in water. At temperatures above the cloud point a two-phase system is formed with a water top phase and a polymer-enriched bottom phase. By adding a few percent of hydroxypropyl starch polymer, Reppal PES 200, to the system, it is possible to change the densities of the phases so the HM-EOPO-enriched phase becomes the top phase and Reppal-enriched phase is the bottom phase. Tryptophan-based peptides strongly preferred the HM-EOPO rich phase. The partitioning was increased with increasing length of the peptides. Full effect of the tag as calculated from peptide partitioning data was not found in the protein partitioning. When a short spacer was introduced between the protein and the tag the partitioning was increased, indicating a better exposure to the hydrophobic core of the polymer micelle. By adding a hydrophilic spacer between the protein and trp-tag, it was possible to increase the partitioning of cutinase 10 times compared to wild-type cutinase partitioning. By lowering the pH of the system and addition of NaCl, the partitioning of tagged protein was further increased towards the HM-EOPO phase. After isolating the HM-EOPO phase, the temperature was increased and the protein was back-extracted from the HM-EOPO phase to a fresh water phase.},
  author       = {Nilsson, Anna and Johansson, Hans-Olof and Mannesse, Maurice and Egmond, Maarten R. and Tjerneld, Folke},
  issn         = {1570-9639},
  keyword      = {Protein-peptide fusion,HM-EOPO,Hydrophobically modified,Partitioning,Aqueous two-phase system,Thermoseparating polymer},
  language     = {eng},
  number       = {2},
  pages        = {138--148},
  publisher    = {Elsevier},
  series       = {Biochimica et Biophysica Acta - Proteins and Proteomics2002-01-01+01:00},
  title        = {Partitioning of peptide-tagged proteins in aqueous two-phase systems using hydrophobically modified micelle-forming thermoseparating polymer},
  url          = {http://dx.doi.org/10.1016/S1570-9639(02)00462-4},
  volume       = {1601},
  year         = {2002},
}