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Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems

Berggren, Kristina; Veide, Andres; Nygren, Per-Åke and Tjerneld, Folke LU (1999) In Biotechnology and Bioengineering 62(2). p.135-144
Abstract
Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the... (More)
Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135-144, 1999. (Less)
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author
organization
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Contribution to journal
publication status
published
subject
keywords
aqueous two-phase systems, partitioning, thermoseparating polymer, fusion proteins
in
Biotechnology and Bioengineering
volume
62
issue
2
pages
135 - 144
publisher
John Wiley & Sons
external identifiers
  • scopus:0033585488
ISSN
1097-0290
language
English
LU publication?
yes
id
5bdabe45-fec8-4f01-8103-46ad971901a0 (old id 125249)
alternative location
http://www3.interscience.wiley.com/cgi-bin/abstract/71003252/ABSTRACT
date added to LUP
2007-07-04 11:34:45
date last changed
2017-01-01 04:30:12
@article{5bdabe45-fec8-4f01-8103-46ad971901a0,
  abstract     = {Genetic engineering has been used for the fusion of peptides, with different length and composition, on a protein to study the effect on partitioning in aqueous two-phase systems containing thermoseparating polymers. Peptides containing 2-6 tryptophan residues or tryptophan plus 1-3 lysine or aspartate residues, were fused near the C-terminus of the recombinant protein ZZT0, where Z is a synthetic IgG-binding domain derived from domain B in staphylococcal protein A. The partitioning behavior of the peptides and fusion proteins were studied in an aqueous two-phase system composed of dextran and the thermoseparating ethylene oxide-propylene oxide random copolymer, EO30PO70. The zwitterionic compound beta-alanine was used to reduce the charge-dependent salt effects on partitioning, and to evaluate the contribution to the partition coefficient from the amino acid residues, Trp, Lys, and Asp, respectively. Trp was found to direct the fusion proteins to the EO-PO copolymer phase, while Asp and Lys directed them to the dextran phase. The effect of sodium perchlorate and triethylammonium phosphate on the partitioning of the fusion proteins was also studied. Salt effects were directly proportional to the net charge of the fusion proteins. Sodium perchlorate was found to be 3.5 times more effective in directing positively charged proteins to the EO-PO copolymer phase compared to the effect of triethyl ammonium phosphate on negatively charged proteins. An empirical correlation has been tested where the fusion protein partitioning is a result of independent contributions from unmodified protein, fused peptide, and salt effects. A good agreement with experimental data was obtained which indicates the possibility, by independent measurements of partitioning of target protein and fusion peptide, to approximately predict the fusion protein partitioning. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 135-144, 1999.},
  author       = {Berggren, Kristina and Veide, Andres and Nygren, Per-Åke and Tjerneld, Folke},
  issn         = {1097-0290},
  keyword      = {aqueous two-phase systems,partitioning,thermoseparating polymer,fusion proteins},
  language     = {eng},
  number       = {2},
  pages        = {135--144},
  publisher    = {John Wiley & Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Genetic engineering of protein-peptide fusions for control of protein partitioning in thermoseparating aqueous two-phase systems},
  volume       = {62},
  year         = {1999},
}