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Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems

Fexby, Sara LU ; Nilsson, Anna LU ; Hambraeus, Gustav LU ; Tjerneld, Folke LU and Bülow, Leif LU (2004) In Biotechnology Progress 20(3). p.793-798
Abstract
The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three... (More)
The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase. (Less)
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Contribution to journal
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published
subject
keywords
INDUCED PHASE-SEPARATION, AMINO-ACID-RESIDUES, ESCHERICHIA-COLI, SURFACE HYDROPHOBICITY, RECOMBINANT PROTEINS, MONOMERIC PROTEINS, BIPHASIC SYSTEMS, PEPTIDE FUSIONS, PURIFICATION, CUTINASE
in
Biotechnology Progress
volume
20
issue
3
pages
793 - 798
publisher
The American Chemical Society
external identifiers
  • wos:000221841800021
  • scopus:2942704125
ISSN
1520-6033
DOI
10.1021/bp034177j
language
English
LU publication?
yes
id
64a9d190-b9b7-471c-89b2-97c075e4796c (old id 126098)
date added to LUP
2007-07-05 08:54:31
date last changed
2017-01-01 06:51:03
@article{64a9d190-b9b7-471c-89b2-97c075e4796c,
  abstract     = {The green fluorescent protein GFPuv has been genetically engineered to investigate the influence of N-terminal tyrosine extensions in aqueous two-phase systems. Fusions in the N-terminus affected the protein expression, and tags containing three tyrosines and prolines influenced the expression favorably. This effect is probably due to changes in mRNA stability, because the amounts of corresponding mRNAs correlated with the amounts of GFPuv proteins. The partitioning was investigated in two different aqueous two-phase systems, a two-polymer system composed of EO30PO70/dextran and a PEG/salt system with potassium phosphate. Partitioning in the PEG/salt system generally was more favorable than in the EO30PO70/dextran system. Tags with three tyrosines resulted in higher partitioning toward the EO30PO70- and PEG-rich phases, respectively. The effect of adding proline residues to the tag was also investigated, and the partitioning effect of the tag was enhanced when prolines were included in the tags with three tyrosines. The best tyrosine tag, Y3P2, increased the partition coefficient 5 times in the PEG/salt system. Thermoseparation of the EO30PO70 phase allowed recovery of 83% Y3P2-GFPuv protein in a water phase.},
  author       = {Fexby, Sara and Nilsson, Anna and Hambraeus, Gustav and Tjerneld, Folke and Bülow, Leif},
  issn         = {1520-6033},
  keyword      = {INDUCED PHASE-SEPARATION,AMINO-ACID-RESIDUES,ESCHERICHIA-COLI,SURFACE HYDROPHOBICITY,RECOMBINANT PROTEINS,MONOMERIC PROTEINS,BIPHASIC SYSTEMS,PEPTIDE FUSIONS,PURIFICATION,CUTINASE},
  language     = {eng},
  number       = {3},
  pages        = {793--798},
  publisher    = {The American Chemical Society},
  series       = {Biotechnology Progress},
  title        = {Partitioning and characterization of tyrosine-tagged green fluorescent proteins in aqueous two-phase systems},
  url          = {http://dx.doi.org/10.1021/bp034177j},
  volume       = {20},
  year         = {2004},
}