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Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.

Becker, Kristian LU ; Hallgren, Elisabeth; Carredano, Enrique; Palmgren, Ronnie and Bülow, Leif LU (2009) In Journal of Molecular Recognition 22(2). p.104-109
Abstract
Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand... (More)
Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R(2) = 0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure. Copyright (c) 2008 John Wiley & Sons, Ltd. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
green fluorescent protein, hydrophobic interaction chromatography, tyrosine tag, pH-responsive, polymer ligand
in
Journal of Molecular Recognition
volume
22
issue
2
pages
104 - 109
publisher
John Wiley & Sons
external identifiers
  • wos:000263566400008
  • pmid:18654996
  • scopus:63449090356
ISSN
1099-1352
DOI
10.1002/jmr.897
language
English
LU publication?
yes
id
4d51ff60-4dd0-4177-8c21-66cff84edecf (old id 1180833)
date added to LUP
2008-10-03 12:44:43
date last changed
2017-01-01 04:25:48
@article{4d51ff60-4dd0-4177-8c21-66cff84edecf,
  abstract     = {Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R(2) = 0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure. Copyright (c) 2008 John Wiley & Sons, Ltd.},
  author       = {Becker, Kristian and Hallgren, Elisabeth and Carredano, Enrique and Palmgren, Ronnie and Bülow, Leif},
  issn         = {1099-1352},
  keyword      = {green fluorescent protein,hydrophobic interaction chromatography,tyrosine tag,pH-responsive,polymer ligand},
  language     = {eng},
  number       = {2},
  pages        = {104--109},
  publisher    = {John Wiley & Sons},
  series       = {Journal of Molecular Recognition},
  title        = {Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.},
  url          = {http://dx.doi.org/10.1002/jmr.897},
  volume       = {22},
  year         = {2009},
}