Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.
(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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1180833
- author
- Becker, Kristian LU ; Hallgren, Elisabeth ; Carredano, Enrique ; Palmgren, Ronnie and Bülow, Leif LU
- organization
- publishing date
- 2009
- 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 Inc.
- external identifiers
-
- wos:000263566400008
- pmid:18654996
- scopus:63449090356
- ISSN
- 1099-1352
- DOI
- 10.1002/jmr.897
- language
- English
- LU publication?
- yes
- additional info
- Published Online: 24 Jul 2008
- id
- 4d51ff60-4dd0-4177-8c21-66cff84edecf (old id 1180833)
- date added to LUP
- 2016-04-01 11:40:24
- date last changed
- 2025-01-14 14:08:10
@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}},
keywords = {{green fluorescent protein; hydrophobic interaction chromatography; tyrosine tag; pH-responsive; polymer ligand}},
language = {{eng}},
number = {{2}},
pages = {{104--109}},
publisher = {{John Wiley & Sons Inc.}},
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}},
doi = {{10.1002/jmr.897}},
volume = {{22}},
year = {{2009}},
}