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Thermostable carbohydrate-binding modules in affinity chromatography

Johansson, Reine ; Cicortas Gunnarsson, Lavinia LU ; Ohlin, Mats LU orcid and Ohlson, Sten (2006) Bioaffinity 2005 19(4). p.275-281
Abstract
Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydratebinding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based olligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline... (More)
Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydratebinding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based olligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-mu M range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates. Copyright (c) 2006 John Wiley & Sons Ltd. (Less)
Please use this url to cite or link to this publication:
author
; ; and
organization
publishing date
type
Chapter in Book/Report/Conference proceeding
publication status
published
subject
keywords
affinity chromatography, thermostable, carbohydrate-binding modules, weak affinity
host publication
Journal of Molecular Recognition
volume
19
issue
4
pages
275 - 281
publisher
John Wiley and Sons
conference name
Bioaffinity 2005
conference location
Upsala, Sweden
conference dates
2005-08-14 - 2005-08-18
external identifiers
  • wos:000239882300005
  • scopus:33747274375
  • pmid:16838297
ISSN
0952-3499
1099-1352
DOI
10.1002/jmr.794
project
Designed carbohydrate binding modules and molecular probes
language
English
LU publication?
yes
id
5ebb7166-a791-4197-b56d-03ef462acf8a (old id 397359)
date added to LUP
2016-04-01 12:37:30
date last changed
2021-02-17 05:35:19
@inproceedings{5ebb7166-a791-4197-b56d-03ef462acf8a,
  abstract     = {Affinity chromatography is routinely used mostly on a preparative scale to isolate different biomolecules such as proteins and carbohydrates. To this end a variety of proteins is in common use as ligands. To extend the arsenal of binders intended for separation of carbohydrates, we have explored the use of carbohydratebinding modules (CBM) in affinity chromatography. The thermostable protein CBM4-2 and two variants (X-6 and A-6) thereof, selected from a newly constructed combinatorial library, were chosen for this study. The CBM4-2 predominantly binds to xylans but also crossreacts with glucose-based olligomers. The two CBM-variants X-6 and A-6 had been selected for binding to xylan and Avicel (a mixture of amorphous and microcrystalline cellulose), respectively. To assess the ability of these proteins to separate carbohydrates, they were immobilized to macroporous microparticulate silica and analyses were conducted at temperatures ranging from 25 to 65 degrees C. With the given set of CBM-variants, we were able to separate cello- and xylo-oligomers under isocratic conditions. The affinities of the CBMs for their targets were weak (in the mM-mu M range) and by adjusting the column temperature we could optimize peak resolution and chromatographic retention times. The access to thermostable CBM-variants with diverse affinities and selectivities holds promise to be an efficient tool in the field of affinity chromatography for the separation of carbohydrates. Copyright (c) 2006 John Wiley & Sons Ltd.},
  author       = {Johansson, Reine and Cicortas Gunnarsson, Lavinia and Ohlin, Mats and Ohlson, Sten},
  booktitle    = {Journal of Molecular Recognition},
  issn         = {0952-3499},
  language     = {eng},
  number       = {4},
  pages        = {275--281},
  publisher    = {John Wiley and Sons},
  title        = {Thermostable carbohydrate-binding modules in affinity chromatography},
  url          = {http://dx.doi.org/10.1002/jmr.794},
  doi          = {10.1002/jmr.794},
  volume       = {19},
  year         = {2006},
}