Rational stabilization of the C-LytA affinity tag by protein engineering

40 p.-8 fig.-4 tab.

Detalhes bibliográficos
Autores: Hernández-Rocamora, V. M., Maestro, Beatriz, Mollá-Morales, Almudena, Sanz, Jesús M.
Formato: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2008
País:España
Recursos:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/344830
Acesso em linha:http://hdl.handle.net/10261/344830
Access Level:acceso abierto
Palavra-chave:Affinity chromatography
Choline-binding proteins
Protein immobilization
Protein stability
Recombinant protein purification
Liquid–liquid extraction
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spelling Rational stabilization of the C-LytA affinity tag by protein engineeringRunning title: Stabilization of C-LytA by protein engineeringHernández-Rocamora, V. M.Maestro, BeatrizMollá-Morales, AlmudenaSanz, Jesús M.Affinity chromatographyCholine-binding proteinsProtein immobilizationProtein stabilityRecombinant protein purificationLiquid–liquid extraction40 p.-8 fig.-4 tab.The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Due to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 ºC thermal stabilization compared to the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at around 65 ºC, whereas C-LytAm7 may stand temperatures up to 90 ºC. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.Spanish Ministerio de Educación y Ciencia (MEC) [CIT-010000-2005-32, FIT-010000-2003-110]; Fundación SALVAT-Inquifarma.Peer reviewedOxford University PressMinisterio de Educación y Ciencia (España)Fundació Salvat-InquifarmaHernández-Rocamora, V. M. [0000-0003-2517-5707]Maestro, Beatriz [0000-0001-5317-650X]Mollá-Morales, Almudena [0000-0001-9465-0490]Sanz, Jesús M. [0000-0002-4421-9376]]202420242008info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/344830reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Ingléshttps://doi.org/10.1093/protein/gzn046Noinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/3448302026-05-22T06:33:51Z
dc.title.none.fl_str_mv Rational stabilization of the C-LytA affinity tag by protein engineering
Running title: Stabilization of C-LytA by protein engineering
title Rational stabilization of the C-LytA affinity tag by protein engineering
spellingShingle Rational stabilization of the C-LytA affinity tag by protein engineering
Hernández-Rocamora, V. M.
Affinity chromatography
Choline-binding proteins
Protein immobilization
Protein stability
Recombinant protein purification
Liquid–liquid extraction
title_short Rational stabilization of the C-LytA affinity tag by protein engineering
title_full Rational stabilization of the C-LytA affinity tag by protein engineering
title_fullStr Rational stabilization of the C-LytA affinity tag by protein engineering
title_full_unstemmed Rational stabilization of the C-LytA affinity tag by protein engineering
title_sort Rational stabilization of the C-LytA affinity tag by protein engineering
dc.creator.none.fl_str_mv Hernández-Rocamora, V. M.
Maestro, Beatriz
Mollá-Morales, Almudena
Sanz, Jesús M.
author Hernández-Rocamora, V. M.
author_facet Hernández-Rocamora, V. M.
Maestro, Beatriz
Mollá-Morales, Almudena
Sanz, Jesús M.
author_role author
author2 Maestro, Beatriz
Mollá-Morales, Almudena
Sanz, Jesús M.
author2_role author
author
author
dc.contributor.none.fl_str_mv Ministerio de Educación y Ciencia (España)
Fundació Salvat-Inquifarma
Hernández-Rocamora, V. M. [0000-0003-2517-5707]
Maestro, Beatriz [0000-0001-5317-650X]
Mollá-Morales, Almudena [0000-0001-9465-0490]
Sanz, Jesús M. [0000-0002-4421-9376]]
dc.subject.none.fl_str_mv Affinity chromatography
Choline-binding proteins
Protein immobilization
Protein stability
Recombinant protein purification
Liquid–liquid extraction
topic Affinity chromatography
Choline-binding proteins
Protein immobilization
Protein stability
Recombinant protein purification
Liquid–liquid extraction
description 40 p.-8 fig.-4 tab.
publishDate 2008
dc.date.none.fl_str_mv 2008
2024
2024
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/344830
url http://hdl.handle.net/10261/344830
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv https://doi.org/10.1093/protein/gzn046
No
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv Oxford University Press
publisher.none.fl_str_mv Oxford University Press
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869412784655040512
score 15,812429

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