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PEDS Advance Access originally published online on October 7, 2008
Protein Engineering Design and Selection 2008 21(12):709-720; doi:10.1093/protein/gzn046
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

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

Víctor M. Hernández-Rocamora, Beatriz Maestro, Almudena Mollá-Morales1 and Jesús M. Sanz2

Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Avda Universidad s/n, Elche 03202, Spain

2 To whom correspondence should be addressed. E-mail: jmsanz{at}umh.es

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing 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°C thermal stabilization compared with 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 ~65°C, whereas C-LytAm7 may stand temperatures up to 90°C. 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.

Keywords: affinity chromatography/choline-binding proteins/protein immobilization/protein stability/recombinant protein purification

Received February 14, 2008; revised July 21, 2008; accepted August 6, 2008.

1 Present address: Instituto de Bioingeniería, Universidad Miguel Hernández, Avda Universidad s/n, Elche 03202, Spain.


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