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Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein co...

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Autores principales: Wiseman, Benjamin, Kilburg, Arnaud, Chaptal, Vincent, Reyes-Mejia, Gina Catalina, Sarwan, Jonathan, Falson, Pierre, Jault, Jean-Michel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269414/
https://www.ncbi.nlm.nih.gov/pubmed/25517996
http://dx.doi.org/10.1371/journal.pone.0114864
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author Wiseman, Benjamin
Kilburg, Arnaud
Chaptal, Vincent
Reyes-Mejia, Gina Catalina
Sarwan, Jonathan
Falson, Pierre
Jault, Jean-Michel
author_facet Wiseman, Benjamin
Kilburg, Arnaud
Chaptal, Vincent
Reyes-Mejia, Gina Catalina
Sarwan, Jonathan
Falson, Pierre
Jault, Jean-Michel
author_sort Wiseman, Benjamin
collection PubMed
description Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.
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spelling pubmed-42694142014-12-26 Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA Wiseman, Benjamin Kilburg, Arnaud Chaptal, Vincent Reyes-Mejia, Gina Catalina Sarwan, Jonathan Falson, Pierre Jault, Jean-Michel PLoS One Research Article Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli. Public Library of Science 2014-12-17 /pmc/articles/PMC4269414/ /pubmed/25517996 http://dx.doi.org/10.1371/journal.pone.0114864 Text en © 2014 Wiseman et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Wiseman, Benjamin
Kilburg, Arnaud
Chaptal, Vincent
Reyes-Mejia, Gina Catalina
Sarwan, Jonathan
Falson, Pierre
Jault, Jean-Michel
Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title_full Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title_fullStr Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title_full_unstemmed Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title_short Stubborn Contaminants: Influence of Detergents on the Purity of the Multidrug ABC Transporter BmrA
title_sort stubborn contaminants: influence of detergents on the purity of the multidrug abc transporter bmra
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269414/
https://www.ncbi.nlm.nih.gov/pubmed/25517996
http://dx.doi.org/10.1371/journal.pone.0114864
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