Stubborn contaminants: Influence of detergents on the purity of the multidrug ABC transporter BmrA
| Autor: | Gina Catalina Reyes-Mejia, Pierre Falson, Jonathan Sarwan, Arnaud Kilburg, Benjamin Wiseman, Jean-Michel Jault, Vincent Chaptal |
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| Přispěvatelé: | Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'enzymologie et biochimie structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS) |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
Models
Molecular Protein Structure Phosphorylcholine [SDV]Life Sciences [q-bio] Detergents Molecular Sequence Data Porins lcsh:Medicine ATP-binding cassette transporter Chemical Fractionation Proteomics Bioinformatics Biochemistry Mass Spectrometry Transmembrane Transport Proteins chemistry.chemical_compound Coli strain Bacterial Proteins Escherichia coli Amino Acid Sequence Maltose Protein Structure Quaternary lcsh:Science ComputingMilieux_MISCELLANEOUS Multidisciplinary lcsh:R Biology and Life Sciences Proteins Transporter Contamination Recombinant Proteins chemistry Membrane protein ATP-Binding Cassette Transporters lcsh:Q Multidrug Resistance-Associated Proteins Protein Structure Determination Crystallization Bacterial outer membrane Bacillus subtilis Research Article |
| Zdroj: | 'PloS One ', vol: 9, pages: e114864-1-e114864-25 (2014) PLoS ONE, Vol 9, Iss 12, p e114864 (2014) PLoS ONE PLoS ONE, 2014, 9 (12), pp.e114864. ⟨10.1371/journal.pone.0114864⟩ PLoS ONE, Public Library of Science, 2014, 9 (12), pp.e114864. ⟨10.1371/journal.pone.0114864⟩ |
| ISSN: | 1932-6203 |
| DOI: | 10.1371/journal.pone.0114864⟩ |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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