Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.
Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2011-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC3094339?pdf=render |
| _version_ | 1819237987491250176 |
|---|---|
| author | Carmen Galián Florence Manon Manuela Dezi Cristina Torres Christine Ebel Daniel Lévy Jean-Michel Jault |
| author_facet | Carmen Galián Florence Manon Manuela Dezi Cristina Torres Christine Ebel Daniel Lévy Jean-Michel Jault |
| author_sort | Carmen Galián |
| collection | DOAJ |
| description | Optimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment. |
| first_indexed | 2024-12-23T13:29:04Z |
| format | Article |
| id | doaj.art-5357d18f2f764c27b551e5c0d62caadb |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-23T13:29:04Z |
| publishDate | 2011-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-5357d18f2f764c27b551e5c0d62caadb2022-12-21T17:45:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032011-01-0165e1967710.1371/journal.pone.0019677Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization.Carmen GaliánFlorence ManonManuela DeziCristina TorresChristine EbelDaniel LévyJean-Michel JaultOptimized protocols for achieving high-yield expression, purification and reconstitution of membrane proteins are required to study their structure and function. We previously reported high-level expression in Escherichia coli of active BmrC and BmrD proteins from Bacillus subtilis, previously named YheI and YheH. These proteins are half-transporters which belong to the ABC (ATP-Binding Cassette) superfamily and associate in vivo to form a functional transporter able to efflux drugs. In this report, high-yield purification and functional reconstitution were achieved for the heterodimer BmrC/BmrD. In contrast to other detergents more efficient for solubilizing the transporter, dodecyl-ß-D-maltoside (DDM) maintained it in a drug-sensitive and vanadate-sensitive ATPase-competent state after purification by affinity chromatography. High amounts of pure proteins were obtained which were shown either by analytical ultracentrifugation or gel filtration to form a monodisperse heterodimer in solution, which was notably stable for more than one month at 4°C. Functional reconstitution using different lipid compositions induced an 8-fold increase of the ATPase activity (k(cat)∼5 s(-1)). We further validated that the quality of the purified BmrC/BmrD heterodimer is suitable for structural analyses, as its reconstitution at high protein densities led to the formation of 2-D crystals. Electron microscopy of negatively stained crystals allowed the calculation of a projection map at 20 Å resolution revealing that BmrC/BmrD might assemble into oligomers in a lipidic environment.http://europepmc.org/articles/PMC3094339?pdf=render |
| spellingShingle | Carmen Galián Florence Manon Manuela Dezi Cristina Torres Christine Ebel Daniel Lévy Jean-Michel Jault Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. PLoS ONE |
| title | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. |
| title_full | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. |
| title_fullStr | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. |
| title_full_unstemmed | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. |
| title_short | Optimized purification of a heterodimeric ABC transporter in a highly stable form amenable to 2-D crystallization. |
| title_sort | optimized purification of a heterodimeric abc transporter in a highly stable form amenable to 2 d crystallization |
| url | http://europepmc.org/articles/PMC3094339?pdf=render |
| work_keys_str_mv | AT carmengalian optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT florencemanon optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT manueladezi optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT cristinatorres optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT christineebel optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT daniellevy optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization AT jeanmicheljault optimizedpurificationofaheterodimericabctransporterinahighlystableformamenableto2dcrystallization |