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.
Pubmed ID: 21602923 RIS Download
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The Center for Biological Sequence Analysis of the Technical University of Denmark conducts basic research in the field of bioinformatics and systems biology and directs its research primarily towards topics related to the elucidation of the functional aspects of complex biological mechanisms. A large number of computational methods have been produced, which are offered to others via WWW servers. Several data sets are also available. The center also has experimental efforts in gene expression analysis using DNA chips and data generation in relation to the physical and structural properties of DNA. The on-line prediction services at CBS are available as interactive input forms. Most of the servers are also available as stand-alone software packages with the same functionality. In addition, for some servers, programmatic access is provided in the form of SOAP-based Web Services. The center also educates engineering students in biotechnology and systems biology and offers a wide range of courses in bioinformatics, systems biology, human health, microbiology and nutrigenomics.
View all literature mentionsA python software package that can identify nucleosome positions given histone-modification ChIP-seq or nucleosome sequencing at the nucleosome level.
View all literature mentionsA portal for bioinformatics analyses, including the following: alignment assembly database display genetics hmm information nucleic phylogeny protein sequence structure
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