MPI für molekulare Genetik / Department of Vertebrate Genomics |
|Homologous high-throughput expression and purification of highly conserved E coli proteins|
|Authors:||Ergin, Asgar; Büssow, Konrad; Sieper, Joachim; Thiel, Andreas; Duchmann, Rainer; Adam, Thomas|
|Date of Publication (YYYY-MM-DD):||2007-06-06|
|Title of Journal:||Microbial Cell Factories|
|Journal Abbrev.:||Microb Cell Fact|
|Copyright:||© 1999-2008 BioMed Central Ltd|
|Review Status:||not specified|
|Abstract / Description:||Background
Genetic factors and a dysregulated immune response towards commensal bacteria contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). Animal models demonstrated that the normal intestinal flora is crucial for the development of intestinal inflammation. However, due to the complexity of the intestinal flora, it has been difficult to design experiments for detection of proinflammatory bacterial antigen(s) involved in the pathogenesis of the disease. Several studies indicated a potential association of E. coli with IBD. In addition, T cell clones of IBD patients were shown to cross react towards antigens from different enteric bacterial species and thus likely responded to conserved bacterial antigens. We therefore chose highly conserved E. coli proteins as candidate antigens for abnormal T cell responses in IBD and used high-throughput techniques for cloning, expression and purification under native conditions of a set of 271 conserved E. coli proteins for downstream immunologic studies.
As a standardized procedure, genes were PCR amplified and cloned into the expression vector pQTEV2 in order to express proteins N-terminally fused to a seven-histidine-tag. Initial small-scale expression and purification under native conditions by metal chelate affinity chromatography indicated that the vast majority of target proteins were purified in high yields. Targets that revealed low yields after purification probably due to weak solubility were shuttled into Gateway (Invitrogen) destination vectors in order to enhance solubility by N-terminal fusion of maltose binding protein (MBP), N-utilizing substance A (NusA), or glutathione S-transferase (GST) to the target protein. In addition, recombinant proteins were treated with polymyxin B coated magnetic beads in order to remove lipopolysaccharide (LPS). Thus, 73% of the targeted proteins could be expressed and purified in large-scale to give soluble proteins in the range of 500 μg.
Here, we report a cost-efficient procedure to produce around 200 soluble recombinant E. coli proteins in large-scale, including removal of LPS by polymyxin B coated beads for subsequent use of the proteins in downstream immunological studies.
|External Publication Status:||published|
|Communicated by:||Hans Lehrach|
|Affiliations:||MPI für molekulare Genetik|
|External Affiliations:||Universitätsmedizin Berlin, Charité, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany;
Deutsches Rheuma-Forschungszentrum, Charitéplatz 1, 10117 Berlin, Germany;
Universitätsmedizin Berlin, Charité, Campus Charité Mitte, Dorotheenstr. 96, 10117 Berlin, Germany
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