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The RD1 proteins of Mycobacterium tuberculosis : expression in Mycobacterium smegmatis and biochemical characterization

Daugelat, S. and Kowall, J. and Mattow, J. and Bumann, D. and Winter, R. and Hurwitz, R. and Kaufmann, S. H. E.. (2003) The RD1 proteins of Mycobacterium tuberculosis : expression in Mycobacterium smegmatis and biochemical characterization. Microbes and Infection, 5 (12). pp. 1082-1095.

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Official URL: http://edoc.unibas.ch/dok/A5259816

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Abstract

A 9.5-kb section of DNA called region of deletion 1 (RD1) is present in virulent Mycobacterium tuberculosis strains but is deleted in all attenuated Mycobacterium bovis BCG vaccine strains. This region codes for at least nine genes. Some or all RD1 gene products may be involved in virulence and pathogenesis, and at least two, ESAT-6 and CFP-10, represent potent T- and B-cell antigens. In order to produce the entire set of RD1 proteins with their natural posttranslational modifications, a robust expression system for M. tuberculosis proteins in the fast-growing saprophytic strain Mycobacterium smegmatis was developed. Our system employs the inducible acetamidase promoter and allows translational fusion of recombinant M. tuberculosis proteins with polyhistidine or influenza hemagglutinin epitope tags for affinity purification. Using eGFP as reporter gene, we showed that the acetamidase promoter is tightly regulated in M. smegmatis and that this promoter is much stronger than the widely used constitutive groEL2 promoter. We then cloned 11 open reading frames (ORFs) found within RD1 and successfully expressed and purified the respective proteins. Sera from tuberculosis patients and M. tuberculosis-infected mice reacted with 10 purified RD1 proteins, thus demonstrating that Rv3871, Rv3872, Rv3873, CFP-10, ESAT-6, Rv3876, Rv3878, Rv3879c and ORF-14 are expressed in vivo. Finally, glycosylation of the RD1 proteins was analyzed. We present preliminary evidence that the PPE protein Rv3873 is glycosylated at its C terminus, thus highlighting the ability of M. smegmatis to produce M. tuberculosis proteins bearing posttranslational modifications.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Infection Biology > Molecular Microbiology (Bumann)
UniBasel Contributors:Bumann, Dirk
Item Type:Article, refereed
Article Subtype:Research Article
Bibsysno:Link to catalogue
Publisher:Elsevier
ISSN:1286-4579
e-ISSN:1769-714X
Note:Publication type according to Uni Basel Research Database: Journal article
Identification Number:
Last Modified:27 Nov 2017 09:26
Deposited On:22 Mar 2012 13:23

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