Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.

Abstract : BACKGROUND: Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map. FINDINGS: For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris-HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein. CONCLUSIONS: An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.
Type de document :
Article dans une revue
BMC Research Notes, BioMed Central, 2013, 6 (1), pp.55. 〈10.1186/1756-0500-6-55〉
Liste complète des métadonnées

Littérature citée [32 références]  Voir  Masquer  Télécharger

http://www.hal.inserm.fr/inserm-00796280
Contributeur : Ed. Bmc <>
Soumis le : samedi 2 mars 2013 - 17:02:09
Dernière modification le : mardi 3 juillet 2018 - 11:31:52
Document(s) archivé(s) le : vendredi 31 mai 2013 - 03:54:49

Fichiers

1756-0500-6-55.pdf
Fichiers éditeurs autorisés sur une archive ouverte

Identifiants

Collections

Citation

Louise Lefrancois, Christelle Bodier, Sophie Lecher, Florence Gilbert, Thierry Cochard, et al.. Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis.. BMC Research Notes, BioMed Central, 2013, 6 (1), pp.55. 〈10.1186/1756-0500-6-55〉. 〈inserm-00796280〉

Partager

Métriques

Consultations de la notice

477

Téléchargements de fichiers

352