title: 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.
  
  
  RESULTS
  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.
authors: INRA, UMR ISP 1282 Infectiologie et Santé Publique, Nouzilly F-37380, France. Franck.Biet@tours.inra.fr
authors: Louise H
authors: LH
authors: Lefrancois
authors: Christelle C
authors: CC
authors: Bodier
authors: Sophie
authors: S
authors: Lecher
authors: Florence B
authors: FB
authors: Gilbert
authors: Thierry
authors: T
authors: Cochard
authors: Grégoire
authors: G
authors: Harichaux
authors: Valérie
authors: V
authors: Labas
authors: Ana Paula
authors: AP
authors: Teixeira-Gomes
authors: Dominique
authors: D
authors: Raze
authors: Camille
authors: C
authors: Locht
authors: Franck
authors: F
authors: Biet
mesh_terms: D000595:Amino Acid Sequence; D018533:Biomass; D019149:Bioreactors; D002850:Chromatography, Gel; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016927:Mycobacterium avium subsp. paratuberculosis / Q000737:chemistry*; D063347:Native Polyacrylamide Gel Electrophoresis; D017386:Sequence Homology, Amino Acid
chemical_list: D037102:Lectins; C104578:heparin-binding hemagglutinin
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