title: Production and Purification of Mycolyl Transferase B of Mycobacterium tuberculosis.
abstract: BACKGROUND
  Antigen 85 complex of Mycobacterium tuberculosis includes three immunogenic proteins which are TB vaccine candidates of great importance. As they are very hard to be achieved in natural form, recombinant production of them fuels immunological experiments. Production of such apolar mycobacterial proteins located in the cell wall faces substantial challenges mainly regarding their solubility. This study reports the production of soluble recombinant Ag85B with an efficient yield.
  
  
  METHODS
  Ag85B gene was cloned in pJET1.2 and subsequently in pET32a (+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with 1mM IPTG. Recombinant Ag85B was purified through dissolving inclusions in 8M urea buffer, absorbing to Ni-NTA resins, washing by buffers with decreasing urea concentrations and finally eluted in imidazole. Western blot analysis was performed using anti-6His tag antibody, rabbit anti- M. tuberculosis polyclonal antibody and serum of hospitalized TB patients.
  
  
  RESULTS
  Ag85B gene was successfully cloned in both plasmid vectors. The recombinant Ag85B was expressed in E. coli host and purified with significant yield.
  
  
  CONCLUSIONS
  Western blot results along with those of sequencing ensured accurate production of recombinant Ag85B and retaining of its antigenic structure.
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Haniyeh
authors.initials: H
authors.lastname: Aghababa
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Ashraf Mohabati
authors.initials: AM
authors.lastname: Mobarez
authors.affiliation: Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Behmanesh
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Nima
authors.initials: N
authors.lastname: Khoramabadi
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Mandana
authors.initials: M
authors.lastname: Mobarhan
keywords: Ag85 complex; Ag85B; Mycobacterium tuberculosis; TB vaccine