title: A comparative approach to strategies for cloning, expression, and purification of Mycobacterium tuberculosis mycolyl transferase 85B and evaluation of immune responses in BALB/c mice.
abstract: Protein antigens have drawn a lot of attention from investigators working on tuberculosis vaccines. These proteins can be used to improve the immunogenicity of the new generation BCG vaccines or even replace them completely. Recombinant technology is used to insure the production of pure mycobacterial antigens in high quantities. Mycolyl transferase 85B (Ag85B) is a potent, mycobacterial antigen that significantly stimulates immune responses. Since Ag85B is an apolar protein, production of the water-soluble antigen is of interest. In this work, we report a systematic optimization strategy concerning cloning systems and purification methods, aiming at increasing the yield of recombinant Ag85B. Our optimized method resulted in a yield of 8 mg of recombinant Ag85B from 1 liter of induced culture (400 μg/ml) by using pET32a(+), Escherichia coli Rosseta-gami™(DE3) pLysS and a Ni-NTA agarose-based procedure and on-column re-solubilization. The purified recombinant Ag85B showed strong immunostimulating properties by inducing high levels of TNF-α, IFN-γ, IL-12, and IgG2a in immunized mice, therefore it can effectively be applied in TB vaccine researches.
authors.affiliation: Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Haniyeh
authors.initials: H
authors.lastname: Aghababa
authors.forename: Ashraf
authors.initials: A
authors.lastname: Mohabati Mobarez
authors.forename: Nima
authors.initials: N
authors.lastname: Khoramabadi
authors.forename: Mehrdad
authors.initials: M
authors.lastname: Behmanesh
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mahdavi
authors.forename: Majid
authors.initials: M
authors.lastname: Tebianian
authors.forename: Mehdi
authors.initials: M
authors.lastname: Nejati
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D015964:Gene Expression Regulation, Bacterial; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D018664:Interleukin-12 / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000276:immunology; D014376:Tuberculosis / Q000188:drug therapy* / Q000235:genetics / Q000517:prevention & control; D032581:Tuberculosis Vaccines / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D007074:Immunoglobulin G; D032581:Tuberculosis Vaccines; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D007371:Interferon-gamma; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis