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I. Schiller, H.M. Vordermeier, W.R. Waters, A.O. Whelan, M. Coad, E. Gormley, B.M. Buddle, M. Palmer, T. Thacker, J. McNair, M. Welsh, R.G. Hewinson, B. Oesch. Bovine tuberculosis: effect of the tuberculin skin test on in vitro interferon gamma responses. Vet Immunol Immunopathol (2010); 136:1-11.

has been cited by the following article:

• TITLE: Successful expression and purification of dppd, using a codon optimized synthetic gene

  AUTHORS: Suely S. Kashino, Antonio Campos-Neto

  KEYWORDS: Mycobacterium tuberculosis; DPPD; Codon Bias

  JOURNAL NAME: Open Journal of Immunology, Vol.1 No.1, June 30, 2011

  ABSTRACT: DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitiv-ity reactions in humans infected with M. tuber-culosis. Therefore DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the di-agnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fu-sion molecule with another Mycobacterium protein. This approach was used because no expression of DPPD could be achieved either as a single molecule or as a fusion protein using a variety of commercially available expression systems. Here, we report the production and purification of rDPPD using a synthetic gene engineered to contain E. coli codon bias. The gene was cloned into pET14b expression vector, which was subsequently used to transform Rosetta 2(DE3)pLysS or BL-21(DE3)pLysS host cells. The recombinant protein was over-ex- pressed after induction with IPTG and its puri-fication was easily achieved at levels of 5 – 10 mg/l of bacterial broth cultures. The purified protein was confirmed to be DPPD by Mass Spectroscopy sequencing analysis. Moreover, purified rDPPD stimulated peripheral blood mononuclear cells of PPD positive blood do-nors to produce high levels of IFN-γ, thus con-firming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacte-rium genus, this highly purified molecule should be useful for the identification of indi-viduals sensitized with tubercle bacilli