OJI  Vol.1 No.1 , June 2011
Successful expression and purification of dppd, using a codon optimized synthetic gene
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
Cite this paper: nullKashino, S. and Campos-Neto, A. (2011) Successful expression and purification of dppd, using a codon optimized synthetic gene. Open Journal of Immunology, 1, 1-7. doi: 10.4236/oji.2011.11001.

[1]   S. Jana, J.K. Deb. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol (2005); 67:289-98.

[2]   W.H. Brondyk. Selecting an appropriate method for expressing a recombinant protein. Methods Enzymol (2009); 463:131-47.

[3]   A. Campos-Neto, V. Rodrigues-Junior, D.B. Pedral-Sam- paio, E.M. Netto, P.J. Ovendale, R.N. Coler, Y.A. Skeiky, R. Badaro, S.G. Reed. Evaluation of DPPD, a single recombinant Mycobacterium tuberculosis protein as an alternative antigen for the Mantoux test. Tuberculosis (Edinb ) (2001); 81:353-8.

[4]   R.N. Coler, Y.A. Skeiky, P.J. Ovendale, T.S. Vedvick, L. Gervassi, J. Guderian, S. Jen, S.G. Reed, A. Campos- Neto. Cloning of a Mycobacterium tuberculosis gene encoding a purified protein derivative protein that elicits strong tuberculosis-specific delayed-type hypersensitivity. J Infect Dis (2000); 182:224-33.

[5]   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.

[6]   E. Lee, R.S. Holzman. Evolution and current use of the tuberculin test. Clin Infect Dis (2002); 34:365-70.

[7]   M.S. Rangel-Frausto, S. Ponce-De-Leon-Rosales, C. Martinez-Abaroa, K. Haslov. Tuberculosis and tuberculin quality: best intentions, misleading results. Infect Control Hosp Epidemiol (2001); 22:481-4.

[8]   M.E. Villarino, M.J. Brennan, C.M. Nolan, A. Catanzaro, L.L. Lundergan, N.N. Bock, C.L. Jones, Y.C. Wang, W.J. Burman. Comparison testing of current (PPD-S1) and proposed (PPD-S2) reference tuberculin standards. Am J Respir Crit Care Med (2000); 161:1167-71.

[9]   D.L. Whipple, M.V. Palmer, R.E. Slaughter, S.L. Jones. Comparison of purified protein derivatives and effect of skin testing on results of a commercial gamma interferon assay for diagnosis of tuberculosis in cattle. J Vet Diagn Invest (2001); 13:117-22.

[10]   C. Liu, E. Flamoe, H.J. Chen, D. Carter, S.G. Reed, A. Campos-Neto. Expression and purification of immunologically reactive DPPD, a recombinant Mycobacterium tuberculosis skin test antigen, using Mycobacterium smegmatis and Escherichia coli host cells. Can J Microbiol (2004); 50:97-105.

[11]   H. Furthmayr, R. Timpl. Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem (1971); 41:510-6.

[12]   J.A. Marrs, G.B. Bouck. The two major membrane skeletal proteins (articulins) of Euglena gracilis define a novel class of cytoskeletal proteins. J Cell Biol (1992); 118:1465-75.

[13]   K.F. Ogle, K.K. Lee, D.C. Krause. Nucleotide sequence analysis reveals novel features of the phase-variable cytadherence accessory protein HMW3 of Mycoplasma pneumoniae. Infect Immun (1992); 60:1633-41.

[14]   T. Proft, H. Hilbert, G. Layh-Schmitt, R. Herrmann. The proline-rich P65 protein of Mycoplasma pneumoniae is a component of the Triton X-100-insoluble fraction and exhibits size polymorphism in the strains M129 and FH. J Bacteriol (1995); 177:3370-8.

[15]   A. Wang, J. Clapper, J.A. Guderian, T.M. Foy, G.R. Fanger, M.W. Retter, Y.A. Skeiky. A novel method for increasing the expression level of recombinant proteins. Protein Expr Purif (2003); 30:124-33.