ISSN 1214-0287 (on-line), ISSN 1214-021X (printed)
J Appl Biomed
Volume 10 (2012) No 4, p 169-176
DOI 10.2478/v10136-012-0013-z

Expression of Mycobacterium tuberculosis proteins MPT63 and MPT83 as a fusion: purification, refolding and immunological characterization

Taras Redchuk, Natalia Korotkevich, Oksana Gorbatiuk, Pavlo Gilchuk, Andrii Kaberniuk, Olena Oliynyk, Denis Kolibo, Serhiy Komisarenko

Address: Taras Redchuk, Palladin Institute of Biochemistry (NASU), 9 Leontovicha Street, Kyiv, 01601, Ukraine
rtakyiv@gmail.com

Received 3rd January 2012.
Revised 20th February 2012.
Published online 22nd February 2012.

Full text article (pdf)
Full text article (html)

SUMMARY
Proteins MPT63 and MPT83 which are common for both Mycobacterium tuberculosis and Mycobacterium bovis, due to their high immunogenicity, are thought to play a promising role in the development of immunodiagnostic reagents and vaccines. To enhance the antigenic and immunogenic properties of these proteins, fragments of the mpt83 and mpt63 genes were fused in tandem. In this article we present an effective method for the MPT63-MPT83 fusion product purification by metal-affinity chromatography and in vitro refolding. Our results demonstrate that the antigenic properties of the recombinant proteins obtained are comparable to their native analogues. The anti-rMPT63 and anti-rMPT83 sera were found to be highly reactive against the rMPT63-MPT83 fusion protein, which suggests that the fusion protein retains the antigenic properties of the parent proteins. Our results may potentially contribute to the development of improved diagnostic tools or vaccines against human and/or cattle tuberculosis.

KEY WORDS
tuberculosis; Mycobacterium tuberculosis; MPT63; MPT83; fusion; antigen

REFERENCES
Chambers MA, Stagg D, Gavier-Widen D, Lowrie D, Newell D, Hewinson RG. A DNA vaccine encoding MPB83 from Mycobacterium bovis reduces M. bovis dissemination to the kidneys of mice and is expressed in primary cell cultures of the European badger (Meles meles). Res Vet Sci. 71: 119-126, 2001.
[CrossRef] [PubMed]

Chambers MA, Gavier-Widen D, Hewinson RG. Antibody bound to the surface antigen MPB83 of Mycobacterium bovis enhances survival against high dose and low dose challenge. FEMS Immunol Med Microbiol. 41: 93-100, 2004.
[CrossRef]

Charlet D, Mostowy S, Alexander D, Sit L, Wiker HG, Behr MA. Reduced expression of antigenic proteins MPB70 and MPB83 in Mycobacterium bovis BCG strains due to a start codon mutation in sigK. Mol Microbiol. 56: 1302-1313, 2005.
[CrossRef] [PubMed]

Clark ED. Protein refolding for industrial processes. Curr Opin Biotechnol. 12: 202-207, 2001.
[CrossRef]

Clemens DL, Horwitz MA. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med. 181: 257-270, 1995.
[CrossRef] [PubMed]

Fleischmann RD, Alland D, Eisen JA, Carpenter L, White O, Peterson J, DeBoy R, Dodson R, Gwinn M, Haft D, Hickey E, Kolonay JF et al. Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol. 184: 5479-5490, 2002.
[CrossRef] [PubMed]

Goulding CW, Parseghian A, Sawaya MR, Cascio D, Apostol MI, Gennaro ML, Eisenberg D. Crystal structure of a major secreted protein of Mycobacterium tuberculosis-MPT63 at 1.5-A resolution. Protein Sci. 11: 2887-2893, 2002.
[CrossRef] [PubMed]

Green LR, Jones CC, Sherwood AL, Garkavi IV, Cangelosi GA, Thacker TC, Palmer MV, Waters WR, Rathe CV. Single-antigen serological testing for bovine tuberculosis. Clin Vaccine Immunol. 16: 1309-1313, 2009.
[CrossRef] [PubMed]

Harboe M, Wiker HG, Ulvund G, Lund-Pedersen B, Andersen AB, Hewinson RG, Nagai S. MPB70 and MPB83 as indicators of protein localization in mycobacterial cells. Infect Immun. 66: 289-296, 1998.
[PubMed]

Horwitz MA, Lee BW, Dillon BJ, Harth G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA. 92: 1530-1534, 1995.
[CrossRef]

Lee BY, Horwitz MA. T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs. Infect Immun. 67: 2665-2670, 1999.
[PubMed]

Li M, Su ZG, Janson JC. In vitro protein refolding by chromatographic procedures. Protein Expr Purif. 33: 1-10, 2004.
[CrossRef] [PubMed]

McShane H, Brookes R, Gilbert SC, Hill AV. Enhanced immunogenicity of CD4(+) T-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect Immun. 69: 681-686, 2001.
[CrossRef] [PubMed]

Mukherjee S, Daifalla N, Liu C, Campos-Neto A. Alternative approach to express Mycobacterium tuberculosis proteins in Escherichia coli. Biotechniques. 35: 34-36, 2003.
[PubMed]

Nagai S, Wiker HG, Harboe M, Kinomoto M. Isolation and partial characterization of major protein antigens in the culture fluid of Mycobacterium tuberculosis. Infect Immun. 59: 372-382, 1991.
[PubMed]

Raja A, Ranganathan UD, Bethunaickan R. Improved diagnosis of pulmonary tuberculosis by detection of antibodies against multiple Mycobacterium tuberculosis antigens. Diagn Microbiol Infect Dis. 60: 361-368, 2008.
[CrossRef] [PubMed]

Song L, Nakaar V, Kavita U, Price A, Huleatt J, Tang J, Jacobs A, Liu G, Huang Y, Desai P, Maksymiuk G, Takahashi V et al. Efficacious recombinant influenza vaccines produced by high yield bacterial expression: a solution to global pandemic and seasonal needs. PLoS One. 3: e2257, 2008.
[CrossRef] [PubMed]

Studier FW. Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif. 41: 207-234, 2005.
[CrossRef] [PubMed]

Tsumoto K, Ejima D, Kumagai I, Arakawa T. Practical considerations in refolding proteins from inclusion bodies. Protein Expr Purif. 28: 1-8, 2003.
[CrossRef]

Vallejo LF, Rinas U. Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins. Microb Cell Fact. 3: 11, 2004.
[CrossRef] [PubMed]

Vosloo W, Tippoo P, Hughes JE, Harriman N, Emms M, Beatty DW, Zappe H, Steyn LM. Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70. Gene. 188: 123-128, 1997.
[CrossRef]

Xue T, Stavropoulos E, Yang M, Ragno S, Vordermeier M, Chambers M, Hewinson G, Lowrie DB, Colston MJ, Tascon RE. RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection. Infect Immun. 72: 6324-6329, 2004.
[CrossRef] [PubMed]
CITED

0


BACK