Successful expression and purification of DPPD using a codon optimized synthetic gene

Area of Science:

• Microbiology
• Immunology
• Protein Expression

Background:

• DPPD (Rv0061) from Mycobacterium tuberculosis is crucial for specific delayed type hypersensitivity reactions.
• Current tuberculin skin tests for tuberculosis diagnosis lack specificity.
• Previous attempts to express DPPD as a single or fusion protein were unsuccessful.

Purpose of the Study:

• To develop a method for producing and purifying recombinant DPPD (rDPPD) as a single molecule.
• To confirm the biological activity and specificity of the purified rDPPD.
• To assess the utility of rDPPD for improved tuberculosis diagnosis.

Main Methods:

• Engineered a synthetic gene with E. coli codon bias for DPPD production.
• Cloned the synthetic gene into the pET14b expression vector.
• Transformed host cells (Rosetta 2(DE3) pLysS or BL-21(DE3)pLysS) and induced protein over-expression.
• Purified rDPPD using standard protocols and confirmed identity via Mass Spectroscopy.
• Assessed biological activity by measuring IFN-γ production in peripheral blood mononuclear cells.

Main Results:

• Successfully produced and purified rDPPD at 5-10 mg/l of bacterial broth.
• Mass Spectroscopy confirmed the purified protein as DPPD.
• Purified rDPPD stimulated significant IFN-γ production in PPD-positive donors, indicating biological activity.
• The DPPD gene is specific to the tuberculosis-complex organisms of the Mycobacterium genus.

Conclusions:

• A novel method using a synthetic gene and E. coli expression system enabled the production of highly purified, biologically active rDPPD.
• This rDPPD molecule shows promise for enhancing the specificity of tuberculosis diagnostic tests.
• rDPPD could be a valuable tool for identifying individuals sensitized to tubercle bacilli.