Abstract
DPPD (Rv0061) is a difficult to express protein of Mycobacterium tuberculosis that elicits strong and specific delayed type hypersensitivity reactions in humans infected with M. tuberculosis. Therefore e DPPD is a molecule that can improve the specificity of the tuberculin skin test, which is widely used as an aid for the diagnosis of tuberculosis. However, a pitfall of our initial studies was that the DPPD molecule used to perform the skin tests was engineered as fusion 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-expressed after induction with IPTG and its purification 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 donors to produce high levels of IFN-γ, thus confirming that this molecule is biologically active. Because of the DPPD gene is restricted to the tuberculosis-complex organisms of Mycobacterium genus, this highly purified molecule should be useful for the identification of individuals sensitized with tubercle bacilli.
Highlights
Over-expression of recombinant proteins in Escherichia coli host cells followed by purification in affinity resins has become a routine and useful procedure to produce a variety of antigenic molecules
DPPD is a small protein composed of 84 amino acids and is a major component of the complex protein mixture present in purified protein derivative (PPD), which is the antigenic preparation used in the skin test performed in humans and cattle for the diagnosis of tuberculosis [5,6,7,8,9]
The M. tuberculosis DPPD full length gene (Rv0061) codes for a typical secreted protein, which includes a signal peptide sequence of 39 amino acids followed by the signal peptidase recognition sequence Ala-Ser-Ala (Figure 1)
Summary
Over-expression of recombinant proteins in Escherichia coli host cells followed by purification in affinity resins has become a routine and useful procedure to produce a variety of antigenic molecules. Not seldom and for not entirely known reasons, the bacterial host cells fail to properly express the recombinant proteins coded for by the genes cloned into a number of different expression vectors. Several possibilities have been raised to explain this difficult-to-express proteins puzzle These include protein toxicity to E. coli, plasmid or protein instability, inefficient transcription or translation, inefficient post-translational modification, and presence in the cloned gene of inadequate or non-used codon sequences by the E. coli host cells [1,2]. Expression of recombinant DPPD in E. coli host cells either as a single molecule or as a fusion protein, using an exhaustive list of commercially available expression systems for this host cells, consistently failed. Attempts to produce a synthetic DPPD polypeptide through several protein manufacturing services were unsuc-
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