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 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.

Figure 1

Deduced amino acid sequence of the protein coded by Mycobacterium tuberculosis Rv0061. The deduced amino acid sequence of the 112 residues of the protein, reveals that the full length gene codes for a typical secretory protein, which includes a signal peptide sequence (illustrated in red letters), the signal peptidase recognition sequence ASA (illustrated in green, bold, italic letters) and the mature sequence of protein (DPPD), which is depicted in black bold letters.

Figure 2

DPPD DNA sequence with optimized codon usage. Codon optimization for protein expression was done using the Blue Heron Expression Optimization tool which minimizes secondary mRNA structure to reduce translational impediments. Optimized DPPD sequence is displayed in bold (upper sequence). Note that an Nde I and a Bam HI restriction enzyme sequences (underline sequences) flanking the 5′ and 3′ ends respectively were incorporated in the optimized sequence. For comparison purposes the Rv0061 gene sequence (coding the mature form of the protein only) is also shown (lower sequence).

Figure 3

Expression of recombinant DPPD in E. coli. Recombinant DPPD was expressed in E. coli with six His-tag amino terminal residues and the protein was purified by affinity chromatography using Ni-NTA agarose matrix. Coomassie blue stained SDS/4–20% polyacrylamide gradient gel of 5μg of purified recombinant DPPD (lane 1). MWM, molecular weight markers; numbers on left are the MW of the markers in kDa.

Figure 4

Western blot analyses of recombinant DPPD expressed by E. coli. Purified rDPPD was initially subjected to SDS-PAGE (gradient gel 4 – 20) performed under reducing (R) and non-reducing (NR) conditions. Protein was transferred to membrane and the presence of the recombinant molecule was identified using a mouse anti- His-tag (terminus) monoclonal antibody. Reactivity was detected using chemo luminescent reagent. Numbers on the left side indicate the molecular weights of the markers.

Figure 5

DPPD peptide sequence identified by mass spectroscopy in purified recombinant protein. The peptide sequence and positioning within the peptide donor protein (DPPD) is highlighted in red/bold/underline. The trypsin cleavage sites (right side of the amino acids R and K) that generated the peptide are illustrated above the molecule.

Figure 6

Recognition of purified recombinant DPPD by human PBMC. IFN-γ production by PBMC from PPD positive healthy donors following stimulation for 72 h with medium, rDPPD (10 μg/ml) or PPD (10 μg/ml) was measured by sandwich ELISA in the culture supernatants. Bars represent the SD of the means calculated from the results of triplicate cultures.