Heterologous Expression, Purification, and Characterization of the HspX, Ppe44, and EsxV Proteins of Mycobacterium tuberculosis

摘要

Background: Subunit vaccines are appropriate vaccine candidates for the prevention of some infections. In thisstudy, three immunogenic proteins of Mycobacterium tuberculosis, including HspX, Ppe44, and EsxV as anew construction, were expressed alone and as a fusion protein to develop a new vaccine candidate againsttuberculosis infection.Methods: To make the fusion protein, the three genes were linked together by AEAAAKEAAAKAlinkers and inserted into pET21b and pET32b vectors. Escherichia coli (E. coli) Top10 cells weretransformed with the plasmid, and the purified plasmid was used to transform E. coli BL21 cells.Protein expression was induced with IPTG. After optimizing protein expression, the recombinantproteins were purified by Ni-NTA chromatography. Protein purification was confirmed by SDS-PAGEand Western blotting with an anti-poly histidine-peroxidase monoclonal antibody against the 6His–tagsat the proteins’ C termini.Results: Directional cloning was confirmed by polymerase chain reaction (PCR), restriction enzymedigestion, and sequencing. The highest expression of the tri-fusion protein and HspX were obtained by theaddition of 0.2 mM of IPTG to E. coli BL-21 cells at 37 ?C and 18 h of incubation. For Ppe44 and EsxV,the optimum expression conditions were 18 ?C and 16 h of incubation. SDS-PAGE and Western blotsconfirmed that the desired proteins were produced.Conclusions: The three desired proteins and the fusion protein were successfully expressed and theconditions for optimum expression determined. These recombinant proteins will be evaluated as vaccinecandidates against tuberculosis. Further studies are needed to evaluate the abilities of these proteins toinduce strong immunological responses.