Cloning and Expression of Mycobacterium Tuberculosis Rv0285 (Pe5) Gene

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Background: PE5 is a member of the PE protein family whose precise function is yet to be understood. There are about 100 members of the PE family proteins in mycobacterium tuberculosis. These glycineand alanine-rich proteins consist of Proline-Glutamate motifs at their N-termini and may be implicated in pathogenesis of the bacilli. Objectives: We aimed to clone and over-express the Rv0285 coding region in the BL21 (DE3) Escherichia coli strain for the future functional investigations. Materials and Methods: The PE5 coding region was cloned into a specific vector containing N-terminal GST tag using ligation independent cloning (LIC) method and then the recombinant vector was transferred into the competent TOP10 E. coli strain. The positive colonies were screened by the colony PCR approach and finally integration of the constructed expression vector was assessed by DNA sequencing. The vectors were then transferred and expressed in E. coli BL21 (DE3) strain and finally the protein expression level was analyzed using SDS-PAGE. Results: In this study, the PE5 (Rv0285) coding region was amplified as a 309 bp DNA fragment from the mycobacterium tuberculosis H37Rv chromosome using specific sets of primers. The amplicon was then cloned into pLEICS-02 and then confirmed by DNA sequencing. The recombinant protein was over-expressed as a ~ 40 kDa tagged protein in E. coli, and finally confirmed by SDS-PAGE analysis. Conclusions: Our data showed that recombinant PE5 coding region was successfully cloned in pLEICS-02 and expressed in BL21 (DE3) E. coli strain as host.