Cloning, optimization of induction conditions and purification of Rv1733c protein expressed in .

BACKGROUND AND OBJECTIVES

Rv1733c is a latency antigen from , a probable integral-membrane protein with promiscuous T-cell and B-cell epitopes, making it a potential vaccine candidate against tuberculosis. This study aimed to clone and optimize the expression of recombinant Rv1733c in for purification.

MATERIALS AND METHODS

Chemically synthesized coding sequence was cloned in pET-23a(+) followed by transforming BL21 (DE3) cells. To evaluate the induction conditions for optimized expression, factorial design of experiments was employed using four different media as well as four levels of isopropyl-b-D-thiogalactopyranosid [IPTG] concentration and duration of induction. The recombinant protein was then purified using a His-tag purification kit and detected through western blotting.

RESULTS

Recombinant Rv1733c (> 24 kDa) was expressed and accumulated in the cytoplasm of the cells. Medium composition showed the most significant effect on the yield of the recombinant protein (P = 0.000). The highest yield of recombinant Rv1733c occurred in the presence of 0.4 mM of IPTG in Terrific Broth medium (containing 1.2% tryptone, 2.4% yeast extract, 72 mM K HPO , 17 mM KH PO and 0.4% glycerol) after 10 h at 37°C. Under these conditions, the expression level was around 0.5 g/L of culture medium. Purified Rv1733c was detected by an anti-polyhistidine antibody and a tuberculosis patient's serum. Systematic optimization of induction conditions gave us high yield of recombinant polyhistidine-tagged Rv1733c in which was successfuly purified.

CONCLUSION

We believe that the purified Rv1733c recombinant protein from might be a good candidate for vaccine production against tuberculosis.