3.2.1 Cloning, expression and purification of recombinant Rv2615c protein
His-tagged recombinant Rv2615c protein was successful with pGEMT easy vector in DH5α followed by pET28a[+] expression vector in BL21DE3 and the clones were also confirmed by sequencing. Rv2615c was successfully expressed and purified from insoluble fraction of transformed BL21 DE3 culture by affinity chromatography. The protein had a molecular weight of 39.3 kDa observed in SDS-PAGE and anti-His antibody on western blotting (Fig S1). One liter of culture yielded approximately 2 mg/mL of protein. The purified protein was passed through polymyxin B-agarose beads to remove bacterial endotoxin and Limulus Amoebocyte Lysate assay (Pierce, USA) was performed in collected fractions which confirmed no endotoxin contamination.