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.