4.2.1 Cloning, expression and purification of recombinant Rv2615c protein
Cloning, expression and purification of Rv2615c protein was performed as described in Medha et al ., (Medha, Joshi, et al. , 2022) (Medha, Priyanka, et al. , 2022).
Briefly, the Rv2615c gene was directly cloned in the pGEM-T Easy vector and its successful sequencing was verified. The full-length gene was then sub-cloned using BamHI and HindIII restriction enzymes in the expression vector pET-28a (+) in frame with a 6x histidine tag at the N-terminus. The target gene containing recombinant plasmid was introduced into Escherichia coli (E. coli) BL21 DE3 for protein over-expression after being induced with 1mM IPTG and maintained at 37° 250 rpm for a couple of hours, then at 20° overnight. Culture was pelleted and re-suspended in 20-30 ml of 1XPBS+1mM PMSF followed by sonication (duty cycle 30; 5-6 cycles). Pellet was washed step wise with 10 ml 2%, 1% TritonX in PBS followed by 10ml 1X PBS and finally dissolved in Buffer (50mM NaH2PO4, 300 mMNacl, 8M Urea) and kept for binding at 4° with Ni-NTA matrix in column. Column was washed thrice with 1X cold PBS and wash fractions collected. Elution was performed with different gradients of immidazole. Elution buffer constituents: 50mM NaH2PO4, 300mM Nacl, 10mM TrisCl, 1mM PMSF, 10% glycerol, βMe, 8mM urea. SDS-PAGE was used to analyse the recombinant protein’s purity, which was then validated by Coomassie Blue staining and immuno-blotting using anti-His antibodies. To remove immidazole and urea, purified protein was dialyzed against 1XPBS and decreasing urea concentrations. Purified recombinant proteins were then incubated for 1 h at 4°C with polymyxinB-agarose beads to remove endotoxin contamination. Limulus Amoebocyte Lysate (LAL) assay was performed using Peirce LAL assay kit following manufacturer’s instructions to ensure negligible endotoxin levels in the purified protein (Pierce LAL Chromogenic Endotoxin Quantitation Kit, Cat no. 88282). The BCA protein assay was used to determine the protein content. Up to usage, the purified protein was kept in tiny aliquots at 80 °C.
4.2.2 Human Cell Culture: THP-1 cells stimulation with recombinant Rv2615c protein
In tissue culture plates, the human monocytic cell line THP-1 (NCCS, Pune) was cultured (1X105cells/ml cells per ml) in RPMI 1640, 10% foetal calf serum and an antibiotic cocktail that included penicillin (10,000 units/ml) and streptomycin (10,000 g/ml). For the purpose of differentiating the monocytic cells into macrophages, 40ng/ml Phorbol-Myristate Acetate (PMA; Sigma) was added to the monocytic cells and left overnight.