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.