The DNA fragments corresponding to rv3874, rv3875 and rv3619c were subsequently cloned into pGES-TH-1 vector as described previously [24] giving constructs pGES-TH/Rv3874, pGES-TH/Rv3875 and pGES-TH/Rv3619c, respectively. To illustrate the cloning, expression and purification procedures, the schematic presentation of Rv3874 is shown as a reference in Fig. 1. The E. coli BL-21 carrying the plasmid pGES-TH-1 was used as a positive control for expression of GST. The untransformed parent E. coli BL-21 served as

a negative control. The growth and induction of expression were carried out as described previously [20, Protein Tyrosine Kinase inhibitor 24]. DNA sequencing.  The rv3874, rv3875 and rv3619c genes cloned in pGES-TH-1 vector were sequenced using CEQ Dye Terminator Cycle Sequencing (DTCS) Quick Start kit (Beckman Coulter, Brea, CA, USA), according to manufacturer’s instruction. The processed DNA was resuspended with 40 μl of Sample Loading Solution and loaded in CEQ sample plate, and the sample was layered with 8 μl of mineral oil (Sigma, St. Louis, MO, USA). The plate was loaded into DNA sequencer https://www.selleckchem.com/products/bgj398-nvp-bgj398.html (Model CEQ 8000, Beckman Coulter), as described by the manufacturer. The DNA sequencing data were analysed by using the BLAST2 program analysis [31]. SDS–PAGE and immunoblotting.  Whole cell or soluble proteins in cell-free extracts of E. coli transformed with recombinant

pGES-TH-1 were separated by 15% SDS–PAGE gels. The resolved proteins were either stained with Coomassie brilliant blue or transferred to nitrocellulose membranes for Western immunoblotting with anti-GST (Amersham-Pharmacia) and anti-penta His antibodies (Qiagen, GmbH, Hilden, Germany), as described previously [24]. Purification of recombinant Rv3874 and Rv3875 proteins.  The growth conditions, induction

of expression and preparation of cell-free extract from cultures of E. coli BL21 cells carrying G protein-coupled receptor kinase the plasmid pGES-TH/Rv3874 and pGES-TH/Rv3875 were performed at 30 °C essentially as described previously [24]. The extract was loaded onto the glutathione-Sepharose column (Amersham-Pharmacia) and equilibrated in PBS containing 5 mm Dithiothreitol at 4 °C. After washing the column extensively with PBS, the column was brought to room temperature, and the Rv3874 and Rv3875 proteins were released by proteolytic cleavage of the respective fusion proteins bound to the column matrix by thrombin protease, as described earlier [20, 24]. Fractions of 1 ml were collected and analysed by SDS–PAGE. The active fractions in PBS containing Rv3874 and Rv3875 were combined, adjusted to 2.5 m NaCl, 300 mm Imidazole, 50 mmβ-marcaptoethanol (buffer I) then loaded on Ni-NTA agarose affinity column (bed volume 2 ml) (Qiagen, Germany) equilibrated with buffer I at 4 °C.