Q-Sepharose, Phenyl Superose, ECL Western blotting detection reagents were obtained from Amersham. All other biochemicals were of the highest grade available. The WHO reference strain of L. donovani (MHOM/IN/80/Dd8) was obtained from Imperial College London (UK) and maintained in vitro as promastigotes in RPMI 1640, supplemented with 10% heat-inactivated fetal bovine serum containing 40 μg mL−1 gentamycin at 25 °C. The TA cloning vector pGEM-T Easy was used to clone the PCR product, and the pET-28(a) vector having both N and C terminal His6-tag was used for expression of the recombinant protein. The recombinant plasmids were transformed into E. coli BL21 (DE3) cells for expression. PCR primers 5′-CATATGGGGTTCTTCTCGGATTCGGTAG-3′

(forward) and 5′-AAGCTTCGCGTGGCCGGCAATCTCCTTG-3′ (reverse) with NdeI restriction INK 128 in vivo site at the forward and HindIII at the reverse end shown as underlined were designed based on the L. major SSN gene (U30455) sequence. Amplification of the SSN gene was carried out using genomic DNA of L. donovani as template. The reaction mixture contained 50 ng of genomic DNA, 1.5 U Taq polymerase, 0.5 μM primer (each) and 200 μM each dNTPs in 50 μL PCR reaction mixture volume. After initial denaturation at 94 °C

for 3 min, the PCR reaction was programmed for 30 selleck inhibitor cycles, with each cycle including denaturation at 94 °C for 30 s, 50 °C annealing temperature for 1 min and extension at 72 °C for 2 min. There was a final extension at 72 °C for 10 min. The amplified product after gel purification was cloned in the pGEM-T-Easy vector and transformed in E. coli DH5α competent cells. Nucleotide sequencing of the recombinant construct was carried out in both directions to confirm the sequence of the amplicon and the sequence was submitted to NCBI GenBank. The recombinant construct was digested with

restriction endonuclease NdeI and HindIII and subcloned into the prokaryotic expression vector pET-28(a) for overexpression and purification of recombinant LdSSN. SSN genes of diverse species at the level of the deduced amino acid sequence from Swiss prot or cAMP protein data bank (http://www.expasy.org/sprot/) were aligned with clustalw, followed by the generation of a phylogenetic tree. The recombinant construct pET-28 (a)-LdSSN was used to transform competent E. coli BL21 (DE3). The plasmid was grown overnight as primary culture. Luria–Bertani broth (1 L) containing 25 μg mL−1 kanamycin was reinoculated at 0.1% cell density and grown at 37 °C under constant shaking. The culture was induced by 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) (OD600 nm 0.4) and incubated at 20 °C for another 12 h under gentle shaking at 120 r.p.m. Uninduced culture was taken out and run as a negative control. The overnight grown culture was harvested at 5000 g for 15 min at 4 °C and suspended in buffer A [20 mM Tris buffer, pH 7.