Computational protocol: Leishmania donovani Nucleoside Hydrolase (NH36) Domains Induce T-Cell Cytokine Responses in Human Visceral Leishmaniasis

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Protocol publication

[…] NH36 [EMBL, Genbank, and DDJB databases, access number AY007193 GENBANK (AY007193 and AAG02281.1 access codes) and in SWISS-PROT (Q8WQX2 UNi-Prot access code)] and its N-terminal (F1, amino acids 1–103), central (F2, amino acids 104–198), and C-terminal (F3, amino acids 199–314) domains were cloned in E. coli (), expressed, and purified as modified from the methods of Rodrigues et al. () and Saini et al. (). Briefly, protein expression was induced in bacterial suspensions with 1 mM IPTG, for 4 h at 37°C. The cells were sonicated, and the insoluble pellets were washed twice with 10 mM Tris–HCl pH 8.0 and 0.5% CHAPS and further treated for 2 h at 37°C, under agitation, with a solubilization buffer composed of 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 10% glycerol, and 8 M urea. Then, the suspension was homogenized by successive passages through 20-ml syringes with 1.2 mm × 40 mm needles followed by centrifugation, for 30 min at 14,000 rpm. The supernatant was loaded on a Ni-NTA chromatography column previously equilibrated with solubilization buffer. After sample application, the column was washed with three volumes of solubilization buffer containing 20 mM imidazole. Next, the column was washed with three volumes of the same buffer containing 5 mM reduced glutathione, 0.1% Triton X-100, and each one of the decreasing concentrations of a urea gradient (6–1 M), and buffer with no urea added for refolding. Elution of the proteins was achieved using 250 mM imidazole in refolding buffer without urea and confirmed by protein assay and 15% SDS-PAGE. The proteins were finally dialyzed against 50 mM Tris–HCl, pH 8, 50 mM NaCl, 50% glycerol, and 0.1 mM DTT and the absence of LPS was confirmed using the LAL QCL-1000 kit (Lonza). The purification process yielded 1 mg protein antigen per liter of bacterial culture. The homology between the sequence of L. donovani NH36 (GenBank: AAG02281.1) and Leishmania infantum chagasi NH (Lch-NH) (GenBank: AAS48353.1) was determined using NIH-NCBI Standard Protein BLAST software. A molecular model was obtained by homology modeling using the Modeller 9.10 software and data for the nucleoside hydrolase from a L. major template (RCSB PDB code: 1EZR; crystal structure of nucleoside hydrolase of L. major) ().For control purposes, in order to further demonstrate that NH36 is a component of SLA, we also assayed if sera of mice vaccinated with three doses of 100 μg of either NH36, F1, F2, or F3 recombinant antigens formulated with 100 μg saponin, with a weekly interval, recognized the SLA of L. infantum chagasi (2 μg/well) in a standard ELISA assay using peroxidase-conjugated protein A ().HLA-DR-binding CD4 epitopes were mapped with the TEPITOPE program. CD8 epitopes were identified using SYFPEITHI software. The predicted epitopes were synthetized by GenScript (NJ, USA). The analysis of the identity of the epitopes in the different leishmanial species was performed using the sequences of nucleoside hydrolase of Leishmania species of PubMed Protein Databank and the sequence of L. amazonensis NH A34480 (). […]

Pipeline specifications

Software tools BLASTP, MODELLER
Databases UniProt SYFPEITHI RCSB PDB
Application Protein structure analysis
Organisms Homo sapiens, Leishmania donovani
Diseases Infection, Leishmaniasis, Leishmaniasis, Visceral