Expression, purification and evaluation of recombinant lipoprotein of Salmonella typhi as a vaccine candidate.

Lipoprotein has been reported as a vaccine candidate against many pathogenic bacteria, it plays direct role as a virulence-associated function. Here the approach is toward the expression of recombinant lipoprotein of Salmonella typhi in prokaryotic host and its evaluation as a vaccine candidate. Lipoprotein gene (lp1) was cloned in pET32a expression vector in addition to Bam HI and Hind III restriction sites, and BL21(pLysS) was used as prokaryotic expression host for transformation. Lipoprotein induction was performed by IPTG and 55 kDa (31 kDa of Gene +24 kDa of vector additional protein with His-tag) was analyzed by 12% SDS-PAGE. The recombinant lipoprotein was purified by Ni-NTA affinity chromatography due to the addition of 6X His-tag in recombinant lipoprotein. Western blot analysis using anti-His tag polyclonal antibodies confirmed the specificity of recombinant lipoprotein. Immunogenicity and protection study of recombinant lipoprotein against S. Typhi was performed in BALB/c mice. Adjuvants IFA and alum salts were used to enhance the immune response. ELISA results proved that biologically active truncated recombinant lipoprotein (31 kDa) is a suitable immunogen. Alum salts used as adjuvant was effective for long-lasting immunity.

Biosynthesis of Low Molecular Weight Antifungal Protein from Aspergillus giganteus in Batch Fermentation and In-Vitro Assay.

　In present study, Taguchi's design of experiment L9 orthogonal array was created using Qualitek-4 software with four most critical factors namely, K2HPO4, MgSO4, CaCl2 and culture pH. Production of a new intracellular antifungal protein in submerged fermentation was optimized with yield of 0.98±0.1 mg/gram dry cell weight mycelia from Aspergillus giganteus MTCC 8408. The average molecular mass of the purified protein was figured as 5.122 kDa using Electro Spray Ionization-Mass Spectrometry. Scanning electron microscopy was used to correlate the effect of selected factors on fungal cell morphology and its metabolite production. In vitro antifungal susceptibility assay was profiled against Aspergillus niger and minimum inhibitory concentrations were in the range 0.3±0.06 µg/ml. The stronger influencing factors on afp production and mycelial biomass were noted with CaCl2 and K2HPO4 respectively. The validation experiments using optimized conditions confirmed an improvement in afp by 3.86 times with mycelial biomass by 1.52 times, compared to the basal medium. The present statistical optimization study revealed an opportunity to promote economical design at the industrial level for future scale up of effective antifungal agent against systemic aspergillosis as well as possible post harvest loss.

Protein-protein docking on molecular models of Aspergillus niger RNase and human actin: novel target for anticancer therapeutics.

The 3D models of human actin protein and A.niger RNase were designed using the templates ACTBIND (PDB ID: 3D3Z) and crystalline profilin-beta-actin (PDB ID: 2BTF), respectively in Modeller9v5. These models are testified using several validation methods including PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The stereo-chemical quality of the models was judged by Ramachandran plot with PROCHECK. The total quality G-factor -0.2, shows a good quality model. The ERRAT score for the human actin and A.niger RNase models are 86.104 and 84.615, respectively, fit well within the range of a high quality model. The ERRAT score for the templates 2BTF and 3D3Z are 91.111 and 97.391, respectively. The WHAT-IF evaluation justifies a reasonable homology model structure as none of the scores for each residue in the homology model is lower than -5.0. The energy-minimized model of human actin with PROSA reveals the Z-score value -10.52 between native conformations of the crystal structures. The VERIFY 3D average score is 0.36. All evidence suggests that the geometric quality of the backbone conformation, the residue interaction, the residue contact and the energy profile of the structures were well within the limits of reliable structures. The interaction energy of docking was calculated using the HEX server. The Etotal, lowest docked energy, and calculated RMSD values were -1.608 kcal mol(-1), -8.369 kcal mol(-1) and 0.617 Å, respectively. The study presented in the current project may be useful to design molecules that may have anticancer activity.