Browsing by Author "Singh, D.D."
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Item Characterization of a Kunitz-type serine protease inhibitor from Solanum tuberosum having lectin activity(Elsevier B.V., 2016) Shah, K.R.; Patel, D.K.; Pappachan, A.; Prabha, C.R.; Singh, D.D.Plant lectins and protease inhibitors constitute a class of proteins which plays a crucial role in plant defense. In our continuing investigations on lectins from plants, we have isolated, purified and characterized a protein of about 20kDa, named PotHg, showing hemagglutination activity from tubers of Indian potato, Solanum tuberosum. De novo sequencing and MS/MS analysis confirmed that the purified protein was a Kunitz-type serine protease inhibitor having two chains (15 kDa and 5 kDa). SDS and native PAGE analysis showed that the protein was glycosylated and was a heterodimer of about 15 and 5kDa subunits. PotHg agglutinated rabbit erythrocytes with specific activity of 640H.U./mg which was inhibited by complex sugars like fetuin. PotHg retained hemagglutination activity over a pH range 4-9 and up to 80?C. Mannose and galactose interacted with the PotHg with a dissociation constant (Kd) of 1.5?10-3M and 2.8?10-3M, respectively as determined through fluorescence studies. Fluorescence studies suggested the involvement of a tryptophan in sugar binding which was further confirmed through modification of tryptophan residues using N-bromosuccinimide. Circular dichroism (CD) studies showed that PotHg contains mostly ? sheets (~45%) and loops which is in line with previously characterized protease inhibitors and modeling studies. There are previous reports of Kunitz-type protease inhibitors showing lectin like activity from Peltophorum dubium and Labramia bojeri. This is the first report of a Kunitz-type protease inhibitor showing lectin like activity from a major crop plant and this makes PotHg an interesting candidate for further investigation. ? 2015 Elsevier B.V.Item Heterologous expression, purification and characterization of L-type lectin homologue from Leishmania donovani(Elsevier, 2015) Singh, V.; Nair, D.N.; Kaushal, R.S.; Kumar, M.; Pappachan, A.; Singh, D.D.Leishmaniasis, a disease of the developing world affects about 12 million people and has limited therapeutic interventions available. L-type lectins, Endoplasmic Reticulum Golgi Intermediate Compartment/Vesicular Integral Proteins (ERGIC-53/VIP36) are involved in protein sorting in luminal compartments of animal cells and are important for parasite biology. A lectin homologue was identified through a bioinformatics analysis of Leishmania genome and it was found to have N-terminal conserved carbohydrate recognition domain (CRD) and a unique C-terminal region rich in repetitive amino acids and a poly glutamine tract. The N-terminal CRD region was cloned and expressed in Escherichia coli, but gave an insoluble expression which was re-solubilized by on column refolding. The fold integrity was checked through CD, fluorescence and functional assay of hemagglutination activity using rabbit erythrocyte. Bioinformatics analysis identified 15 members from Tritryps (Leishmania spp., Trypanosoma spp.) and they separate out as a distinct clade in the global phylogenetic analysis of all ERGIC-53/VIP36 sequences downloaded from Uniprot. Our analysis shows that the extended C-terminal regions with repeats is unique to Tritryps and this repeat pattern is different in sequences from Leishmania spp. and Trypanosoma spp. and all these features make this protein an interesting candidate for further detailed studies. ? 2015 The Author. Published by Elsevier B.V.