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Heterologous expression, purification and characterization of L-type lectin homologue from Leishmania donovani.

ABSTRACT: 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.

SUBMITTER: Singh V

PROVIDER: S-EPMC4980737 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Heterologous expression, purification and characterization of L-type lectin homologue from <i>Leishmania donovani</i>.

Singh Vijay V Nair Divya N DN Kaushal Radhey Shyam RS Kumar Manoj M Pappachan Anju A Singh Desh Deepak DD

Biotechnology reports (Amsterdam, Netherlands) 20151003

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 <i>Leishmania</i> genome and it was found to have N-terminal conserve ...[more]

PMID: 28352576

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Project description:BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn???) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp???-Val??? peptide was shortened to Asp???-Arg??? in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (?86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Dataset Information

Heterologous expression, purification and characterization of L-type lectin homologue from Leishmania donovani.

Publications

Heterologous expression, purification and characterization of L-type lectin homologue from <i>Leishmania donovani</i>.

Similar Datasets

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