3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item South African cassava mosaic virus movement and nuclear shuttle proteins: uncovering their structures(2022) Nankoo, NikitaSouth African cassava mosaic virus (SACMV) is a plant-infecting circular ssDNA bipartite begomovirus, whose genome comprises of DNA-A (encodes six genes) and DNA-B [encodes BC1 cell-to-cell movement (MP) and BV1 nuclear shuttle protein (NSP)]. Expression of these viral proteins in vitro and their purification have not been achieved to date and this study aimed to achieve this for a truncated region of the MP (N-ter) and NSP (C-ter), respectively. Furthermore, SACMV movement and nuclear shuttle proteins were truncated due to the difficulties in working with the full-length proteins; and the secondary structures of the truncated proteins were then investigated. Computational characterization and homology modelling were also undertaken. The truncated MP (amino acid 1-258) and NSP (amino acid 171-258) were cloned into a pCOLDI expression vector and transformed into BL21 (DE3) pLYSs and BL21 (DE3) E. coli respectively. Optimal conditions for the induced expression of the MP were found to be 0.50 mM IPTG at an OD600 of 0.45 expressed for 24 h. In contrast, the optimal conditions of the expression of the truncated NSP were found to be 0.25 mM IPTG at an OD600 of 0.40 expressed for 24 h. 8-Anilinonaphthalene-1- sulfonic acid (ANS) has been used as a folding/unfolding monitoring tool. This study showed that both proteins bind ANS with greater affinity in the denatured form, indicating that there are more hydrophobic regions for the polar fluorescent dye to bind. Both truncated proteins do not have the ability to bind ATP, and this was determined by using the MANT-ATP intrinsic fluorescence assay. In order to confirm that both proteins were refolded and did not bind ATP, intrinsic tryptophan fluorescence was assessed. This assay confirmed that both proteins were refolded due to the observed difference in spectra profiles. The assay also confirmed that ATP caused no conformational changes in both proteins in the native or denatured forms. Secondary structure analysis employing Circular Dichroism confirmed that the truncated NSP and MP contained predominantly β-sheet secondary structures. The estimated molecular weights by SDS-PAGE, were determined to be 23 kDa and 13kDa for MP and NSP, respectively. The employment of Mass Spectrometry in future studies could assist in confirming the exact molecular weight of the protein as Size Exclusion High Performance Liquid Chromatography did not yield desirable results as 2 the proteins eluted at the incorrect retention times possibly due to the proteins being expressed in the insoluble form.Item The recombinant expression and structural characterization of movement protein BC1 from South African cassava mosaic virus(2018) Nankoo, NikitaSouth African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises of DNA A (encodes six genes) and DNA B (encodes BC1 (cell-to-cell movement protein) and BV1 (nuclear shuttle protein)). Begomoviruses cause cassava mosaic disease (CMD) resulting in substantial root yield losses impacting farmers and potential starch yields for industrial purposes. The structural and physiochemical characteristics of begomoviruses have not been elucidated to date. Additionally, it is crucial to identify protein-protein interactions between SACMV BC1 and cassava host proteins that facilitate SACMV movement. In this study, in silico characterization studies for BC1 were undertaken. The FASTA amino acid sequence of BC1 was uploaded onto a webserver (Predict Protein (https://www.predictprotein.org)) and resulted in the predicted secondary structure of BC1 as well as predicted protein binding sites. BC1 was cloned into a pET-28a(+) expression vector and transformed into host cells E.coli BL21 (DE3). The optimal conditions for the expression of BC1 protein were found to be induction with 0.25 mM IPTG at an OD600 of ~0.6 expressed at 37 °C for four hours. The protein was refolded using stepwise dialysis. The molecular weight of BC1 was 35 kDa (SDS-PAGE). The secondary structure of BC1 was confirmed to be predominantly β-strands (CD). An ANS (1-anilino-8-naphthalene sulphonate) binding assay confirmed that BC1 possesses hydrophobic pockets with the ability to bind ligands such as ATP. A 2’ (3’)-N-methylanthraniloyl-ATP (MANT-ATP) assay showed binding of MANTATP to BC1. Intrinsic tryptophan fluorescence studies indicated significant conformational change in the denatured form of BC1 in the presence of ATP compared to in the absence of ATP. Literature and the PredictProtein webserver were employed to in silico predict suitable cassava host prey proteins for the yeast-two-hybrid (Y2H) system. Cloning of host proteins (HSP70 and Histone H3) into pDEST™22 plasmids was done at GenScript USA. The in silico study and structural characterization of BC1 provide insights to the structural characteristics of BC1 and further explains its functionality. Completion of Y2H assays will confirm or refute that cassava host proteins Histone H3 and HSP70 interact with the cell-to-cell movement protein of SACMV