Nankoo, Nikita2019-05-202019-05-202018Nankoo, Nikita, (2018) The recombinant expression and structural characterisation of movement protein BC1 from South African cassava mosaic virus, University of the Witwatersrand, Johannesburg, https://hdl.handle.net/10539/27066.https://hdl.handle.net/10539/27066A dissertation submitted to the Faculty of Science at the University of Witwatersrand in fulfillment of the requirements for the degree of Master of Science in the School of Molecular and Cell biology. June 2018South 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 SACMVOnline resource (87 leaves)enCassava mosaic diseaseMosaic diseases--South AfricaPlant viruses--South AfricaThesis