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Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein

South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristic...

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Autores principales: Nankoo, Nikita, Achilonu, Ikechukwu Anthony, Rey, Marie Emma Christine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221656/
https://www.ncbi.nlm.nih.gov/pubmed/35735627
http://dx.doi.org/10.3390/cimb44060186
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author Nankoo, Nikita
Achilonu, Ikechukwu Anthony
Rey, Marie Emma Christine
author_facet Nankoo, Nikita
Achilonu, Ikechukwu Anthony
Rey, Marie Emma Christine
author_sort Nankoo, Nikita
collection PubMed
description South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD(600) of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS–PAGE. The secondary structure of SACMV MP presented as predominantly β-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2′ (3′)-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity.
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spelling pubmed-92216562022-06-24 Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein Nankoo, Nikita Achilonu, Ikechukwu Anthony Rey, Marie Emma Christine Curr Issues Mol Biol Article South African cassava mosaic virus (SACMV) is a circular ssDNA bipartite begomovirus, whose genome comprises DNA-A (encodes six genes) and DNA-B (encodes BC1 cell-to-cell movement and BV1 nuclear shuttle proteins) components. A few secondary and tertiary structural and physicochemical characteristics of partial but not full-length begomovirus proteins have been elucidated to date. The full-length codon-optimised SACMV BC1 gene was cloned into a pET-28a (+) expression vector and transformed into expression host cells E. coli BL21 (DE3). The optimal expression of the full-length BC1-encoded movement protein (MP) was obtained via induction with 0.25 mM IPTG at an OD(600) of ~0.45 at 37 °C for four hours. Denatured protein fractions (dialysed in 4 M urea), passed through an IMAC column, successfully bound to the nickel resin, and eluted using 250 mM imidazole. The protein was refolded using stepwise dialysis. The molecular weight of MP was confirmed to be 35 kDa using SDS–PAGE. The secondary structure of SACMV MP presented as predominantly β-strands. An ANS (1-anilino-8-naphthalene sulphonate)-binding assay confirmed that MP possesses hydrophobic pockets with the ability to bind ligands such as ANS (8-anilino-1-naphthalenesulphonic acid). A 2′ (3′)-N-methylanthraniloyl-ATP (mant-ATP) assay showed binding of mant-ATP to MP and indicated that, while hydrophobic pockets are present, MP also exhibits hydrophilic regions. Intrinsic tryptophan fluorescence indicated a significant conformational change in the denatured form of BC1 in the presence of ATP. In addition, a phosphatase assay showed that MP possessed ATPase activity. MDPI 2022-06-15 /pmc/articles/PMC9221656/ /pubmed/35735627 http://dx.doi.org/10.3390/cimb44060186 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Nankoo, Nikita
Achilonu, Ikechukwu Anthony
Rey, Marie Emma Christine
Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title_full Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title_fullStr Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title_full_unstemmed Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title_short Expression, Purification, and Characterisation of South African Cassava Mosaic Virus Cell-to-Cell Movement Protein
title_sort expression, purification, and characterisation of south african cassava mosaic virus cell-to-cell movement protein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9221656/
https://www.ncbi.nlm.nih.gov/pubmed/35735627
http://dx.doi.org/10.3390/cimb44060186
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