Refolding, purification and characterization of replication-initiator protein from soybean-infecting geminivirus

Girish, K.R.; Palanivelu, Sowmiya Balasubramanian and Peramachi; Kumar, Prem; Usha, R.

doi:10.1016/j.jviromet.2006.05.007

Cited by 3 publications

(2 citation statements)

References 35 publications

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“…However, ToLCKeV AC1 could not be expressed as a soluble protein in either case (data not shown). A similar case was observed in case of MYMIV-sp [MP] Rep protein [26]. So, we cloned the Rep protein as an MBP fusion and purified it as the soluble protein (Fig 1c).…”

Section: Resultssupporting

confidence: 73%

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1)

Pasumarthy

Choudhury

Mukherjee

2010

Virol J

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BackgroundGeminiviruses are emerging plant viruses that infect a wide variety of vegetable crops, ornamental plants and cereal crops. They undergo recombination during co-infections by different species of geminiviruses and give rise to more virulent species. Antiviral strategies targeting a broad range of viruses necessitate a detailed understanding of the basic biology of the viruses. ToLCKeV, a virus prevalent in the tomato crop of Kerala state of India and a member of genus Begomovirus has been used as a model system in this study.ResultsAC3 is a geminiviral protein conserved across all the begomoviral species and is postulated to enhance viral DNA replication. In this work we have successfully expressed and purified the AC3 fusion proteins from E. coli. We demonstrated the higher order oligomerization of AC3 using sucrose gradient ultra-centrifugation and gel-filtration experiments. In addition we also established that ToLCKeV AC3 protein interacted with cognate AC1 protein and enhanced the AC1-mediated ATPase activity in vitro.ConclusionsHighly hydrophobic viral protein AC3 can be purified as a fusion protein with either MBP or GST. The purification method of AC3 protein improves scope for the biochemical characterization of the viral protein. The enhancement of AC1-mediated ATPase activity might lead to increased viral DNA replication.

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Section: Resultssupporting

confidence: 73%

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1)

Pasumarthy

Choudhury

Mukherjee

2010

Virol J

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“…The sensitive antibody binding activity of MBP-HPV16 L1 against HPV16 L1-specific monoclonal antibody shows that MBP-HPV16 L1 may prove an appropriate ELISA coating protein in HPV16 L1-based vaccines. Similar to what was observed with MBP-HPV16 L1, GST-fused HPV16 L1 proteins were demonstrated to retain their antigenic property for ELISA-based antibody detection [26].…”

Section: Discussionsupporting

confidence: 70%

Expression, Purification, and Antibody Binding Activity of Human Papillomavirus 16 L1 Protein Fused to Maltose Binding Protein

Cho

Hahm²,

Kim³

et al. 2007

PPL

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Genetic human papillomavirus type 16 L1 (HPV16 L1) has been widely studied for cervical cancer vaccine development. For the enzyme-linked immunosorbent assay (ELISA) screening of these vaccines, HPV16 L1 protein, which is required as a coating protein, has previously been expressed from costly and laborious recombinant baculovirus-infected insect cells. For a novel HPV16 L1 expression system characterized by a high yield of soluble form with simple purification steps, we have cloned and expressed two different types of HPV16 L1, both fused to maltose binding protein (MBP) or glutathione-S-transferase (GST) in Escherichia coli. The yield of soluble HPV16 L1 was influenced by the cultivation temperature. The yield of soluble form in the total MBP-fused HPV16 L1 protein (MBP-HPV16 L1) was 35% at 37 degrees C, but increased to 85% at 22 degrees C. Among the fusion partners, MBP provided higher yields of total and soluble HPV16 L1 than did GST. MBP-HPV16 L1 showed a 4.9-fold higher yield of the soluble form over insoluble inclusion bodies under optimized culture conditions. The soluble form of MBP-HPV16 L1 was purified via MBP affinity chromatography in a recovery yield of 9.7%. After fusion with MBP, HPV16 L1 showed binding activity to HPV16 L1-specific monoclonal antibody comparable to HPV16 L1 from the insect cells in ELISA tests. These results demonstrate that the use of MBP as a fusion partner may generate a high yield of soluble HPV16 L1 under optimized temperature conditions, and that MBP-fused HPV16 L1 might be applied further in evaluations of the immune responses of HPV16 L1-based cervical cancer vaccines.

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Begomovirus research in India: A critical appraisal and the way ahead

Borah

Dasgupta

2012

J Biosci

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Begomoviruses are a large group of whitefly-transmitted plant viruses containing single-stranded circular DNA encapsidated in geminate particles. They are responsible for significant yield losses in a wide variety of crops in India. Research on begomoviruses has focussed on the molecular characterization of the viruses, their phylogenetic analyses, infectivities on host plants, DNA replication, transgenic resistance, promoter analysis and development of virus-based gene silencing vectors. There have been a number of reports of satellite molecules associated with begomoviruses. This article aims to summarize the major developments in begomoviral research in India in the last approximately 15 years and identifies future areas that need more attention.

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Refolding, purification and characterization of replication-initiator protein from soybean-infecting geminivirus

Cited by 3 publications

References 35 publications

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1)

Tomato leaf curl Kerala virus (ToLCKeV) AC3 protein forms a higher order oligomer and enhances ATPase activity of replication initiator protein (Rep/AC1)

Expression, Purification, and Antibody Binding Activity of Human Papillomavirus 16 L1 Protein Fused to Maltose Binding Protein

Begomovirus research in India: A critical appraisal and the way ahead

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