Abstract
The replication-initiator protein (Rep) from a soybean-infecting geminivirus was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). In spite of the presence of the highly soluble MBP as the fusion partner, the overexpressed MBP-Rep fusion protein formed insoluble inclusion bodies. The protein was solubilized from the inclusion bodies and refolded. The refolded MBP-Rep protein was purified using ion exchange and amylose affinity chromatography. The activity of the purified MBP-Rep was assessed using an in vitro cleavage assay. Soluble and stable MBP-Rep protein was obtained in high abundance, providing the feasibility of large-scale production of active Rep protein for functional characterization and X-ray crystallographic structure determination.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Chromatography, Affinity
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Chromatography, Ion Exchange
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DNA Helicases / genetics
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DNA Helicases / isolation & purification*
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DNA Helicases / metabolism*
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / isolation & purification*
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DNA-Binding Proteins / metabolism*
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / genetics
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Geminiviridae / genetics*
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Glycine max / virology*
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Inclusion Bodies
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Protein Folding*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism
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Trans-Activators / genetics
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Trans-Activators / isolation & purification*
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Trans-Activators / metabolism*
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Viral Proteins / genetics
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Viral Proteins / isolation & purification*
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Viral Proteins / metabolism*
Substances
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DNA-Binding Proteins
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Recombinant Fusion Proteins
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Trans-Activators
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Viral Proteins
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replication initiator protein
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DNA Helicases