TURNIP CRINKLE VIRUS COAT PROTEIN EXPRESSION AND PURIFICATION
Dori, Kathleen E
MetadataShow full item record
Turnip crinkle virus is a (+) sense single strand RNA plant virus consisting of a single copy of the genome surrounded by 180 copies of the 38 kDa coat protein (CP) that comprise the virion capsid. As a structural protein, the CP's main function is assembly of the virion but it has also been linked to suppression of virus-induced gene silencing. The CP gene was cloned and protein expressed in Escherichia coli. Protein formed insoluble aggregates packaged into inclusion bodies located in the cell cytoplasm. Cells were mechanically disrupted/lysed and inclusion bodies isolated via 8 M urea extraction. Soluble TCV CP was purified by ion-exchange chromatography using a CM Sepharose column under denaturing conditions. Subsequent step-wise dialysis out of urea and into a high salt buffer refolded the denatured protein, allowing the assembly of a series of aggregates. The purified protein was partially characterized through sedimentation and circular dichroism measurements.