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Expression, purification and properties of papaya mosaic virus capsid protein in Escherichia coli

University of British Columbia

Potexviruses are composed of a single species of capsid (coat) protein (CP) which encapsidates a single positive-sense genomic RNA (gRNA) and is also implicated in cell-to cell movement. Several potexviruses have been reconstituted from their individual purified components and, of these, papaya mosaic virus (PMV) has been most extensively characterized. Capsid protein mutants that affect encapsidation and/or movement usually cannot, however, be efficiently recovered from plants. Accordingly, an alternative system was designed to permit the generation of a broad range of mutant CPs for the systematic investigation of the role of potexviral CP in assembly which would otherwise be impossible in planta. To test the general feasibility of this approach, I have overexpressed PMV CP and CYMV CP in E. coli. The PMV CP has been purified to approximately 90 % purity using only four steps: centrifugation at 30,000 x g, ammonium sulphate precipitation to 40 % saturation, anion exchange chromatography and gel filtration chromatography. Recombinant CP was assayed for its in vitro reconstitutive ability with genomic PMV RNA via electron microscopy and local lesion host infectivity. Reconstitution was demonstrated, but it was suboptimal as evidenced by the formation of partial rods and the inability of reconstituted viral particles exposed to Tl RNase to infect a local lesion host.

Thesis/Dissertation

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2009-03-09

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http://hdl.handle.net/2429/5783

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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