RAPID, EFFICIENT, LARGE-SCALE PURIFICATION OF UNFUSED, NON-DENATURED E7 PROTEIN OF COTTONTAIL RABBIT PAPILLOMAVIRUS

Cottontail rabbit papillomavirus (CRPV) E7 protein is one of the 'high risk' papillomavirus E7 oncoproteins that are partially insoluble in aqueous solution. An Escherichia coli expression system was used for p urification of CRPV E7 protein in quantities sufficient for immunologi c studies and structural analysis. A glutathione S-transferase (GST)-C RPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione S epharose beads. The CRPV E7 portion was cleaved from the column with t hrombin at a thrombin cleavage site between the fused partners. Thromb in was removed subsequently by adsorption to benzamidine. This method is rapid, requiring just one week, and efficient, yielding 3 mg of pur e CRPV E7 protein per liter of bacterial culture. It produced a protei n product that was about 95% pure. High-titered polyclonal antisera ge nerated to the product recognized CRPV E7 but not GST. Purified CRPV E 7 protein exhibited the ability to bind pRB, making it the first unfus ed, non-denatured CRPV E7 product reported to do so. This attribute co uld facilitate structure-function studies of CRPV E7-pRB interactions.