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 purification of CRPV E7 protein in quantities sufficient for immunologic studies and structural analysis. A glutathione S-transferase (GST)-CRPV E7 fusion protein was solubilized in the presence of non-ionic and ionic detergents, and isolated on an affinity column of glutathione Sepharose beads. The CRPV E7 portion was cleaved from the column with thrombin at a thrombin cleavage site between the fused partners. Thrombin was removed subsequently by adsorption to benzamidine. This method is rapid, requiring just one week, and efficient, yielding 3 mg of pure CRPV E7 protein per liter of bacterial culture. It produced a protein product that was about 95% pure. High-titered polyclonal antisera generated to the product recognized CRPV E7 but not GST. Purified CRPV E7 protein exhibited the ability to bind pRB, making it the first unfused, non-denatured CRPV E7 product reported to do so. This attribute could facilitate structure-function studies of CRPV E7-pRB interactions.
Sundaram
P
P
Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520-8016, USA.
Brandsma
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D002645:Chickens; D004279:DNA, Viral; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification
D000914:Antibodies, Viral; D000956:Antigens, Viral; D004279:DNA, Viral; C079183:E7 protein, cottontail rabbit papillomavirus; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins