A GENERAL PURIFICATION PROTOCOL FOR E7 PROTEINS FROM HIGH-RISK AND LOW-RISK HUMAN PAPILLOMAVIRUS TYPES EXPRESSED IN THE YEAST SCHIZOSACCHAROMYCES-POMBE

A purification protocol was developed to obtain human papillomavirus ( HPV) type 16 E7 protein expressed in the yeast Schizosaccharomyces pom be. Only three chromatographic steps were necessary to purify the unfu sed HPV 16 E7 protein to homogeneity (95-99%) as shown by silver stain ing after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purif ied HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rbl and p107 in vitro and induc tion of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological acti vities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV ty pes such as HPV 18 and 11. (C) 1997 Academic Press.