A general purification protocol for E7 proteins from "high- 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 pombe. Only three chromatographic steps were necessary to purify the unfused HPV 16 E7 protein to homogeneity (95-99%) as shown by silver staining after polyacrylamide gel electrophoresis. Approximately 0.8 mg of highly purified E7 was obtained from 5 x 10(10) yeast cells. The purified HPV 16 E7 phosphoprotein (Ser 31/32) was refolded and assayed for functionality. Binding to the proteins Rb1 and p107 in vitro and induction of DNA synthesis after microinjection into serum-deprived NIH 3T3 cells suggest that the E7 protein retains some of its biological activities. Most importantly, the purification strategy is also applicable for different HPV 16 E7 mutants and for E7 proteins from other HPV types such as HPV 18 and 11.
Braspenning
J
J
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Manetti
R
R
Zumbach
K
K
Meschede
W
W
Gissmann
L
L
Tommasino
M
M
D000818:Animals; D003218:Condylomata Acuminata / Q000821:virology*; D004247:DNA / Q000096:biosynthesis; D005260:Female; D006801:Humans; D051379:Mice; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D027383:Papillomaviridae / Q000737:chemistry* / Q000472:pathogenicity; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000821:virology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics* / Q000821:virology; D014412:Tumor Virus Infections / Q000821:virology*; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; C059731:oncogene protein E7, Human papillomavirus type 16; D004247:DNA