Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16.
E7 is the major oncogenic protein produced in cervical cancer-associated human papillomavirus type 16 (HPV16). This protein was expressed in Escherichia coli as a glutathione-S-transferase (GST) fusion protein. E7-enriched inclusion bodies were collected from bacterial lysates, were solubilized in 10 M urea, and the protein was purified using anion exchange column chromatography. After removal of endotoxin with serial Triton X-114 extractions, material of high purity (about 90%) was obtained, which is suitable for use in a human clinical trial. This material was immunogenic, and when used as a vaccine, protected mice against challenge with an HPV16 E7 DNA transfected tumour cell line. Based on this observation, the E7GST fusion protein is currently being used in a human clinical trial of a vaccine against HPV16-induced cervical cancer. This fusion protein could be cleaved with thrombin to remove the GST fusion part and further purified by preparative SDS gel electrophoresis to obtain free E7 with > 98% purity.
Fernando
G J
GJ
University of Queensland, Department of Medicine, Princess Alexandra Hospital, Brisbane, Australia.
Murray
B
B
Zhou
J
J
Frazer
I H
IH
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D001483:Base Sequence; D019496:Cancer Vaccines / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009856:Oncogene Proteins, Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D027383:Papillomaviridae / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000150:complications / Q000517:prevention & control; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D014412:Tumor Virus Infections / Q000150:complications / Q000517:prevention & control; D002583:Uterine Cervical Neoplasms / Q000209:etiology / Q000517:prevention & control / Q000821:virology*; D014765:Viral Vaccines / Q000494:pharmacology
D000914:Antibodies, Viral; D019496:Cancer Vaccines; D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011993:Recombinant Fusion Proteins; D014765:Viral Vaccines; C059731:oncogene protein E7, Human papillomavirus type 16