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Published on: July 24, 2010
Expression, purification and immunological characterization of the transforming protein E7, from cervical
G J Fernando1, B Murray, J Zhou
1University of Queensland, Department of Medicine, Princess Alexandra Hospital, Brisbane, Australia.
Abstract:
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.
