“Expression and purification of the Human Papillomavirus (HPV) E6 protein for antibody isolation and therapeutic vaccination purposes”

ILLIANO ELENA; VILLANOVA, GABRIELA VANINA,; GARDIOL, DANIELA; DUPRÈ S.; FRANCONI ROSSELLA

Riccione, Italia

Congreso; Reunión anual de la Sociedad Italiana de Bioquímica SIB 2004; 2004

Sociedad Italiana de Bioquímica

The high risk human papillomaviruses (HPVs, mainly types 16 and 18) are accepted as the primary etilologic agents of cervical cancer. cervical citology screening has resulted in a significant decline in mortality in developed countries. The HPV 16 E6 and E7 oncoproteins are responsible for the onset and maintenance of the transformed state, and, therefor, represent appropiate targets for therapeutic vaccines as well as early diagnostic markers. The E6 protein interferes with the normal function of several cellular proteins that regulate cell proliferation , differentiation and apoptosis and it is thought to promote tumorigenesis mainly by stimulating cellular degradation of the tumor suppressor p53. recombinant E6 protein is extremely difficult to produce and purify in a soluble and active form. Fusions of E6 to either GST or MBP are expressed in a soluble form in E. coli but proteolytic removal of GST or MBP leads to rapid precipittation of the E6 protein, demonstrating that fusion to the C-terminus of a carrier protein does not guarantee proper folding and activity. As a consecuence , E6-related biophysical and structural data as well as good molecular tools for basic research and diagnostic purposes are missing. The aim of our project is to obtain the E6 protein in a soluble form to be used as a therapeutic vaccine and/or to select recombinant antibodies from a synthetic phage display antibody library. To achieve this goal, in this work we tested different strategies based both on using different expression vectors, optimization of the induction and purification procediures as well as testing of a new carrier protein, alternative to GST or MBP.