Biophysical characterization of the recombinant HPV18 E7 oncoprotein

DANTUR, K., SMAL., C., GARCIA ALAI, M.M. AND PRAT GAY, G. DE

Cambridge, United Kingdom, 19 al 24 de julio de 2005

Congreso; DNA Tumour Virus Meeting 2005; 2005

Cancer Research UK and University of California, San Francisco

BIOPHYSICAL CHARACTERIZATION OF THE RECOMBINANT HPV18 E7 ONCOPROTEIN K. Dantur, C. Smal, M. Garcia-Alai, and G. de Prat-Gay Instituto Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, and CONICET. Patricias Argentinas 435, (1405) Buenos Aires, Argentina. HPV18 E7 was cloned from HeLa cells and expressed in bacteria. Large amounts of highly purified protein were obtained aiming to an initial biophysical characterization. Unlike its HPV16 counterpart, purified HPV18 E7 showed conformational homogeneity in gel filtration chromatography and ran as a 54 kDa species against the 44 kDa specie reported for the extended HPV16 dimer. The far UV CD spectra resembles that of strain HPV16 but with different ellipticity values suggesting a possible different molecularity, with well defined _-helical bands at 208 and 222 nm. The fluorescence spectrum shows a buried tryptophan, indicative of a compact folded protein, which shifts together with the CD signal to spectra of unfolded protein by treatment with 6 M Gdm.Cl. Temperature denaturation indicates it is less stable than the hyperthermostable HPV16 E7, with a conformational transition around 40°C. However, similar to the HPV16 countermpart, treatment with a chelating agent causes HPV18 E7 to form large soluble, yet undefined oligomers, which bind Thioflavin T, a fluorophore that indicates repetitive _-sheet structure. Thus, we are able to draw comparisons on the behaviour of the two transforming proteins from the main high risk HPV strains in solution.