Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy

Journal of Translational Medicine

Research

Paola Di Bonito¹  Carlo Zanotto²  Carlo De Giuli Morghen³  Antonia Radaelli⁴  Elena Illiano⁵  Aldo Venuti⁶  Rosella Franconi⁷  Olivia Costantina Demurtas⁸  Silvia Massa⁸  Roberta Chiaraluce⁹  Valerio Consalvi⁹ 

[1] Department of Infectious Diseases, Istituto Superiore Sanità, Viale Regina Elena 299, 00185, Rome, Italy;Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129, Milan, Italy;Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129, Milan, Italy;Catholic University ‘Our Lady of Good Counsel’, Tirana, Albania;Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Balzaretti 9, 20133, Milan, Italy;Cellular and Molecular Pharmacology Section, CNR Institute of Neurosciences, University of Milan, 20129, Milan, Italy;Department of Pharmacological and Biomolecular Sciences, University of Milan, Via Balzaretti 9, 20133, Milan, Italy;Laboratory of Biomedical Technologies (SSPT-TECS-TEB), Department for Sustainability, Division of Health Protection Technologies, Italian National Agency for New Technologies, Energy and the Environment (ENEA), ‘Casaccia’ Research Centre, Via Anguillarese 301, 00123, Rome, Italy;HPV-UNIT, Ridait Department, Regina Elena National Cancer Institute, Via E. Chianesi 53, 00144, Rome, Italy;Laboratory of Biomedical Technologies (SSPT-TECS-TEB), Department for Sustainability, Division of Health Protection Technologies, Italian National Agency for New Technologies, Energy and the Environment (ENEA), ‘Casaccia’ Research Centre, Via Anguillarese 301, 00123, Rome, Italy;Laboratory of Biotechnology (SSPT-BIOAG-BIOTEC), Department for Sustainability, Division Biotechnology and Agroindustry, Italian National Agency for New Technologies, Energy and the Environment (ENEA), ‘Casaccia’ Research Centre, Via Anguillarese 301, 00123 Rome, Italy;‘A. Rossi Fanelli’ Department of Biochemical Sciences, University of Rome ‘La Sapienza’, P.le Aldo Moro 5, 00185, Rome, Italy;

关键词: HPV;    E6 oncoprotein;    Biomarker;    Vaccines;    Diagnostic;   

DOI  :  10.1186/s12967-016-0978-6

 received in 2016-04-20, accepted in 2016-07-13,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundHigh-risk human papillomaviruses (HR-HPVs) types 16 and 18 are the main etiological agents of cervical cancer, with more than 550,000 new cases each year worldwide. HPVs are also associated with other ano-genital and head-and-neck tumors. The HR-HPV E6 and E7 oncoproteins are responsible for onset and maintenance of the cell transformation state, and they represent appropriate targets for development of diagnostic and therapeutic tools.MethodsThe unmutated E6 gene from HPV16 and HPV18 and from low-risk HPV11 was cloned in a prokaryotic expression vector for expression of the Histidine-tagged E6 protein (His6-E6), according to a novel procedure. The structural properties were determined using circular dichroism and fluorescence spectroscopy. His6-E6 oncoprotein immunogenicity was assessed in a mouse model, and its functionality was determined using in vitro GST pull-down and protein degradation assays.ResultsThe His6-tagged E6 proteins from HPV16, HPV18, and HPV11 E6 genes, without any further modification in the amino-acid sequence, were produced in bacteria as soluble and stable molecules. Structural analyses of HPV16 His6-E6 suggests that it maintains correct folding and conformational properties. C57BL/6 mice immunized with HPV16 His6-E6 developed significant humoral immune responses. The E6 proteins from HPV16, HPV18, and HPV11 were purified according to a new procedure, and investigated for protein–protein interactions. HR-HPV His6-E6 bound p53, the PDZ1 motif from MAGI-1 proteins, the human discs large tumor suppressor, and the human ubiquitin ligase E6-associated protein, thus suggesting that it is biologically active. The purified HR-HPV E6 proteins also targeted the MAGI-3 and p53 proteins for degradation.ConclusionsThis new procedure generates a stable, unmutated HPV16 E6 protein, which maintains the E6 properties in in vitro binding assays. This will be useful for basic studies, and for development of diagnostic kits and immunotherapies in preclinical mouse models of HPV-related tumorigenesis.

【 授权许可】

CC BY   
© The Author(s) 2016