title: Production of functional, stable, unmutated recombinant human papillomavirus E6 oncoprotein: implications for HPV-tumor diagnosis and therapy.
abstract: High-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.
  
  
  
  The 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.
  
  
  
  The 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.
  
  
  
  This 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.
authors.affiliation: 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.
authors.forename: Elena
authors.initials: E
authors.lastname: Illiano
authors.affiliation: 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.
authors.forename: Olivia Costantina
authors.initials: OC
authors.lastname: Demurtas
authors.affiliation: 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.
authors.forename: Silvia
authors.initials: S
authors.lastname: Massa
authors.affiliation: Department of Infectious Diseases, Istituto Superiore Sanità, Viale Regina Elena 299, 00185, Rome, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Di Bonito
authors.affiliation: 'A. Rossi Fanelli' Department of Biochemical Sciences, University of Rome 'La Sapienza', P.le Aldo Moro 5, 00185, Rome, Italy.
authors.forename: Valerio
authors.initials: V
authors.lastname: Consalvi
authors.affiliation: 'A. Rossi Fanelli' Department of Biochemical Sciences, University of Rome 'La Sapienza', P.le Aldo Moro 5, 00185, Rome, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Chiaraluce
authors.affiliation: Department of Medical Biotechnologies and Translational Medicine, University of Milan, Via Vanvitelli 32, 20129, Milan, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Zanotto
authors.affiliation: 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.
authors.forename: Carlo
authors.initials: C
authors.lastname: De Giuli Morghen
authors.affiliation: 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.
authors.forename: Antonia
authors.initials: A
authors.lastname: Radaelli
authors.affiliation: HPV-UNIT, Ridait Department, Regina Elena National Cancer Institute, Via E. Chianesi 53, 00144, Rome, Italy. venuti@ifo.it.
authors.forename: Aldo
authors.initials: A
authors.lastname: Venuti
authors.affiliation: 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. rosella.franconi@enea.it.
authors.forename: Rosella
authors.initials: R
authors.lastname: Franconi
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D003902:Detergents / Q000494:pharmacology; D005260:Female; D006801:Humans; D056724:Immunity, Humoral / Q000187:drug effects; D008810:Mice, Inbred C57BL; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis* / Q000628:therapy* / Q000821:virology; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000628:therapy*; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012995:Solubility
chemical_list: D004268:DNA-Binding Proteins; D003902:Detergents; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; D018832:Molecular Chaperones; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins
keywords: Biomarker; Diagnostic; E6 oncoprotein; HPV; Vaccines