title: Expression and purification of His-tagged HPV16 E7 protein active in pRb binding.
abstract: Human papillomavirus type 16 (HPV16) protein E7 is the major oncogenic factor associated with the development of human cervical cancer. The transforming activity of the E7 protein is linked to its interaction with host regulatory proteins such as the retinoblastoma tumor suppressor protein. The recombinant production of E7 protein is a prerequisite for its structural and functional characterization as well as for the development of various preventive and therapeutic strategies. We present an approach to enhance the soluble expression of His-tagged E7 protein by optimization of the E7 gene and the expression conditions in the host Escherichia coli. We also report a detailed protocol for the purification of E7 protein by standard chromatographic methods. The binding of E7 protein to the recombinant non-phosphorylated form of retinoblastoma protein was examined by ELISA and surface plasmon resonance analysis. These studies confirm that the recombinant His-tagged E7 protein retains its conformational properties and biological activity.
authors.affiliation: Institut für Biotechnologie, Martin-Luther-Universität Halle/Wittenberg, Halle (Saale), Germany.
authors.forename: Ewa A
authors.initials: EA
authors.lastname: Mirecka
authors.forename: Rainer
authors.initials: R
authors.lastname: Rudolph
authors.forename: Thomas
authors.initials: T
authors.lastname: Hey
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*
chemical_list: D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16; D006639:Histidine