Structural and functional characterization of the HPV16 E7 protein expressed in bacteria.
The E7 gene of the human papillomaviruses (HPV) encodes a 98-amino acid, multifunctional nuclear phosphoprotein with functional and structural similarities to adenovirus E1A and the papovavirus T antigens. E7 is a viral oncoprotein, which will cooperate with an activated ras oncogene to transform primary rodent cells, and can cooperate with the HPV E6 protein for the efficient immortalization of primary human keratinocytes. Due to the compelling epidemiological and experimental association between HPV infection and cervical cancer, we have undertaken a detailed study of the structure of the HPV16 E7 protein. The E7 protein was expressed in Escherichia coli as a native, unfused polypeptide, and soluble protein was purified by conventional chromatographic techniques. The purified protein was assessed for various biochemical and biophysical properties. Purified E7 binds the retinoblastoma protein avidly and specifically, and it can dissociate the E2F transcription factor when assayed in vitro. Circular dichroism spectroscopy indicated that E7 reversibly binds Zn2+ and Cd2+, resulting in a substantial increase in the alpha-helical content of the metal-bound E7 consistent with the stabilization of a hydrophobic core in the COOH terminus of the protein.
Pahel
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G
Wellcome Research Laboratories, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
Aulabaugh
A
A
Short
S A
SA
Barnes
J A
JA
Painter
G R
GR
Ray
P
P
Phelps
W C
WC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005260:Female; D005814:Genes, Viral; D007700:Kinetics; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000150:complications; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014412:Tumor Virus Infections / Q000150:complications; D002583:Uterine Cervical Neoplasms / Q000382:microbiology; D014709:Vero Cells
D017931:DNA Primers; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D010455:Peptides; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16