Characterization of functional HPV-16 E7 protein produced in Escherichia coli.
Human papillomaviruses (HPVs) are the etiologic agents responsible for genital warts and are contributing factors in the pathogenesis of human cervical cancer. The HPV E7 gene is transcriptionally active in these diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherichia coli. The isolated E7 protein contains zinc in a 1:1 molar ratio. X-ray absorption fine structure studies demonstrated that the zinc is coordinated by 4 sulfur ligands. We sequentially derivatized the E7 cysteines to differentiate between solvent-exposed, metal-bound, and disulfide-associated cysteines. Our results demonstrate that Cys24 and Cys68 are accessible to solvent, while cysteines in the two conserved Cys-X-X-Cys motifs are likely involved in binding zinc. We observed no evidence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested.
Patrick
D R
DR
Department of Cancer Research, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486.
Zhang
K
K
Defeo-Jones
D
D
Vuocolo
G R
GR
Maigetter
R Z
RZ
Sardana
M K
MK
Oliff
A
A
Heimbrook
D C
DC
D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors; D007460:Iodoacetamide / Q000737:chemistry; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D016160:Retinoblastoma Protein / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16; D015032:Zinc; D003545:Cysteine; D007460:Iodoacetamide