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 hum an cervical cancer. The HPV E7 gene is transcriptionally active in the se diseases and has been shown to transform mammalian cells in vitro. We have expressed and purified the HPV-16 E7 gene product in Escherich ia coli. The isolated E7 protein contains zinc in a 1:1 molar ratio. X -ray absorption fine structure studies demonstrated that the zinc is c oordinated by 4 sulfur ligands. We sequentially derivatized the E7 cys teines to differentiate between solvent-exposed, metal-bound, and disu lfide-associated cysteines. Our results demonstrate that Cys24 and Cys 68 are accessible to solvent, while cysteines in the two conserved Cys -X-X-Cys motifs are likely involved in binding zinc. We observed no ev idence for the existence of disulfide bonds in recombinant E7 protein under the conditions tested.