Masanobu Chinami

Functional oligomerization of purified human papillomavirus types 16 and 6b E7 proteins expressed in Escherichia coli

Purified non-fused soluble human papillomavirus type 16 and 6b E7 proteins expressed in Escherichia coli were found to form oligomers. For both proteins, several degrees of oligomerization were demonstrated by gel filtration, dynamic laser light scattering and scanning electron microscopy. Oligomerization was dependent on the concentration of E7 protein. Oligomerized E7 proteins were able to bind the retinoblastoma gene product pRB and stimulated DNA synthesis when introduced into cells.

Refolding and purification of human papillomavirus type 16 E7-IacZ fusion protein expressed in Escherichia coli

Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration. The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif (C. Edmond and K. H. Vousden, 1989, J. Virol. 63, 2650-2656) and supporting the data of J. A. Rawls, R. Pusztai, and M. Green (1990, J. Virol. 64, 6121-6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion.

Nucleic acid binding by zinc finger-like motif of human papillomavirus type 16 E7 oncoprotein

Human papillomavirus (HPV) types 16 and 6b E7 proteins and their chimeric or mutant proteins were analyzed for oligonucleotide-binding activity by surface plasmon resonance-based biomolecular interaction analysis. The results indicated that type 16 E7 protein has stronger nucleic acid-binding activity than that of type 6b E7 protein. In addition, the results also indicated that the zinc finger-like motif in the C-terminal region of the type 16 E7 protein plays an important role in this activity.

Human papillomavirus DNA from warts for typing by endonuclease restriction patterns: purification by alkaline plasmid methods

SummaryThe alkaline plasmid DNA extraction method of Birnboim and Doly was applied for the isolation of human papillomavirus (HPV) from warts. Tissue from common and plantar warts was digested with proteinase K, and the extrachromosomal circular covalently-closed form of HPV-DNA was rapidly extracted by alkaline sodium dodecyl sulphate and phenol-chloroform treatment. Recovery of HPV-DNA from the tissue was sufficient for determination of endonuclease restriction patterns by agarose gel electrophoresis.