One-step purification of recombinant human papillomavirus type 16 E7 oncoprotein and its binding to the retinoblastoma gene product.
Six derivatives of the human papillomavirus type 16 E7 gene were constructed, each of which encoded a peptide at the amino terminus of the E7 protein capable of chelating metal ions, for use in subsequent purification of the proteins by chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC). Five of the six chelating peptides examined contained six amino acids, and each varied in the number of histidine and tryptophan residues. The CP-E7 proteins were expressed in Escherichia coli, and the cysteine residues were blocked in the form of S-sulfonate groups. This series of CP-E7 proteins was purified in a single chromatographic step using CP-IMAC. The efficiency of purification correlated with the number of histidine and tryptophan residues present in the CP. The purified CP-E7 proteins bound to the human retinoblastoma gene product, pRB, in in vitro co-immunoprecipitation assays and immobilized CP-E7 binding assays. The efficiency of E7-pRB binding was not altered by the presence of a CP at the N-terminus of E7 nor by the S-sulfonate groups within E7.
Kasher
M S
MS
Virology Research Division, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285-0438.
Wakulchik
M
M
Cook
J A
JA
Smith
M C
MC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D001709:Biotechnology; D002846:Chromatography, Affinity / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005069:Evaluation Studies as Topic; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D050725:Papillomavirus E7 Proteins; D010957:Plasmids / Q000235:genetics; D011233:Precipitin Tests; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism*
D004274:DNA, Recombinant; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D011994:Recombinant Proteins; D016160:Retinoblastoma Protein; C059731:oncogene protein E7, Human papillomavirus type 16