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.