EXPRESSION AND CHARACTERIZATION OF A CYTOTOXIC HUMAN-FROG CHIMERIC RIBONUCLEASE - POTENTIAL FOR CANCER-THERAPY

Onconase is a cytotoxic ribonuclease with antitumor properties. A semi synthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 1 04 amino acid residues to a genomic clone that encoded the remaining a mino acid residues. Additionally, the 15 N-terminal amino acid residue s of onconase were replaced with the first 21 amino acid residues of t he homologous human RNase, eosinophil-derived neurotoxin, EDN. Two ver sions of the hybrid EDN-onconase protein were cloned, expressed and pu rified. The chimera that contained a glycine in lieu of the aspartic a cid present in native onconase (position 26 in the chimera) exhibited enzymatic activity more characteristic of EDN than native onconase and was considerably more active with respect to both RNase activity and cellular cytotoxicity than recombinant onconase. In contrast to native or recombinant onconase, the EDN chimera was recognized by anti-EDN p olyclonal antibodies, demonstrating that the chimera also shared struc tural antigenic determinants to the human enzyme. These results demons trate that a chimeric ribonuclease has cytotoxicity comparable to onco nase in two out of four cell lines tested. The implications with regar d to cancer therapy are presented.