Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy.
Onconase is a cytotoxic ribonuclease with antitumor properties. A semisynthetic gene encoding the entire protein sequence was constructed by fusing oligonucleotides coding for the first 15 and last six of the 104 amino acid residues to a genomic clone that encoded the remaining amino acid residues. Additionally, the 15 N-terminal amino acid residues of onconase were replaced with the first 21 amino acid residues of the homologous human RNase, eosinophil-derived neurotoxin, EDN. Two versions of the hybrid EDN-onconase protein were cloned, expressed and purified. The chimera that contained a glycine in lieu of the aspartic acid 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 polyclonal antibodies, demonstrating that the chimera also shared structural antigenic determinants to the human enzyme. These results demonstrate that a chimeric ribonuclease has cytotoxicity comparable to onconase in two out of four cell lines tested. The implications with regard to cancer therapy are presented.
Newton
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Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702, USA.
Xue
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Boqué
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Wlodawer
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Kung
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Rybak
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D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D004354:Drug Screening Assays, Antitumor; D004527:Egg Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D004591:Electrophoresis, Polyacrylamide Gel; D047208:Eosinophil-Derived Neurotoxin; D006801:Humans; D008715:Methionine / Q000737:chemistry / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D011894:Rana pipiens; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000627:therapeutic use; D012984:Software; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D004527:Egg Proteins; D009498:Neurotoxins; D011993:Recombinant Fusion Proteins; D008715:Methionine; D047208:Eosinophil-Derived Neurotoxin; D012260:Ribonucleases; C058703:ranpirnase