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Expression and characterization of a cytotoxic human-frog chimeric ribonuclease: potential for cancer therapy
1Intramural Research Support Program, SAIC Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, MD 21702, USA.
Abstract:
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.
Insights
A novel chimeric ribonuclease, combining elements of onconase and human eosinophil-derived neurotoxin (EDN), shows significant cytotoxic activity against cancer cells. This engineered protein offers potential for new cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Onconase is a cytotoxic ribonuclease with demonstrated antitumor properties.
- Engineering novel proteins can enhance therapeutic efficacy.
Purpose of the Study:
- To create and characterize a hybrid ribonuclease by combining onconase and human eosinophil-derived neurotoxin (EDN).
- To evaluate the enzymatic activity, cellular cytotoxicity, and antigenic properties of the engineered chimera.
Main Methods:
- Construction of a semisynthetic gene encoding the hybrid EDN-onconase protein.
- Cloning, expression, and purification of two versions of the chimera.
- Assessment of RNase activity, cellular cytotoxicity, and immunoreactivity against anti-EDN antibodies.
Main Results:
- The EDN-onconase chimera with a glycine substitution exhibited enhanced RNase activity and cellular cytotoxicity compared to recombinant onconase.
- The chimera was recognized by anti-EDN antibodies, indicating shared antigenic determinants with human EDN.
- The chimeric ribonuclease demonstrated cytotoxicity comparable to onconase in two of four tested cell lines.
Conclusions:
- The developed chimeric ribonuclease possesses potent cytotoxic properties and retains structural similarities to human EDN.
- This engineered enzyme represents a promising candidate for further investigation in cancer therapy development.