Single amino acid substitutions at the N-terminus of a recombinant cytotoxic ribonuclease markedly influence biochemical and biological properties

Biochemistry
D L NewtonS M Rybak

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 the last 6 of the 104 amino acids to a genomic clone that encoded the remaining amino acid residues [Newton, D. L., et al. (1997) Protein Eng. 10, 463-470]. The resulting protein product expressed in Escherichia coli exhibited little enzymatic or cytotoxic activity due to the unprocessed N-terminal Met amino acid residue. In this study, we demonstrate that modification of the 5'-region of the gene to encode [Met-(-1)]Ser or [Met-(-1)]Tyr instead of the native pyroglutamate results in recombinant onconase derivatives with restored activities. [Met-(-1)]rOnc(E1S) was more active than [Met-(-1)]rOnc(E1Y) in all assays tested. Consistent with the action of native onconase, [Met-(-1)]rOnc(E1S) was a potent inhibitor of protein synthesis in the cell-free rabbit reticulocyte lysate assay, degrading tRNA at concentrations that correlated with inhibition of protein synthesis. An interesting difference between the recombinant onconase derivatives and the native protein was their susceptibility to inhibition by the major intracellular RNase inhibito...Continue Reading

Citations

Jun 22, 2001·Critical Reviews in Oncology/hematology·D L NewtonS M Rybak
Aug 2, 2006·Molecular BioSystems·Antoni BenitoMaria Vilanova
Mar 7, 2002·The Biochemical Journal·Aurora BracaleGiuseppe D'Alessio
Dec 5, 1998·Journal of the National Cancer Institute·D L Newton, S M Rybak
Feb 5, 2008·Protein Engineering, Design & Selection : PEDS·Robert F GahlHarold A Scheraga
May 10, 2012·Protein Expression and Purification·Minmin LiuSusan S Wallace
Oct 20, 2007·The FEBS Journal·Cindy SchulenburgUlrich Arnold
Dec 10, 1999·Experimental Cell Research·S M Rybak, D L Newton
Sep 17, 2005·Progress in Nucleic Acid Research and Molecular Biology·Kimberly A DicksonRonald T Raines
Dec 3, 2003·The Journal of Biological Chemistry·Vitaliy Y GorbatyukTai-huang Huang
Oct 26, 2005·Protein Engineering, Design & Selection : PEDS·Heidi A EricksonChristopher A Pennell
May 1, 1999·The Journal of Biological Chemistry·K W Walker, R A Bradshaw
Feb 25, 2005·The Journal of Biological Chemistry·Antonello MerlinoFilomena Sica
Mar 25, 2004·Journal of the American Chemical Society·Ervin WelkerHarold A Scheraga

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