Creation of a ribonuclease abzyme through site-directed mutagenesis

Abstract

The development of abzymes (antibody/enzymes) is one method of creating reagents with novel catalytic activity. To date, most abzymes have been obtained by immunization with transition state analogs. We have chosen to start with an existing antibody and convert it into an enzyme by the addition of catalytic residues to the binding site. We have introduced a histidine residue into antibody Jel 103 and converted it into an abzyme that cleaves poly(rI) with a kinetic efficiency of about 100 M–1sec–1.

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Figure 1: Proposed mechanism of ribonuclease action for the His96H mutant, based on the mechanism of naturally occurring ribonucleases12,13.
Figure 2: Fluorescence polarization measurements of the binding of Jel 103 (▪), scFv.103 (), and the His98H mutant scFv (♦) to fluorescein-labeled (rI)20, and scFv.103 () to fluorescein-labeled (rC)20.
Figure 3: (A) Digestion of fluorescein-labeled (rI)20 assessed by fluorescence polarization. scFv.103 (▪), mutant His98H (), His96H ().
Figure 4: Gel electrophoretic analysis of fluorescein-labeled (rI)20 incubated with His96H.
Figure 5: Digestion of poly(rI) by His96H ().

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Acknowledgements

This work was funded by Medical Research Council by grants to J.S.L. and by Health Services Utilization and Research Commission for fellowships for M.C.F. and A.K.

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Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5.

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