Protein engineering with artificial chemical nucleases

Abstract

Herein we report the application of oxidative artificial chemical nucleases as novel agents for protein engineering. The complex ion [Cu(Phen)2(H2O)](2+) (CuPhen; Phen = 1,10-phenanthroline) was applied under Fenton-type conditions against a recombinant antibody fragment specific for prostate-specific antigen (PSA) and compared against traditional DNA shuffling using DNase I for the generation of recombinant mutagenesis libraries. We show that digestion and re-annealment of single chain variable fragment (scFv) coding DNA is possible using CuPhen. Results indicate recombinant library generation in this manner may generate novel clones—not accessible through the use of DNase I—with CuPhen producing highly PSA-specific binding antibodies identified by surface plasmon resonance.