Protein engineering with artificial chemical nucleases

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.