title: 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.
authors.affiliation: Biomedical Diagnostics Institute, Dublin City University, Dublin 9, Ireland.
authors.forename: Ruth
authors.initials: R
authors.lastname: Larragy
authors.forename: Jenny
authors.initials: J
authors.lastname: Fitzgerald
authors.forename: Andreea
authors.initials: A
authors.lastname: Prisecaru
authors.forename: Vickie
authors.initials: V
authors.lastname: McKee
authors.forename: Paul
authors.initials: P
authors.lastname: Leonard
authors.forename: Andrew
authors.initials: A
authors.lastname: Kellett
mesh_terms: D000595:Amino Acid Sequence; D003300:Copper / Q000737:chemistry*; D004247:DNA / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D007501:Iron / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019151:Peptide Library; D010618:Phenanthrolines / Q000737:chemistry*; D017430:Prostate-Specific Antigen / Q000276:immunology; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology
chemical_list: C045076:Fenton's reagent; D019151:Peptide Library; D010618:Phenanthrolines; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D003300:Copper; D004247:DNA; D006861:Hydrogen Peroxide; D007501:Iron; D003850:Deoxyribonuclease I; D017430:Prostate-Specific Antigen; C025205:1,10-phenanthroline