title: Identification and repair of positive binding antibodies containing randomly generated amber codons from synthetic phage display libraries.
abstract: Phage display technology allows for the rapid isolation and characterization of monoclonal antibodies that have vast potential for therapeutic and diagnostic applications. However, the panning process, which utilizes a host strain that suppresses termination by the amber codon, has an inherent bias toward clones containing randomly generated amber stop codons, complicating identification of positive binding antibodies when the antibody genes are finally expressed in a nonsupressor host. Here, we perform biopanning against a Histone 2A peptide using streptavidin- or anti-biotin-coated beads. After four rounds, a dominant clone is characterized but contains a spurious amber stop codon. A protocol is given that readily corrects the amber codon, allowing for soluble antibody production once the phagemid is transformed into a nonsuppressor bacterial strain. This work also highlights the ability to isolate antibodies against a protein antigen by using only a small peptide (15 amino acids) representing a portion of the antigen.
authors.affiliation: Department of Physics and Astronomy, Arizona State University, Tempe, Arizona 85287, USA. warren.marcus@asu.edu
authors.forename: Warren D
authors.initials: WD
authors.lastname: Marcus
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Lindsay
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sierks
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000937:Antigen-Antibody Reactions; D003062:Codon; D016153:Genes, Suppressor; D006657:Histones / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry*; D019151:Peptide Library; D011485:Protein Binding; D019809:Streptavidin / Q000737:chemistry
chemical_list: D000911:Antibodies, Monoclonal; D003062:Codon; D006657:Histones; D010446:Peptide Fragments; D019151:Peptide Library; D019809:Streptavidin