title: An improved helper phage system for efficient isolation of specific antibody molecules in phage display.
abstract: Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F+ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.
authors.affiliation: IG Therapy Co., Nong 3rd Building 112, Kangwon National University, Chunchon 200-701, South Korea.
authors.forename: Hyunjung
authors.initials: H
authors.lastname: Baek
authors.forename: Kyoung-ho
authors.initials: KH
authors.lastname: Suk
authors.forename: Yong-hwan
authors.initials: YH
authors.lastname: Kim
authors.forename: Sanghoon
authors.initials: S
authors.lastname: Cha
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity; D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D036022:Capsid Proteins; D004268:DNA-Binding Proteins / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology; D015151:Immunoblotting; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000302:isolation & purification; D014169:Transformation, Bacterial; D014760:Viral Fusion Proteins / Q000235:genetics
chemical_list: D000906:Antibodies; D036022:Capsid Proteins; D004268:DNA-Binding Proteins; D018840:HSP70 Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins