title: The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3.
abstract: BACKGROUND
  Filamentous phage display has become an ordinary tool to engineer antibody fragments. Several capsid proteins have been applied for displaying antibodies, of which gene III (p3) protein is used the most followed by experiments with gene IX (p9) protein. Despite the popularity, there are no library scale studies to objectively compare differences in the selection performance of the libraries, when displayed via different capsid proteins.
  
  
  RESULTS
  In this study, an identical antibody repertoire was displayed as Fab fragments on p9, p3 and truncated p3 (p3Δ). In addition, the library clones were displayed as ScFv fragments on p3Δ and the Fab-p3 display valency was modulated by hyperphage and VCS-M13 superinfections. The selection performances of the libraries were followed in repeated parallel panning reactions against streptavidin (STR) and digoxigenin (DIG). Selection was successful with all display formats, but the enrichment of specific clones from Fab-p9 library was clearly less efficient than from the other libraries. The most diverse outputs were obtained from p3Δ display and the highest affinity anti-DIG antibodies from the ScFv repertoire. Unfortunately, the number of retrieved specific clones was too low for explicit analysis of the differences in the number of obtained unique clones from each library. However, severe reduction in sequence diversity was observed in p3-Fab libraries prior to panning, which in turn, materialized as a low number of unique specific clones. Oligovalent display by hyperphage resulted in a higher number of unique clones, but the same highest affinity anti-DIG Fab was recovered also by VCS-M13 superinfection.
  
  
  CONCLUSIONS
  The compromised enrichment of the target-specific clones from the Fab repertoire as a fusion to p9 capsid protein in our experiments, the significant loss of functional diversity in Fab-p3 library after single phage packing cycle and the retrieval of higher affinity anti-digoxigenin clones as ScFv molecules than as Fab molecules from the same source repertoire indicate that the chosen display format may have a significant impact on the selection outcome. This study demonstrates that in addition to library content, also display related issues, should be taken into consideration when planning directed evolution experiments.
authors.affiliation: Department of Biochemistry, University of Turku, Turku 20520, Finland. tuohuo@utu.fi.
authors.forename: Tuomas
authors.initials: T
authors.lastname: Huovinen
authors.forename: Markku
authors.initials: M
authors.lastname: Syrjänpää
authors.forename: Hanna
authors.initials: H
authors.lastname: Sanmark
authors.forename: Titta
authors.initials: T
authors.lastname: Seppä
authors.forename: Sultana
authors.initials: S
authors.lastname: Akter
authors.forename: Liton Md Ferdhos
authors.initials: LM
authors.lastname: Khan
authors.forename: Urpo
authors.initials: U
authors.lastname: Lamminmäki
mesh_terms: D000915:Antibody Affinity; D017104:Bacteriophage M13 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D004076:Digoxigenin / Q000276:immunology / Q000378:metabolism; D005818:Genetic Engineering; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019809:Streptavidin / Q000276:immunology / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D019809:Streptavidin; D004076:Digoxigenin