The selection performance of an antibody library displayed on filamentous phage coat proteins p9, p3 and truncated p3

Abstract

BackgroundFilamentous 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.ResultsIn 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.ConclusionsThe 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.

Multivalent pIX phage display selects for distinct and improved antibody properties

“…However, the most striking observation was the strong dependence on HV display. This is the first report of multivalent antibody pIX display in de novo library selection, explaining why the superior properties of pIX have not been revealed previously16.…”

“…Early studies showed that an N -terminal GST fusion prevented pIX display, as pIX was found aggregated in the cytosol of the E. coli host14. Therefore, ompA and pelB signal sequences were added with the aim to facilitate antibody display15, but later reports are conflicting with respect to how well such pIX display performs in comparison to display on pIII91617. We have developed a display system, where pIX is used as display capsid without addition of a signal sequence to the fusion protein1819.…”

Adding Functions to Biomaterial Surfaces through Protein Incorporation

“…ScFv molecules are expressed at the bacteriophage tip as a fusion with the pIII minor capsid protein, [141] typically resulting in libraries of up to 10 10 phage-displayed antibody fragments which can be screened on immobilized antigens to isolate binding partners. [142] Repetitive cycles of this selection and infection of E. coli cells to amplify phages (and generate increased antibody diversity for the next round) can yield high affinity, highly specific binders in 2-4 weeks. [140] Antibody libraries can also be displayed on bacterial, yeast or mammalian cells by genetic fusion to a cell surface-anchored …”

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique

“…Among the five coat proteins of filamentous M13, f1, or fd bacteriophage, the p3 and p8 proteins are the most frequently used carriers in the phage display technology [ 13 ]. The bacteriophage p3 protein is exploited for the N-terminus or, less frequently, for the C-terminus fusion with the peptide, scFv or cDNA libraries [ 14 , 15 , 16 , 17 ]. The fusion with the p8 protein, in contrast to the p3 protein, makes it possible to present thousands copies of ligands on a single phage particle.…”