Expanding the Versatility of Phage Display II: Improved Affinity Selection of Folded Domains on Protein VII and IX of the Filamentous Phage

Abstract

BackgroundPhage display is a leading technology for selection of binders with affinity for specific target molecules. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). Whereas pVIII display suffers from drawbacks such as heterogeneity in display levels and polypeptide fusion size limitations, toxicity and infection interference effects have been described for pIII display. Thus, display on other coat proteins such as pVII or pIX might be more attractive. Neither pVII nor pIX display have gained widespread use or been characterized in detail like pIII and pVIII display.Methodology/Principal FindingsHere we present a side-by-side comparison of display on pIII with display on pVII and pIX. Polypeptides of interest (POIs) are fused to pVII or pIX. The N-terminal periplasmic signal sequence, which is required for phage integration of pIII and pVIII and that has been added to pVII and pIX in earlier studies, is omitted altogether. Although the POI display level on pIII is higher than on pVII and pIX, affinity selection with pVII and pIX display libraries is shown to be particularly efficient.Conclusions/SignificanceDisplay through pVII and/or pIX represent platforms with characteristics that differ from those of the pIII platform. We have explored this to increase the performance and expand the use of phage display. In the paper, we describe effective affinity selection of folded domains displayed on pVII or pIX. This makes both platforms more attractive alternatives to conventional pIII and pVIII display than they were before.

Next generation phage display by use of pVII and pIX as display scaffolds

“…Thus, the signal sequence is indeed dispensable for display both of short peptides and larger folded domains. In line with the initial observation by Gao et al using pIX for scFv selection [88], we also found the selection efficiency of pVII and pIX to be superior that of pIII [101].…”

“…use of IPTG induction during lacPO driven POI-capsid expression in many full-length pIII systems is known to severely distort this packaging preference in disfavor of the phagemid and even lead to cell death [106,107]. We also found this effect to be present for pIX, and in particular pVII display [101]. However, we consistently observed a strong preference for phagemid packaging even after IPTG induction of the POI-capsid fusion expression once the use of the N-terminal signal sequence was abandoned [101].…”

“…Thus, our observation argues in favor of a low display level as a way to achieve efficient affinity selection [72]. Particularly for pIX display, this low display level was directly coupled to the lack of an N-terminal signal sequence on the POI fusion, as the inclusion of the pelB signal sequence significantly increased the apparent display level, but decreased the efficiency of selection [101].…”

“…We also made an additional and rather unexpected observation when pVII and pIX were used without the N-terminal signal sequence [101]. In general, the use of interference resistant helper phages should ensure preferential phagemid packaging, which is necessary to ensure that the displayed phenotype is included in the phage genome within the virion particle [46].…”

“…Actually, not only pVII, but also the pIX capsid, may offer some distinct advantages as display scaffold for folded domains. We have recently shown that both pVII and pIX may support effective antibody fragment display exploiting the ''N-terminal signal sequence-independent'' nature of the native capsids for phage integration [101]. Thus, the signal sequence is indeed dispensable for display both of short peptides and larger folded domains.…”