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

Geir Åge Løset,Inger Sandlie,Norbert Roos,Bjarne Bogen,Cameron Neylon

doi:10.1371/journal.pone.0017433

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.

Highlights

Phage display is a leading technology for selection of binders with affinity for specific target molecules [1]
All are integral E. coli inner membrane proteins before virion assembly [6], but only protein III (pIII) and protein VIII (pVIII) are synthesized as precursors containing classical N-terminal signal sequences [4]. pVII and pIX appear to be synthesized without such signal peptides and do not undergo post-translational processing [4]
Functional Display of Folded Domains on pVII and pIX To study how pVII and pIX perform in phage display of folded domains, we tested display of three different folded domains and compared with conventional pIII display

Summary

Introduction

Phage display is a leading technology for selection of binders with affinity for specific target molecules [1]. Libraries of polypeptides are created as fusions to phage coat proteins that are solvent exposed. The wt filamentous phage virions of M13, fd and f1 are composed of a small genome surrounded by a cylinder of coat proteins that measures about 1 mm in length and 8–10 nm in diameter, and has a total of about 2,700 copies of the major coat protein pVIII. All are integral E. coli inner membrane proteins before virion assembly [6], but only pIII and pVIII are synthesized as precursors containing classical N-terminal signal sequences [4]. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII) or the minor coat protein III (pIII). 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

Methods

Results

Conclusion