The enrichment of open reading frames (ORFs) from large gene libraries and the presentation of the corresponding polypeptides on filamentous phage M13 (phage display) is frequently used to identify binding partners of unknown ORFs. In particular, phage display is a valuable tool for the identification of pathogen-related antigens and a first step for the development of new diagnostics and therapeutics. Here, we introduce a significant improvement of phage-based ORF enrichment by using Hyperphage, a helperphage with a truncated gIII. The methods allow both the enrichment of ORFs from cDNA libraries and the display of the corresponding polypeptides on phage, thus combining ORF enrichment with a screening for binding in one step without any further subcloning steps. We demonstrated the benefits of the method by isolating the sequences encoding two predicted immunogenic epitopes of the outer membrane protein D encoding gene (ompD) of Salmonella typhimurium. Here, we showed that when using a mixture of three constructs with only one containing an ORF, solely this correct construct could be reisolated in phage particles. Further, both epitopes were detected by enzyme-linked immunosorbent assay (ELISA), demonstrating correct translation of fusion proteins. Furthermore, the enrichment system was evaluated by the enrichment of ORFs from total cDNA of lymphocytes. Here, we could show that 60% of the phage contained ORFs, which is an increase of an order of magnitude compared with conventional phage expression system. Together, these data show that the Hyperphage-based enrichment system significantly improves the enrichment of ORFs and directly allows the display of the corresponding polyp eptide on bacteriophage M13.

Introduction

Phage display was initially described by G.P. Smith (1) as a powerful tool for the presentation of recombinant polypeptides on filamentous phage M13 and the selection of the encoding sequences from expression libraries. The genotype and phenotype of polypeptides are linked by fusing the corresponding gene fragments to the phage gene gIII encoding the minor coat protein III (pIII) of the filamentous bacteriophage M13. At first, the gene fragments were directly inserted into the phage genome fused to the wild-type gIII gene. Today, most successful phage display systems uncouple polypeptide expression from phage propagation by providing the genes encoding the polypeptide::pIII fusion proteins on a separate plasmid (phagemid), containing a phage morphogenetic signal for packaging into the assembled phage particles (2). A large variety of phagemids has been constructed for M13 phage display (for an overview of phagemids used for the display of antibody fragments see Reference (3). The in vitro procedure for isolating polypeptides or peptides by their binding activity was called panning (4).

The display and selection of antibody fragments currently is the major application of phage display technology. However, another important field is the presentation of polypeptides encoded by cDNA or genomic DNA. This approach was used for the identification of allergens (5), identification of binding epitopes of antibodies (6), identification of ligands in protein-ligand interactions (7), or identification of tumor polypeptides eliciting immune responses (8) among others. For an overview of phage displayed polypeptides derived from cDNA, see Reference (9). Phage display-based methods can also be used in high-throughput setup for parallel selection of binders (10,11).

A disadvantage of current approaches to phage-based polypeptide expression results from the necessity of the in-frame insertion between signal sequence and the gIII of the phage to obtain expression. As a consequence, only 6.25%—the rate of nondirectional cloning—of the cloned DNA fragments represent true open reading frames (ORFs). Furthermore, stop codons in the gene fragments can abrogate the translation of the gene::gIII fusion required for the enrichment. Due to the selection pressure, phage display vectors without inserted fragments or with defective inserts are more efficiently propagated than vectors containing a substantial ORF insert. This effect further hampers the selection of binders by increasing the number of junk clones in a selection to 90%–99%. Consequently, it would be helpful to enrich for ORFs. Therefore, the gene fragments were cloned as fusions with the ampicillin resistance (β-lactamase gene to promote the enrichment of gene fragments that are in-frame with the selection marker (12). However, a combination of ORF enrichment based on the ampicillin resistance gene with presentation of the corresponding polypeptide on phage requires the removal of the (β-lactamase gene fragment after antibiotic enrichment. This removal was achieved either by subcloning of the ORF fragment (13) or by constructing a (β-lactamase gene fragment flanked by loxP sites that could then be removed in vivo by Cre recombinase (14). Both systems still require subcloning of the ORF-enriched libraries before further functional selection, thus leading to a loss of complexity.

Here, we present a novel method for the enrichment of ORFs combined with the presentation of the corresponding polypeptides on M13 phage. The helperphage Hyperphage (15) has a truncated gIII on the phage genome, so that the pIII fusion protein encoded on the phagemid is the only source of intact pIII, which is an essential coat protein for the assembly of infective phage. The resulting phage contain only phagemid encoded polypeptide::pIII fusion, thereby providing oligovalent display. In contrast, when using M13K07 for packaging, two sources of pIII are available, and only 1%–10% of the phage display a polypeptide (15). In the Hyperphage-based enrichment system, the DNA fragments are cloned upstream of gIII and packaged with Hyperphage. Only if the DNA fragment is cloned in-frame with the pelB leader sequence and gIII, functional polypeptide::pIII fusion protein can be produced and consequently functional phage can be assembled. Further, the encoded polypeptides are displayed on the phage particles without any further cloning step, avoiding loss of library diversity.