Expanding the versatility of phage display II: improved affinity selection of folded domains on protein VII and IX of the filamentous phage

Background

Phage 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 Findings

Here 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/Significance

Display 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.     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Preparation of peptide-targeted phagemid particles using a protein III-modified helper phage

Ligand or peptide-targeted phagemid particles are being pursued as vehicles for receptor-mediated gene delivery. Here we describe a helper phage in which the protein III (pIII) protein is modified by the addition of a ligand peptide sequence at the amino terminus. Phagemid particles can be prepared with the help of this modified helper phage and should display the ligand peptide in most of the pIII proteins on the phagemid surface. Using such a method, it is not necessary for the phagemid to encode the pIII protein, which leaves a larger space for cloning genes of interest. In addition, the technique should allow for the rapid testing of peptide ligands selected from phage display libraries using phagemids encoding various reporter genes (e.g., green fluorescent protein, luciferase, beta-galactosidase) and therapeutic genes.     

Phage display of cDNA libraries: enrichment of cDNA expression using open reading frame selection

Phage display technologies are powerful tools for selecting binding ligands against purified molecular targets, live cells, and organ vasculature. However, the selection of natural ligands using phage display has been limited because of significant problems associated with the display of complex cDNA repertoires. Here we describe the use of cDNA fragmentation and open reading frame (ORF) selection to display a human placental cDNA library on the pIII coat protein of filamentous phage. The library was enriched for ORFs by selecting cDNA-beta-lactamase fusion proteins on ampicillin, resulting in a cDNA population having 97% ORFs. The ORF-selected cDNAs were fused to pIII in the phagemid vector, pUCMG4CT-198, and the library was rescued with a pIII-deleted helper phage for multivalent display. The resulting phagemid particle library consisted of 87% ORFs, compared to only 6% ORFs when prepared without ORF selection. Western blot analysis indicated cDNA-pIII fusion protein expression in eight out of nine ORF clones tested, and seven of the ORF encoded peptides were displayed multivalently. The high level of cDNA expression obtained by ORF selection suggests that ORF-enriched phage cDNA libraries prepared by these methods will be useful as functional genomics tools for identifying natural ligands from various source tissues.     

Adapter-Directed Display: A Modular Design for Shuttling Display on Phage Surfaces

A novel adapter-directed phage display system was developed with modular features. In this system, the target protein is expressed as a fusion protein consisting of adapter GR1 from the phagemid vector, while the recombinant phage coat protein is expressed as a fusion protein consisting of adapter GR2 in the helper phage vector. Surface display of the target protein is accomplished through specific heterodimerization of GR1 and GR2 adapters, followed by incorporation of the heterodimers into phage particles. A series of engineered helper phages were constructed to facilitate both display valency and formats, based on various phage coat proteins. As the target protein is independent of a specific phage coat protein, this modular system allows the target protein to be displayed on any given phage coat protein and allows various display formats from the same vector without the need for reengineering. Here, we demonstrate the shuttling display of a single-chain Fv antibody on phage surfaces between multivalent and monovalent formats, as well as the shuttling display of an antigen-binding fragment molecule on phage coat proteins pIII, pVII, and pVIII using the same phagemid vectors combined with different helper phage vectors. This adapter-directed display concept has been applied to eukaryotic yeast surface display and to a novel cross-species display that can shuttle between prokaryotic phage and eukaryotic yeast systems.     

An improved helper phage system for efficient isolation of specific antibody molecules in phage display

Phage display technology has been applied in many fields of biological and medical sciences to study molecular interactions and especially in the generation of monoclonal antibodies of human origin. However, extremely low display level of antibody molecules on the surface of phage is an intrinsic problem of a phagemid-based display system resulting in low success rate of isolating specific binding molecules. We show here that display of single-chain antibody fragment (scFv) generated with pIGT3 phagemid can be increased dramatically by using a genetically modified Ex-phage. Ex-phage has a mutant pIII gene that produces a functional wild-type pIII in suppressing Escherichia coli strains but does not make any pIII in non-suppressing E.coli strains. Packaging phagemids encoding antibody-pIII fusion in F⁺ non-suppressing E.coli strains with Ex-phage enhanced the display level of antibody fragments on the surfaces of recombinant phage particles resulting in an increase of antigen-binding reactivity >100-fold compared to packaging with M13KO7 helper phage. Thus, the Ex-phage and pIGT3 phagemid vector provides a system for the efficient enrichment of specific binding antibodies from a phage display library and, thereby, increases the chance of obtaining more diverse antibodies specific for target antigens.     

Expanding the versatility of phage display I: efficient display of peptide-tags on protein VII of the filamentous phage

Background

Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. 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 allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display.

Methodology/Principal Findings

Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS₆ or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether.

Conclusions/Significance

Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations.     

Enhancing phage display of antibody fragments using gIII-amber suppression

The effect of utilizing Ex12 helper phage, a mutant M13K07 helper having two amber codons at the gIII (gIII-amber), in combination with Escherichia coli host strains belonging to the supE genotype on improving the phage display of antibody fragments was investigated. Because of an inefficient read-through of the UAG codons, Ex12 helper phage produced approximately 10% of the intracellular wt pIII in the supE host cells compared to M13K07. The phage progenies rescued from the supE XL-1 Blue MRF' strain carrying the recombinant phagemid, pCMTG-SP112, by Ex12 helper phage displayed both antibody-DeltapIII fusion and wt pIII at a ratio of 1:1.5, and achieved a 50-fold greater display of the antibody-DeltapIII compared to those obtained by a conventional phage rescue using M13K07. Additionally observed were a 100-fold increase in antigen-binding functionality and a drastic improvement on antigen-specific panning efficiency by the phage progenies. Our approach permits the display of at least one antibody fragment as well as more than one copy of wt pIII on the surface of recombinant phages, and this would make the phagemid-based phage display technology more practical and reliable.     

Construction and exploitation in model experiments of functional selection of a landscape library expressed from a phagemid

Phage display has evolved during the past 15 years as a powerful technique to select, from libraries of peptides or proteins, binders for various targets or to evolve new functions in proteins. In recent years, the knowledge acquired in phage display technology was exploited to engineer phages as vehicles for receptor-mediated gene delivery. The first vectors generated provided the proof of the concept that development of gene delivery vehicles based on phages was feasible. Results obtained showed that the level of receptor ligand display was an essential factor that determines the efficiency of transduction and suggested that phagemids might be more appropriate than phages for gene delivery. However, due to the limitations of the existing display systems, vectors constructed up to now allowed only relatively low levels of ligand display. The transduction efficiency of these vectors was relatively poor. Here, we describe the construction and optimization of a new phagemid display system that was designed to allow the functional selection of peptides that promote gene delivery from phagemids in a high display format. Peptides are displayed on every copy of the major coat protein pVIII and are expressed from the phagemid itself. The phagemid is rescued as particles by a modified R408 helper phage, deficient in pVIII production. Besides an expression cassette for pVIII, the phagemid also contains the SV40 origin of replication, the GFP gene and the neomycin resistance marker. As a model we constructed a library of octapeptides and showed that the library is amenable to selection on cos-7 cells. Several selection approaches were investigated and a preliminary analysis of the peptides selected was carried out.     

Phage versus phagemid libraries for generation of human monoclonal antibodies

Non-immune (na?ve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported na?ve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a na?ve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.     

Eliminating helper phage from phage display

Phage display technology involves the display of proteins or peptides, as coat protein fusions, on the surface of a phage or phagemid particles. Using standard technology, helper phage are essential for the replication and assembly of phagemid particles, during library production and biopanning. We have eliminated the need to add helper phage by using 'bacterial packaging cell lines' that provide the same functions. These cell lines contain M13-based helper plasmids that express phage packaging proteins which assemble phagemid particles as efficiently as helper phage, but without helper phage contamination. This results in genetically pure phagemid particle preparations. Furthermore, by using constructs differing in the form of gene 3 that they contain, we have shown that the display, from a single library, can be modulated between monovalent (phagemid-like) and multivalent display (phage-like) without any further engineering. These packaging cells eliminate the use of helper phage from phagemid-based selection protocols; reducing the amount of technical preparation, facilitating automation, optimizing selections by matching display levels to diversity, and effectively using the packaged phagemid particles as means to transfer genetic information at an efficiency approaching 100%.     

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.     

辅助噬菌体在噬菌体展示中的应用及研究进展

噬菌体展示技术是一种将外源肽或蛋白质与特定噬菌体衣壳蛋白相融合,展示于噬菌体表面来构建蛋白质或多肽文库,并从中筛选目的蛋白、多肽或抗体的基因工程高新技术。噬菌粒/辅助噬菌体系统是最常用的噬菌体展示系统,此系统中辅助噬菌体对噬菌粒的复制和组装发挥着至关重要的作用。本文结合当今该领域的最新研究动态,概述了噬菌粒和辅助噬菌体双基因组系统,着重介绍了不同辅助噬菌体的特点及其突变机制,并对其应用前景进行了展望,以期为该技术的进一步完善提供一定的借鉴作用。     

Protein III-based single-chain antibody phage display using bacterial cells bearing an additional genome of a gene-III-lacking helper phage

We present herein a novel method of pIII-based antibody phage display using Hpd3cells--bacterial cells bearing the genome of a gene-III-lacking helper phage (VCSM13d3). A high level of single-chain variable fragments (scFvs) was displayed in the consequent phagemid particles using Hpd3cells to rescue the phagemid encoding scFv-pIII. Hpd3cells considerably improved the specific enrichment factor when used for constructing an immunized antibody library. In addition, using Hpd3cells could overcome pill resistance and can contribute to the efficient enrichment of specific binding antibodies from a phage display library, thereby increasing the chance of obtaining more diverse antibodies specific for target antigens.     

External surface display of proteins linked to DNA-binding domains.

A novel system (DBDX) was developed which allows the external surface display on filamentous bacteriophage of proteins fused to either the N- or the C-terminus of a DNA-binding protein. In conjunction with helper phage infection, expression of proteins fused to the estrogen receptor DNA-binding domain (DBD) in a phagemid vector containing the DNA sequence recognized by the DBD resulted in the production of phage particles which display the fusion protein through the phage pVIII coat on the external surface of the particle. The viability of the technique was established with several model systems: particles displaying the C-terminal domain of N-cadherin or the biotinylation domain of propionyl coenzyme A carboxylase fused to the C-terminus of the DBD were found to be bound specifically by antibody or streptavidin, respectively. Human kappa constant region cDNA was selected from a N-terminal DBD fusion lymphocyte cDNA library after two rounds of selection with anti-kappa antibody. This display system may complement currently available bacterial selection techniques.     

A helper phage to improve single-chain antibody presentation in phage display

We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage). Hyperphage have a wild-type pIII phenotype and are therefore able to infect F(+) Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII-antibody fusion is the sole source of pIII in phage assembly. This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface. Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle. When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin. After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used. Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.     

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.     

Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype

Phage display holds a key position in the use of combinatorial library approaches for the purpose of protein engineering and discovery. However, modifying the pIII protein of the phage can severely and negatively influence the infectiousness of the phage particle. This concern is particularly relevant when large pIII fusions in combination with multivalent display systems are in use. We here describe the use of trypsin to restore wild-type pIII phenotype as a small modification to the standard titration protocol. The results show that the trypsin treatment has a very large but heterogeneous effect on the phage infection efficiency, depending on the pIII fusion domain and the valence of display.     

On the influence of vector design on antibody phage display

Phage display technology is an established technology particularly useful for the generation of monoclonal antibodies (mAbs). The isolation of phagemid-encoded mAb fragments depends on several features of a phage preparation. The aims of this study were to optimize phage display vectors, and to ascertain if different virion features can be optimized independently of each other. Comparisons were made between phagemid virions assembled by g3p-deficient helper phage, Hyperphage, Ex-phage or Phaberge, or corresponding g3p-sufficient helper phage, M13K07. All g3p-deficient helper phage provided a similar level of antibody display, significantly higher than that of M13K07. Hyperphage packaged virions at least 100-fold more efficiently than did Ex-phage or Phaberge. Phaberge's packaging efficiency improved by using a SupE strain. Different phagemids were also compared. Removal of a 56 base pair fragment from the promoter region resulted in increased display level and increased virion production. This critical fragment encodes a lacZ'-like peptide and is also present in other commonly used phagemids. Increasing display level did not show statistical correlation with phage production, phage infectivity or bacterial growth rate. However, phage production was positively correlated to phage infectivity. In summary, this study demonstrates simultaneously optimization of multiple and independent features of importance for phage selection.     

De Novo Selection of High-Affinity Antibodies from Synthetic Fab Libraries Displayed on Phage as pIX Fusion Proteins

Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V_L and three V_H domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V_H by keeping V_L in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V_H diversity was sufficient for isolating Fabs to most antigens, whereas variability in V_L was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V_L diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.