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.     

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.     

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.     

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.     

Multipurpose vectors for peptide expression on the M13 viral surface.

We have developed a set of three cloning vectors for the expression of polypeptides on the surface of the M13 viral coat. The M13mp8 genome has been engineered for expression of foreign protein sequences near the NH2-terminus of the mature pIII protein, which is present in five copies on the outside of each M13 viral particle. All three of the vectors carry the same two useful restriction sites for directed cloning of inserts in the pIII coding region; in addition, one vector carries the bacterial gene conferring resistance to the antibiotic tetracycline, and another expresses the lacZ' polypeptide that allows functional complementation of beta-galactosidase activity within the host bacterial cell. All of these vectors propagate well in E. coli DH5 alpha F' cells and do not require helper phage. We demonstrate that a bacteriophage, expressing an eleven amino acid epitope (from human c-myc) at the NH2-terminus of pIII in one of our vectors, can be purified from a vast mixture of other M13 phage through panning techniques. In particular, we find that the c-myc-expressing viral particles can be easily recovered from phage mixtures with the biotinylated form of the monoclonal antibody, 9E10, and streptavidin-coated MagneSphere beads.     

Ex12 helper phage improves the quality of a phage-displayed antibody library by ameliorating the adverse effect of clonal variations

The quality of a phage-displayed antibody library deteriorates with clonal variations, which are caused by differentially expressed Escherichia coli antibody genes. Using the human Fab SP114 against the pyruvate dehydrogenase complex-E2 (PDCE2), we created four E. coli TOP10F' clones with a pCMTG phagemid encoding Fab-pIII (pCMTG-Fab), Fd (V(H)+C(H1))-pIII (pCMTG-Fd), or light chain (L) (pCMTG-L), or the vector only (pCMTG-?Fab) to investigate the effect of clonal variations in a defined manner. Compared to the others, the E. coli clone with pCMTG-Fab was growth retarded in liquid culture, but efficiently produced phage progenies by Ex12 helper phage superinfection. Our results suggest that an antibody library must be cultured for a short duration before helper phage superinfection, and that the Ex12 helper phage helped to alleviate the detrimental effect of clonal variation, at least in part, by preferentially increasing functional phage antibodies during phage amplification.     

利用噬菌体抗体库筛选U251细胞血清应答基因蛋白特异抗体

利用噬菌体表面展示抗体库对不同血清处理U251细胞吸附的抗体进行差异筛选,筛选获得血清饥饿细胞吸附的阳性噬菌体克隆96个和血清饥饿后恢复血清培养细胞吸附的阳性噬菌体克隆82个。细胞免疫组化检测发现应答反应差异较大的抗体2个,即血清饥饿培养细胞特异反应的抗体1个（11号抗体）和血清饥饿后恢复血清培养细胞特异反应的抗体1个（2号抗体）,其中2号抗体在恢复血清培养细胞中的应答反应强于血清饥饿培养细胞,是一个血清应答基因蛋白特异抗体,且在血清饥饿后恢复血清培养不同时间的U251细胞中具有一定的特异性反应。该研究为寻找与细胞周期调控有关的因子奠定了基础,同时对肿瘤的诊断和治疗研究也有重要意义。     

抗松材线虫纤维素酶单链抗体库的构建及筛选

构建鼠源性松材线虫纤维素酶（Bursaphelenchus xylophilus cellulase, BXC）的噬菌体单链抗体库,从中筛选特异性BXC的单链抗体。以BXC为抗原免疫BALB/C小鼠,从脾脏提取总RNA,用RT-PCR技术扩增小鼠抗体重链（V_H）和轻链(V_L)可变区基因。经重叠PCR(SOE-PCR)在体外将V_H和V_L连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TG1,经辅助噬菌体超感染,成功构建了库容为5×10⁴的Anti-BXC单链抗体库,并从该抗体库中初步筛选到了特异性识别BXC的噬菌体单链抗体scFv。将表面展示单链抗体的单克隆噬菌体转化大肠杆菌HB2151进行可溶性表达,SDS-PAGE及Western blot分析结果显示,可溶性scFv获得表达,且与BXC具有结合活性,为松材线虫的检验检疫以及病理学研究奠定了基础。     

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.     

Immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage

Repeat regions of the circumsporozoite protein gene of Plasmodium falciparum were cloned into the pIII gene of a filamentous phage. These genetically engineered filamentous phage display the recombinant proteins on their surface. We demonstrate that they are both antigenic and immunogenic in rabbits. The recombinant phage were shown to be useful as a source of antigen for this scarce malaria protein, for producing carrier-hapten conjugates for obtaining immunological reagents in rabbits, and for B epitope mapping. In addition, in mice the antibody response to the cloned antigens seems to be controlled by immune response genes. Therefore this system also has the potential for use in helper T cell epitope mapping using inbred mouse strains. This advantage will be of use in vaccine development.     

SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli

Antibody phage display is a key technology for the generation of recombinant (human) antibodies for research, diagnostics and therapy. Most antibody fragments can only be folded correctly in the oxidizing environment of the periplasm of Escherichia coli. A multitude of leader peptides has been used for secretion of antibody::pIII fusion proteins into the periplasm, but a systematic study of their impact on the performance of antibody phage display systems has not been reported so far. In this work we have analysed the influence of various leader peptides on antibody phage display efficiency and production yields of soluble antibody fragments. Four leader peptides using the Sec pathway (PelB, OmpA, PhoA and pIII) and three using the SRP pathway (DsbA, TorT and TolB) were compared. Both pathways are compatible with antibody phage display and the production of soluble antibody fragments. The applicability of the SRP pathway to antibody phage display and the production of functional scFvs is shown here for the first time.     

DeltaPhage--a novel helper phage for high-valence pIX phagemid display

Phage display has been instrumental in discovery of novel binding peptides and folded domains for the past two decades. We recently reported a novel pIX phagemid display system that is characterized by a strong preference for phagemid packaging combined with low display levels, two key features that support highly efficient affinity selection. However, high diversity in selected repertoires are intimately coupled to high display levels during initial selection rounds. To incorporate this additional feature into the pIX display system, we have developed a novel helper phage termed DeltaPhage that allows for high-valence display on pIX. This was obtained by inserting two amber mutations close to the pIX start codon, but after the pVII translational stop, conditionally inactivating the helper phage encoded pIX. Until now, the general notion has been that display on pIX is dependent on wild-type complementation, making high-valence display unachievable. However, we found that DeltaPhage does facilitate high-valence pIX display when used with a non-suppressor host. Here, we report a side-by-side comparison with pIII display, and we find that this novel helper phage complements existing pIX phagemid display systems to allow both low and high-valence display, making pIX display a complete and efficient alternative to existing pIII phagemid display systems.     

The electrooptical parameters of suspensions of Escherichia coli XL-1 cells interacting with helper phage M13K07

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.     

Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries

Abstract Phagemid vectors for display of proteins/peptides on the surface of filamentous phage utilize a plasmid genome carrying the phage origin of replication, along with the gene fused to a fragment of gene III. Generation of phage particles displaying the fusion protein also requires superinfection of the host bacterium with a helper virus. We describe here the construction of a new gene III mutant of M13 KO7 bacteriophage and compare its ability to act as helper phage with two mutants derived from Fd tet (fKN 16 and fCA 55). Furthermore, we investigate their capability to act as helper phages in SAP selection, where non-infectious helper phage, expressing antibody fragments but not protein 3, can still infect by first reacting with a soluble antigen-protein 3 fusion protein. Gene III mutants were found to be non-infectious, and high titers of infective particles were obtained only when the helper phage was grown in cells harbouring a gene III-containing plasmid. An amplification of the phage titer of 10⁶× was achieved in M13-derived phages, when used for the selection of specific antibody fragments.     

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.     

Electrooptical analysis of the Escherichia coli-phage interaction

This article describes electrooptical (EO) characterization of biospecific binding between the bacterium Escherichia coli XL-1 and the phage M13K07. The electrooptical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for EO measurements. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with the phage M13K07 is based on registration of changes of optical parameters of bacterial suspensions. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of E. coli with phage M13K07 induces a strong and specific EO signal as a result of substantial changes of the EO properties of the E. coli XL-1 suspension infected by the phage M13K07. The signal was specific in the presence of foreign microflora (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the EO approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the infection of phage to bacteria is specific, (3) exogenous substrates and mediators are not required for detection, and (4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.     

High-density functional display of proteins on bacteriophage lambda

We designed a bacteriophage lambda system to display peptides and proteins fused at the C terminus of the head protein gpD of phage lambda. DNA encoding the foreign peptide/protein was first inserted at the 3' end of a DNA segment encoding gpD under the control of the lac promoter in a plasmid vector (donor plasmid), which also carried lox P(wt) and lox P(511) recombination sequences. Cre-expressing cells were transformed with this plasmid and subsequently infected with a recipient lambda phage that carried a stuffer DNA segment flanked by lox P(wt) and lox P(511) sites. Recombination occurred in vivo at the lox sites and Amp(r) cointegrates were formed. The cointegrates produced recombinant phage that displayed foreign protein fused at the C terminus of gpD. The system was optimised by cloning DNA encoding different length fragments of HIV-1 p24 (amino acid residues 1-72, 1-156 and 1-231) and the display was compared with that obtained with M13 phage. The display on lambda phage was at least 100-fold higher than on M13 phage for all the fragments with no degradation of displayed products. The high-density display on lambda phage was superior to that on M13 phage and resulted in selective enrichment of epitope-bearing clones from gene-fragment libraries. Single-chain antibodies were displayed in functional form on phage lambda, strongly suggesting that correct disulphide bond formation takes place during display.This lambda phage display system, which avoids direct cloning into lambda DNA and in vitro packaging, achieved cloning efficiencies comparable to those obtained with any plasmid system. The high-density display of foreign proteins on bacteriophage lambda should be extremely useful in studying low-affinity protein-protein interactions more efficiently compared to the M13 phage-based system.     

Application of phage-displayed single chain antibodies in western blot

A phage display single chain fragment variable library constructed on pⅢ protein of M13 filamentous phage was screened using B-lymphocyte stimulator and FP248 as selective molecules. After four rounds of panning, there was a remarkable enrichment in the titer of bound phages. Twenty phage clones were selected from the last round and screened by means of phage-ELISA. With the antibody phages as primary antibodies in Western blot, we developed a method for detecting the specific antigen. The dilutions of antibody phages depend on the affinity between antibody-displayed phage particles and antigens.     

Construction of high-density display of CD147 ectodomain on VCSM13 phage via gpVIII: effects of temperature, IPTG, and helper phage infection-period

Production of VCSM13 phage displaying a high density of CD147 ectodomain (CD147Ex) was achieved when culturing conditions were modulated. A phagemid expressing CD147Ex was constructed and used to produce phage display CD147Ex gpVIII fusion protein in TG1 Escherichia coli. Displaying of CD147Ex via gpVIII was successfully increased when growing the transformed TG1 at 25 degrees C with IPTG-stimulation. In addition to temperature and IPTG-stimulation, the VCSM13 helper phage infection-period particularly affected the insertion of CD147Ex into phage progeny. By sandwich ELISA, incorporation of the CD147Ex into phage particle was confirmed. The correct size of the CD147Ex-gpVIII fusion protein at 28kDa was demonstrated by Western immunoblotting. Multivalent display of CD147Ex on phage particles will be valuable in discovering its ligand partner.     

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.