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.     

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.     

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.     

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.     

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 phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.     

Phage display as a novel screening method to identify extracellular proteins

Extracellular proteins are involved in many diverse and essential cell functions and in pathogenic bacteria, and they may also serve as virulence factors. Therefore, there is a need for methods that identify the genes encoding this group of proteins in a bacterial genome. Here, we present such a method based on the phage display technology. A novel gene III-based phagemid vector, pG3DSS, was constructed that lacks the signal sequence which normally orientates the encoded fusion protein to the Escherichia coli cell membrane, where it is assembled into the phage particle. When randomly fragmented DNA is inserted into this vector, only phagemids containing an insert encoding a signal sequence will give rise to phage particles displaying a fusion protein. These phages also display an E-tag epitope in fusion with protein III, which enables isolation of phages displaying a fusion protein, using antibodies against the epitope. From a library constructed from Staphylococcus aureus chromosomal DNA, genes encoding secreted as well as transmembrane proteins were isolated, including adhesins, enzymes and transport proteins.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

大分子量重组蛋白在丝状噬菌体表面的展示表达及其与小分子相互作用的初步研究

利用pHEN1KM13噬菌粒系统表达融合蛋白,进而确定大分子量重组蛋白在丝状噬菌体表达的部位及其表达后的生物活性。通过蛋白酶切处理前后噬菌体侵染细菌能力的变化快速地检测大分子蛋白质能否在噬菌体表面展示表达;比较了谷胱甘肽S转移酶及其与三个不同长度连接臂融合的外源蛋白在噬菌体表面的表达和组装,确定了不大于40kD的重组蛋白分子能展示表达在丝状噬菌体表面;并利用已知的小分子化合物与蛋白质的相互作用证明了组装在噬菌体表面的谷胱甘肽S转移酶重组蛋白仍保持其天然的结合活性,为利用噬菌体展示系统研究蛋白质与小分子化合物的相互作用建立了基础。     

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.     

Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.     

Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.

Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.     

Twin-arginine signal peptide attributes effective display of CD147 to filamentous phage

A novel phagemid (pTat8) was constructed in this study to improve the quality of a molecule displayed on filamentous phage. The twin-arginine translocation (Tat) pathway was chosen for transporting and integrating a CD147 molecule into a phage particle via gpVIII. The parent vector pComb8-CD147Ex was modified by substituting a Sec signal sequence (PelB) with a twin-arginine signal sequence from trimethylamine N-oxide reductase (TorA). The characteristics of the CD147 displayed on the phage particle were evaluated by Sandwich ELISA and Western immunoblotting. A Tat-dependent leader was found to be superior to the Sec leader for the phage display of CD147. Our findings further support the involvement of an Escherichia coli Tat translocase in mediating the integration of a hydrophobic transmembrane protein into the inner membrane. This modified phagemid will be useful in phage display technique when the correctly folded structure is required (i.e., antibody libraries and ligand–receptor tracing). This work was supported by the Thailand Research Fund and the National Center for Genetic Engineering and Biotechnology. R.J.T. thanks the Canadian Institutes of Health Research for funding support.     

Construction and characterization of phage-displayed leukocyte surface molecule CD99

The phage display technique has been described for the production of various recombinant molecules. In the present report, we used this technique to display a leukocyte surface molecule, CD99. PCR subcloning of CD99 cDNA from the mammalian expression vector pCDM8 to the phagemid expression vector pComb3HSS was performed. The resulting phagemid, pComb3H-CD99, was transformed into Escherichia coli XL-1 Blue. CD99 was displayed on the phage particles following infection of the transformed E. coli with the filamentous phage VCSM13. Using sandwich ELISA, the filamentous phage-displayed CD99 was captured by a CD99 monoclonal antibody (mAb) then detected with anti-M13 conjugated to horseradish peroxidase, confirming that the CD99 molecule was displayed on the phage particles. The CD99-phages inhibited induction of Jurkat cell aggregation by CD99 mAb MT99/1. Proper folding of the displayed CD99 bioactive domain was inferred from this finding. Our results demonstrate that the phage display technique can be applied to the generation of full-length CD99 molecules. The phage carrying this cell surface protein will be useful for identification of its counter receptor or ligand.     

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.     

Prokaryotic expression of antibodies and affibodies

Recent advances have been made in the development of systems for the display and expression of recombinant antibodies and affibodies in filamentous phages, Escherichia coli and other prokaryotic cells. Emphasis has been placed on improving phage and phagemid vectors, alternative systems for expression in different cellular compartments (e.g. the outer membrane, periplasm, cytoplasm and extracellular secretion) and novel multimerization systems for generating bivalent or multivalent binding molecules.     

A twin-arginine translocation (Tat)-mediated phage display system

The major limitation of conventional phage display is caused by its dependence on the Sec translocation pathway. All proteins displayed on filamentous phages must first be transported into the bacterial periplasm in an unfolded state via the Sec translocation machinery. Proteins that require a cytoplasmic environment and/or cytoplasmic components for folding, or that contain "stop transfer" signals, or reach their native state before they interact with the Sec proteins are not compatible with the Sec pathway. They can never be presented using conventional phage display. We have developed an alternative phage display system, termed the TPD system, which overcomes these limitations of conventional phage display by exploiting the properties of the twin-arginine translocation (Tat) pathway. The Tat pathway only exports folded proteins that have already attained their native conformation in the cytoplasm. We investigated the functional efficiency of the TPD system by displaying and panning for a mutant of the green fluorescent protein.     

The construction of bifunctional fusion proteins consisting of MutS and GFP

MutS as a mismatch binding protein is a promising tool for SNP detection. Green fluorescent protein (GFP) is known as an excellent reporter domain. We constructed chimeric proteins consisting of MutS from Thermus thermophilus and GFPuv from Aequorea victoria by cloning the GFPuv gene into the plasmid vectors carrying the mutS gene. The GFPuv domain fused to the N-terminus of MutS (histag-GFP-MutS) exhibited the same level of green fluorescence as free GFPuv. To obtain the fluorescing histag-GFP-MutS protein the expression at 30 degrees C was required, while free GFPuv fluoresces when expressed both at 30 and 37 degrees C. The chimeric protein where the GFPuv domain was fused to the C-terminus of MutS exhibited much weaker green fluorescence (20-25% compared with those of histag-GFP-MutS or free GFPuv). The insertion of (ProGly)5 peptide linker between the MutS and GFP domains resulted in no significant improvement in GFP fluorescence. No shifts in the excitation and emission spectra have been observed for the GFP domain in the fusion proteins. The fusion proteins with GFP at the N- and C-terminus of MutS recognised DNA mismatches similarly like T. thermophilus MutS. The fluorescent proteins recognising DNA mismatches could be useful for SNP scanning or intracellular DNA analysis. The fusion proteins around 125 kDa were efficiently expressed in E. coli and purified in milligram amounts using metal chellate affinity chromatography.     

Effect of DNA copy number on genetic stability of phage-displayed peptides

A small model peptide, the FLAG epitope, was cloned into two filamentous phage display vectors, f88-4 and fd88-4, creating phages f88-FLAG and fd88-FLAG, respectively. Both vectors have a gene VIII display cassette (in addition to their normal phage gene VIII) and display the cloned peptide on a few percent of the virion's 3000-4000 pVIII (major coat protein) subunits. Vector f88-4 has a replication defect and attains low DNA copy number in infected cells, while vector fd88-4 has no replication defect and attains the normal, high DNA copy number characteristic of wild-type filamentous phage. Almost no loss of displayed peptide was observed during six rounds of propagation of low copy number f88-FLAG phage. In contrast, when high copy number fd88-FLAG phage was similarly propagated, variant clones that did not display the FLAG epitope accumulated gradually. The loss of displayed peptide from the high copy number vector is undoubtedly slow enough to be overcome by even weak affinity selection, and high copy number vectors have important advantages that make their use worth considering, at least when the displayed peptides are small.