Reconstitution of the M13 major coat protein and its transmembrane peptide segment on a DNA template

The major coat protein (pVIII) of M13 phage is of particular interest to structure biologists since it functions in two different environments: during assembly and infection, it interacts with the bacterial lipid bilayer, but in the phage particle, it exists as a protein capsid to protect a closed circular, single-stranded DNA (ssDNA) genome. We synthesized pVIII and a 32mer peptide consisting of the transmembrane and DNA binding domains of pVIII. The 32mer peptide displays typically an alpha-helical structure in trifluroethanol or 0.2 M octylglucoside solutions similar to pVIII. Attachment of polyethylene glycol (PEG) onto the N-terminal of 32mer increased the alpha-helical content and the peptide thermal stability. The peptides were reconstituted with DNA from a detergent solution into a discrete (<200 nm diameter) nanoparticle on both linear double-stranded DNA (dsDNA) and linear ssDNA, where the linear dsDNA is used to mimic the closed circular, ssDNA in M13 phage, upon removal of the detergent. The peptide/DNA particle was an irregular and not a rod-shaped aggregate when imaged by atomic force microscopy. All three peptides underwent a structural transition from alpha-helix to beta-sheet within approximately 1 h of DNA addition to the detergent solution. There was a further decrease in alpha-helical content when the detergent was removed. The presence of anionic (such as octanoic acid) or cationic (such as 1,5-diaminopentane) molecules in the detergent mixture resulted in the retention of the peptide alpha-helical structure. Thus the interaction between the peptide and DNA in octylglucoside is driven by electrostatic forces, and peptide-peptide interactions are responsible for the transition from alpha-helix to beta-sheet conformation in pVIII and its analogues. These results suggest that the assembly process to form a rod-shaped phage is a delicate balance to maintain pVIII in an alpha-helical conformation that requires either an oriented bilayer to solubilize pVIII prior to interaction with the DNA or other phage proteins to nucleate pVIII in the alpha-helical conformation on the DNA.     

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.     

Bottom-up assembly of hydrogels from bacteriophage and Au nanoparticles: the effect of cis- and trans-acting factors

Hydrogels have become a promising research focus because of their potential for biomedical application. Here we explore the long-range, electrostatic interactions by following the effect of trans-acting (pH) and cis-acting factors (peptide mutation) on the formation of Au-phage hydrogels. These bioinorganic hydrogels can be generated from the bottom-up assembly of Au nanoparticles (Au NP) with either native or mutant bacteriophage (phage) through electrostatic interaction of the phage pVIII major capsid proteins (pVIII). The cis-acting factor consists of a peptide extension displayed on the pVIII that mutates the phage. Our results show that pH can dictate the direct-assembly and stability of Au-phage hydrogels in spite of the differences between the native and the mutant pVIII. The first step in characterizing the interactions of Au NP with phage was to generate a molecular model that identified the charge distribution and structure of the native and mutant pVIII. This model indicated that the mutant peptide extension carried a higher positive charge relative to the native pVIII at all pHs. Next, by monitoring the Au-phage interaction by means of optical microscopy, elastic light scattering, fractal dimension analysis as well as Uv-vis and surface plasmon resonance spectroscopy, we show that the positive charge of the mutant peptide extension favors the opposite charge affinity between the phage and Au NP as the pH is decreased. These results show the versatility of this assembly method, where the stability of these hydrogels can be achieved by either adjusting the pH or by changing the composition of the phage pVIII without the need of phage display libraries.     

The TolQRA Proteins Are Required for Membrane Insertion of the Major Capsid Protein of the Filamentous Phage f1 during Infection

Infection of Escherichia coli by the filamentous bacteriophage f1 is initiated by interaction of the end of the phage particle containing the gene III protein with the tip of the F conjugative pilus. This is followed by the translocation of the phage DNA into the cytoplasm and the insertion of the major phage capsid protein, pVIII, into the cytoplasmic membrane. DNA transfer requires the chromosomally encoded TolA, TolQ, and TolR cytoplasmic membrane proteins. By using radiolabeled phages, it can be shown that no pVIII is inserted into the cytoplasmic membrane when the bacteria contain null mutations in tolQ, -R and -A. The rate of infection can be varied by using bacteria expressing various mutant TolA proteins. Analysis of the infection process in these strains demonstrates a direct correlation between the rate of infection and the incorporation of infecting bacteriophage pVIII into the cytoplasmic membrane.     

Cobalt ion mediated self-assembly of genetically engineered bacteriophage for biomimetic Co-Pt hybrid material

Biological scaffolds are used for the synthesis of inorganic materials due to their ability to self-assemble and nucleate crystal formation. We report the self-assembly of engineered M13 bacteriophage as a template for Co-Pt crystals. A M13 phage library with an octapeptide library on the major coat protein (pVIII) was used for selection of binders to cobalt ions. Fibrous structures with directionally ordered M13 phage were obtained by interaction with cobalt ions. Co-Pt alloys were synthesized on the fibrous scaffold, and their magnetic properties were characterized. The mineralization showed organized nanoparticles on fibrous bundles. This approach using the phage pVIII library allows for genetic selection that both induces assembly of the phage and directs mineralization of the selected inorganic material.     

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.     

Engineering M13 for phage display

Phage display is achieved by fusing polypeptide libraries to phage coat proteins. The resulting phage particles display the polypeptides on their surfaces and they also contain the encoding DNA. Library members with particular functions can be isolated with simple selections and polypeptide sequences can be decoded from the encapsulated DNA. The technology's success depends on the efficiency with which polypeptides can be displayed on the phage surface, and significant progress has been made in engineering M13 bacteriophage coat proteins as improved phage display platforms. Functional display has been achieved with all five M13 coat proteins, with both N- and C-terminal fusions. Also, coat protein mutants have been designed and selected to improve the efficiency of heterologous protein display, and in the extreme case, completely artificial coat proteins have been evolved specifically as display platforms. These studies demonstrate that the M13 phage coat is extremely malleable, and this property can be used to engineer the phage particle specifically for phage display. These improvements expand the utility of phage display as a powerful tool in modern biotechnology.     

Identification of immunogenic polypeptides from a <Emphasis Type="Italic">Mycoplasma hyopneumoniae</Emphasis> genome library by phage display

The identification of immunogenic polypeptides of pathogens is helpful for the development of diagnostic assays and therapeutic applications like vaccines. Routinely, these proteins are identified by two-dimensional polyacrylamide gel electrophoresis and Western blot using convalescent serum, followed by mass spectrometry. This technology, however, is limited, because low or differentially expressed proteins, e.g. dependent on pathogen-host interaction, cannot be identified. In this work, we developed and improved a M13 genomic phage display-based method for the selection of immunogenic polypeptides of Mycoplasma hyopneumoniae, a pathogen causing porcine enzootic pneumonia. The fragmented genome of M. hyopneumoniae was cloned into a phage display vector, and the genomic library was packaged using the helperphage Hyperphage to enrich open reading frames (ORFs). Afterwards, the phage display library was screened by panning using convalescent serum. The analysis of individual phage clones resulted in the identification of five genes encoding immunogenic proteins, only two of which had been previously identified and described as immunogenic. This M13 genomic phage display, directly combining ORF enrichment and the presentation of the corresponding polypeptide on the phage surface, complements proteome-based methods for the identification of immunogenic polypeptides and is particularly well suited for the use in mycoplasma species.     

Design and evolution of artificial M13 coat proteins

Using simple design and selective pressure, we have evolved an artificial M13 bacteriophage coat protein. M13 coat proteins first reside in the bacterial inner membrane and subsequently surround the DNA core of the assembled virus. The artificial coat protein (ACP) was designed and evolved to mimic both functions of the natural M13 coat proteins, but with an inverted orientation. ACP is a non-functional coat protein because it is not required for the production of phage particles. Instead, it incorporates into a phage coat which still requires all the natural coat proteins for structural integrity. In contrast with other M13 coat proteins, which can display polypeptides as amino-terminal fusions, ACP permits the carboxy-terminal display of large polypeptides. The results suggest that viruses can co-opt host membrane proteins to acquire new coat proteins and thus new functions. In particular, M13 bacteriophage can be engineered for new functions, such as carboxy-terminal phage display.     

Use of bioinformatics to predict a function for the GS element in Mycobacterium avium subspecies paratuberculosis

Filamentous bacteriophages are nonlytic, male-specific bacteriophages which infect Escherichia coli carrying an F-episome. The molecular mechanism of infection remains elusive, including the role of the major coat protein pVIII. In order to evaluate the contributions of major coat protein pVIII in the process of infection, two phage display libraries were generated. One library consisted of random amino acids at positions 2, 4, 5, 8, 11 and 12 of the N-terminus of major coat protein pVIII. The second library was generated by randomizing these positions as well as position 1. All these residues were previously shown to be exposed at the surface of the virions by being accessible to ligands. The infectivity of randomly selected mutant phages was analyzed. The present results demonstrate that phages modified at these positions can be correctly assembled and secreted into the exoplasm, although the efficiency was slightly lower than that of wild-type phage. Their infectivity varied greatly, and a general structural pattern underlying infectivity did not emerge. However, residual differences were observed between infectious and defective phage; in general, uncharged polar amino acids present at positions 5 and 11 of the N-terminus of pVIII reduced phage infectivity, whereas polar residues at position 8 facilitated infection. The first position of pVIII is remarkably critical for infection; when this alanine was substituted with other residues, most of the phages lost their infectivity. These results shed new light on the true complexity of random peptide pVIII phage display libraries.     

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.     

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.     

Protection against Influenza A Virus Challenge with M2e-Displaying Filamentous Escherichia coli Phages

Human influenza viruses are responsible for annual epidemics and occasional pandemics that cause severe illness and mortality in all age groups worldwide. Matrix protein 2 (M2) of influenza A virus is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain of M2 (M2e) is conserved in human and avian influenza A viruses and is being pursued as a component for a universal influenza A vaccine. To develop a M2e vaccine that is economical and easy to purify, we genetically fused M2e amino acids 2–16 to the N-terminus of pVIII, the major coat protein of filamentous bacteriophage f88. We show that the resulting recombinant f88−M2e2-16 phages are replication competent and display the introduced part of M2e on the phage surface. Immunization of mice with purified f88−M2e2-16 phages in the presence of incomplete Freund’s adjuvant, induced robust M2e-specific serum IgG and protected BALB/c mice against challenge with human and avian influenza A viruses. Thus, replication competent filamentous bacteriophages can be used as efficient and economical carriers to display conserved B cell epitopes of influenza A.     

A comprehensive analysis of filamentous phage display vectors for cytoplasmic proteins: an analysis with different fluorescent proteins

Filamentous phage display has been extensively used to select proteins with binding properties of specific interest. Although many different display platforms using filamentous phage have been described, no comprehensive comparison of their abilities to display similar proteins has been conducted. This is particularly important for the display of cytoplasmic proteins, which are often poorly displayed with standard filamentous phage vectors. In this article, we have analyzed the ability of filamentous phage to display a stable form of green fluorescent protein and modified variants in nine different display vectors, a number of which have been previously proposed as being suitable for cytoplasmic protein display. Correct folding and display were assessed by phagemid particle fluorescence, and with anti-GFP antibodies. The poor correlation between phagemid particle fluorescence and recognition of GFP by antibodies, indicates that proteins may fold correctly without being accessible for display. The best vector used a twin arginine transporter leader to transport the displayed protein to the periplasm, and a coil-coil arrangement to link the displayed protein to g3p. This vector was able to display less robust forms of GFP, including ones with inserted epitopes, as well as fluorescent proteins of the Azami green series. It was also functional in mock selection experiments.     

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.     

Functional selection of phage displayed peptides for facilitated design of fusion tags improving aqueous two-phase partitioning of recombinant proteins.

Aqueous two-phase systems allow for the unequal distribution of proteins and other molecules in water-rich solutions containing phase separating polymers or surfactants. One approach to improve the partitioning properties of recombinant proteins is to produce the proteins as fused to certain peptide tags. However, the rational design of such tags has proven difficult since it involves a compromise between multivariate parameters such as partitioning properties, solvent accessibility and production/secretion efficiency. In this work, a novel approach for the identification of suitable peptide tag extensions has been investigated. Using the principles of selection, rather than design, peptide sequences contributing to an improved partitioning have been identified using phage display technology. A 40 million member phagemid library of random nona-peptides, displayed as fusion to the major coat protein pVIII of the filamentous phage M13, was employed in the selection of top-phase partitioning phage particles in a PEG/sodium phosphate system. After multiple cycles of selection by partitioning, peptides with high frequencies of both tyrosine and proline residues were found to be over represented in selected clones. The identified peptide sequences, or derivatives thereof, were subsequently individually analyzed for their partitioning behavior as displayed on phage, as free synthetic peptides and as genetically fused to a recombinant model target protein. The results showed that novel peptide sequences capable of enhancing top-phase partitioning without interfering with protein production and secretion indeed could be identified for the aqueous two-phase system investigated.     

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.     

Structure of virions of the M13 phage containing chimeric B-protein molecules]

We have studied the virion structure of M13 strains (M13B1, M13BOM1, M13BOM2, M13BOL1) with chimeric variants of B-protein. Data concerning the spatial structure of chimeric B-protein molecules and their interaction with intraphage DNA were obtained. The phage contour lengths were measured under electron microscope and the DNA/protein ratios were obtained by spectrophotometry. These data testified that the insertion of foreign peptide affected neither DNA packaging nor the compactness of molecular arrangement of proteins in the virion. By linear dichroism and fluorescence spectra of phages it was determined, that the insert can influence the polarity of amino acid environment and the orientation of amino acids in the B-protein central part. It was shown by quenching of phage fluorescence by KI that the inward or outward amino acids location in the capsid is invariable. The carboxyl residues have been titrated in the phage strains by Auramine O. It was shown that there is no correlation between the number of the titrated carboxyl groups and the number of the carboxyl groups as a whole.     

Use of the arabinose p(bad) promoter for tightly regulated display of proteins on bacteriophage

Phage display is a widely used method to optimize the binding characteristics of protein-ligand interactions. In addition, it has been used to clone genes from genomic and cDNA libraries based on their ligand-binding characteristics. One difficulty often encountered when expressing heterologous proteins by phage display is the toxicity of the protein on the Escherichia coli host. Previous studies have shown that heterologous protein expression can be tightly controlled using plasmids with the P(BAD) promoter of the arabinose operon of E. coli, and the araC gene, which is both a positive and negative regulator of the promoter. We constructed a set of phage display vectors that utilize the P(BAD) promoter to control the expression of proteins on the surface of the M13 bacteriophage. These vectors exhibit tightly controlled expression of proteins on the surface of the phage. In addition, the amount of protein displayed on the phage is modulated by the amount of arabinose present in the growth medium during phage propagation. This may be useful for altering the stringency of binding enrichment during phage display.