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Bacteriophages as Scaffolds for Bipartite Display: Designing Swiss Army Knives on a Nanoscale
Abstract
Bacteriophages have been exploited as cloning vectors and display vehicles for decades owing to their genetic and structural simplicity. In bipartite display setting, phage takes on the role of a handle to which two modules are attached, each endowing it with specific functionality, much like the Swiss army knife. This concept offers unprecedented potential for phage applications in nanobiotechnology. Here, we compare common phage display platforms and discuss approaches to simultaneously append two or more different (poly)peptides or synthetic compounds to phage coat using genetic fusions, chemical or enzymatic conjugations, and in vitro non-covalent decoration techniques. We also review current reports on design of phage frameworks to link multiple effectors, and their use in diverse scientific disciplines. Bipartite phage display had left its mark in development of biosensors, vaccines, and targeted delivery vehicles. Furthermore, multi-functionalized phages have been utilized to template assembly of inorganic materials and protein complexes, showing promise as scaffolds in material sciences and structural biology, respectively.
... Self-assembling structures open up exciting opportunities for the development of various tools, including biosensors, energy storage devices, drug delivery systems and nanobiopolymeric scaffolds [4][5][6][7]. Therefore, it is not surprising that viruses, including bacteriophages, have been used for the preparation of nanoscaled materials [8][9][10][11][12][13][14][15]. ...
... Thus, the results of this study suggest novel ways to construct hybrid self-assembling nanostructures, particularly ones appropriately prearranged on surfaces. It has been demonstrated that self-assembling tubular nanostructures made of peptides, proteins or filamentous bacteriophages have been used for both fundamental studies and practical applications, including in the construction of biosensors, energy storage devices, drug delivery systems or tissue engineering [12][13][14]75,76]. Given the fact that the polysheaths, formed of the wild-type gp053 or its mutants, possess physicochemical properties which are very similar to the properties of self-assembling nanostructures formed of biomolecules mentioned above (well-defined shape and dimensions in the nanoscale, robust, ordered and intrinsically monodisperse structures), it is likely that gp053 polysheaths can be used for the same practical applications as tubular nanostructures made of peptides, proteins or filamentous bacteriophages. ...
The recombinant phage tail sheath protein, gp053, from Escherichia coli infecting myovirus vB_EcoM_FV3 (FV3) was able to self-assemble into long, ordered and extremely stable tubular structures (polysheaths) in the absence of other viral proteins. TEM observations revealed that those protein nanotubes varied in length (~10–1000 nm). Meanwhile, the width of the polysheaths (~28 nm) corresponded to the width of the contracted tail sheath of phage FV3. The formed protein nanotubes could withstand various extreme treatments including heating up to 100 °C and high concentrations of urea. To determine the shortest variant of gp053 capable of forming protein nanotubes, a set of N- or/and C-truncated as well as poly-His-tagged variants of gp053 were constructed. The TEM analysis of these mutants showed that up to 25 and 100 amino acid residues could be removed from the N and C termini, respectively, without disturbing the process of self-assembly. In addition, two to six copies of the gp053 encoding gene were fused into one open reading frame. All the constructed oligomers of gp053 self-assembled in vitro forming structures of different regularity. By using the modification of cysteines with biotin, the polysheaths were tested for exposed thiol groups. Polysheaths formed by the wild-type gp053 or its mutants possess physicochemical properties, which are very attractive for the construction of self-assembling nanostructures with potential applications in different fields of nanosciences.
... Whole phages can be used as precision antimicrobials (Wittebole, De Roock, & Opal, 2013) and biosensors for pathogen detection in food and the environment (Singh, Poshtiban, & Evoy, 2013). Phage display of peptides or other conjugates on their capsids has enabled targeted drug delivery, vaccine development, and affinity screening of random peptides (Molek & Bratkovič, 2015). Phages have even been used as scaffolds to build nanoscale devices (Molek & Bratkovič, 2015). ...
... Phage display of peptides or other conjugates on their capsids has enabled targeted drug delivery, vaccine development, and affinity screening of random peptides (Molek & Bratkovič, 2015). Phages have even been used as scaffolds to build nanoscale devices (Molek & Bratkovič, 2015). Furthermore, phage-derived enzymes, such as T4 DNA ligase and T7 RNA polymerase, have been used for decades as molecular tools and continue to benefit molecular genetics research. ...
Phages are the most abundant entities in the biosphere and profoundly impact the bacterial populations within and around us. They attach to a specific host, inject their DNA, hijack the host's cellular processes, and replicate exponentially while destroying the host. Historically, phages have been exploited as powerful antimicrobials, and phage-derived proteins have constituted the basis for numerous biotechnological applications. Only in recent years have metagenomic studies revealed that phage genomes harbor a rich reservoir of genetic diversity, which might afford further therapeutic and/or biotechnological value. Nevertheless, functions for the majority of phage genes remain unknown, and due to their swift and destructive replication cycle, many phages are intractable by current genetic engineering techniques. Whether to advance the basic understanding of phage biology or to tap into their potential applications, efficient methods for phage genetic engineering are needed. Recent reports have shown that CRISPR–Cas systems, a class of prokaryotic immune systems that protect against phage infection, can be harnessed to engineer diverse phages. In this chapter, we describe methods to genetically manipulate virulent phages using CRISPR–Cas10, a Type III-A CRISPR–Cas system native to Staphylococcus epidermidis. A method for engineering phages that infect a CRISPR-less Staphylococcus aureus host is also described. Both approaches have proved successful in isolating desired phage mutants with 100% efficiency, demonstrating that CRISPR–Cas10 constitutes a powerful tool for phage genetic engineering. The relatively widespread presence of Type III CRISPR–Cas systems in bacteria and archaea imply that similar strategies may be used to manipulate the genomes of diverse prokaryotic viruses.
... Therefore, the M13 bacteriophage (M13 phage) carrying a mimic antigen epitope and multiple enzymes was proposed as a promising surrogate for competing antigens. The filamentous M13 is noninfectious to humans and composed of 2700 copies of the major p8 proteins and 3-5 copies of minor p3, p6, p7, and p9 proteins at the two ends [32]. It can be easily modified to mimic various target analytes, such as small molecules or proteins, by integrating mimotopes at the N-terminal of p3 proteins through a gene modification-fusion expression process [33]. ...
“Point of care” (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL−1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL−1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.
... M13 bacteriophage (M13), a filamentous virus with 2700 identical copies of the major p8 protein and 3-5 copies of minor p3, p6, p7, and p9 proteins at both ends, is an attractive biological material that can be a solution for the above problems [21]. Through a gene modification-fusion expression process, mimotopes can be integrated on the N-terminals of p3 proteins; this process endows an M13 phage with mimicking ability for target antigen [22][23][24]. ...
Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.
... For example, M13 bacteriophage (M13 phage) displaying a mimic antigen epitope and loaded with enzymes has been regarded as a promising alternative competing antigen. It is a 900-nanometer-long and 6.5-nanometer-thick filamentous virus [22] that is noninfectious to humans and composed of 2700 copies of the major p8 protein and 3-5 copies of minor p3, p6, p7 and p9 proteins at both ends [23,24]. It can be easily modified so that it can mimic small molecules or protein compounds by integrating mimotopes or ligands on the N-terminal of p3 protein via a gene modificationfusion expression process [25,26]. ...
Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.
... However, the sensitivity of the above nanozyme-based immunosensor is only slightly superior to the conventional HRP-based ELISA, and the usage of nanozyme-based immunosensor for analyses with trace concentration still faces numerous challenges. M13 bacteriophage (M13) is a filamentous virus with a length and diameter of 900 and 6.5 nm, respectively [16]. M13 consists of 2700 identical copies of the major p8 protein and 3-5 copies of minor p3, p6, p7, and p9 proteins at both ends of the virus [17]. ...
Herein, a sulfydryl modification of M13 bacteriophage was introduced as bio-functional component including biological recognition, and nanozyme container for enhancing sensitivity of colorimetric immunsensor, where the seven peptide sequence fused on the p3 protein of M13 was used to mimic deoxynivalenol (DON) antigen for recognizing the anti-DON monoclonal antibody, while the sulfydryl groups modified on capsid proteins were used to load numerous Ag coated Au nanoparticles ([email protected]) for improving the peroxidase-like activity of [email protected] Owing its great loading capacity, the M13 bacteriophage assembled [email protected] nanocomposites (M13DON@[email protected]) showed approximately 48- and 105-fold enhanced catalytic efficacy to hydrogen peroxide and tramethylbenzidine than those of natural horse radish peroxidase (HRP). Using the M13DON@[email protected] as signal amplifier, the proposed immunosensor exhibits a very high sensitivity for DON detection with the 50% competitive inhibition concentration (IC50) and detection limit (LOD) of 2.03 ng/mL and 13.67 pg/mL, respectively. These values are about 26- and 947-fold lower than those of conventional HRP based ELISA method (IC50 and LOD values =52.43 ng/mL and 12.95 ng/mL, respectively). In addition, the proposed method also showed good specificity and accepted accuracy for DON detection in real corn samples. Moreover, the reliability of this novel strategy was further confirmed through compared with the high performance liquid chromatography method (HPLC). All in all, the M13 bacteriophage exhibits a promising potential as nanozyme container for enhancing the sensitivity of immunosensor, and this novel signal amplification system can be easily extended for highly sensitive detection of other analytes by altering specific mimic peptide sequence.
... Bacteriophages and phage display have been widely used and exploited in cancer research, neurosciences, regenerative medicine, microbiology, infections, and other medical fields, contributing to the discovery and generation of a wide range of therapeutic peptide and protein ligands with therapeutic, diagnostic, and imaging applications. Their biomedical applications range from the development of probes for sensing, imaging, diagnosis, and detection, the development of gene and therapeutic drug carriers for targeted delivery, the development of new vaccine vectors, and also the development of new biomaterials as supramolecular building blocks ( Fig. 9.2) (Farr et al., 2014;Ferreira and Martins, 2016;Henry et al., 2015;Kelly et al., 2006;Li et al., 2010;Martins et al., 2016;Molek and Bratkovic, 2015;Omidfar and Daneshpour, 2015;Tan et al., 2016;Wu et al., 2016). ...
... Such viral rod-like particles can be seen both as elementary building blocks monodisperse in size and shape, and as versatile scaffolds for biological engineering and chemical functionalization of their coat proteins. [30][31][32] Prior studies have reported the use of bacteriophages, mainly modified by phage display, that self-assemble into chains of various length, 25,33 rings 34 or radial structures. 9,10 These works are based on the genetic engineering of viruses with mutated tip proteins (minor proteins p3 and p9, corresponding respectively to the two extremities of the phage) onto which specific polypeptidic sequences are fused. ...
We report on the construction of multiarm colloidal molecules by tip-linking filamentous bacteriophages, functionalized either by biological engineering or chemical conjugation. The affinity for streptavidin of a genetically modified vector phage displaying Strep-tags fused to one end of the viral particle is measured by determining the dissociation constant, Kd. In order to improve both the colloidal stability and the efficiency of the self-assembly process, a biotinylation protocol having a chemical yield higher than 90% is presented to regioselectively functionalize the cystein residues located at one end of the bacteriophages. For both viral systems, a theoretical comparison is performed by developing a quantitative model of the self-assembly and interaction of the modified viruses with streptavidin compounds, which accurately accounts for our experimental results. Multiarm colloidal structures of different valencies are then produced by conjugation of these tip-functionalized viruses with streptavidin activated nanoparticles. We succeed to form stable virus based colloidal molecules, whose number of arms, called valency, is solely controlled by tuning the molar excess. Thanks to a fluorescent labeling of the viral arms, the dynamics of such systems is also presented in real time by fluorescence microscopy.
... Peptides [2][3][4][5], proteins [6,7] and nucleotides [8][9][10] serve as a scaffold material for self-assembling nanostructures. A more complex type of self-assembling structures is virus-based nanoparticles (VNPs) [11][12][13][14][15][16]. The icosahedral nanostructures have been the most extensively produced and studied [17][18][19]. ...
Nucleotides, peptides and proteins serve as a scaffold material for self-assembling nanostructures. In this study, the production of siphovirus vB_EcoS_NBD2 (NBD2) recombinant tail tube protein gp39 reached approximately 33% and 27% of the total cell protein level in Escherichia coli and Saccharomyces cerevisiae expression systems, respectively. A simple purification protocol allowed us to produce a recombinant gp39 protein with 85%–90% purity. The yield of gp39 was 2.9 ± 0.36 mg/g of wet E. coli cells and0.85 ± 0.33 mg/g for S. cerevisiae cells. The recombinant gp39 self-assembled into well-ordered tubular structures (polytubes) in vivo in the absence of other phage proteins. The diameter of these structures was the same as the diameter of the tail of phage NBD2 (~12 nm). The length of these structures varied from 0.1 µm to >3.95 µm, which is 23-fold the normal NBD2 tail length. Stability analysis demonstrated that the polytubes could withstand various chemical and physical conditions. These polytubes show the potential to be used as a nanomaterial in various fields of science.
... 102 Detailed description of different display technologies is beyond the scope of this review; the reader is referred to recent reviews. 103,104 Such approaches led to discovery of several small peptide Fc ligands (listed in Table 3), with the potential to replace Protein A in various applications. 105 Dendrimeric protein A mimetic peptide, or PAM for short (also termed TG19318), is a tripeptide tetramer with chemical formula (Arg−Thr−Tyr) 4 −Lys 2 −Lys−Gly ( Figure 4A). ...
The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. Especially the development of better affinity chromatography matrices, supporting robust, time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.
... The M13 phage is the Ff phage most widely used in phage display experiments where, by DNA engineering, a peptide or protein can be site-specifically displayed at the tip and/or along the side wall of the phage through fusion, leading to the display of a peptide or protein on the surface of a virus particle. The foreign gene sequence is spliced between genes encoding a phage signal peptide and a portion of the coat protein, which ensures that the foreign protein is produced as a fusion with the coat protein (Molek and Bratkovic, 2015) Despite being successfully employed to identify peptide ligands for a wide variety of targets, which can often be accessed free of charge in online databases (Huang et al., 2011(Huang et al., , 2012, phage display has recently broadened its application, by taking advantage of the surface display and self-assembly abilities of the phage particles, for the development of (i) biologically active molecules, (ii) probes for sensing and imaging, and (iii) target drug and gene delivery vehicles (Petrenko, 2008;Farr et al., 2014). Further details are described in the following sections. ...
This chapter intends to provide an overview of the use of viruses for medical applications. The first part describes the basic characteristics of viral particles regarding their chemical composition and size, as well as their structure and assembly/disassembly abilities. Next, a discussion about the newest and most important strategies for virus particles modification including genetic, chemical, and self-assembly/encapsulation engineering towards the development of new virus particles for biomedical applications, including targeted delivery and therapy, molecular imaging for disease detection, vaccine development, and bacterial infection control.
... This can either be achieved through combining modified genes, or through modified phage propagation protocols utilizing double virus infection, 12 as reviewed by Bratkovič and coworkers. 13 ...
Long fascinating to biologists, viruses offer nanometer-scale benchtops for building molecular-scale devices and materials. Viruses tolerate a wide-range of chemical modifications including reaction conditions, pH values, and temperatures. Recent examples of non-genetic manipulation of viral surfaces have extended viruses into applications ranging from biomedical imaging, drug delivery, tissue regeneration, and biosensors to materials for catalysis and energy generation. Chemical reactions on the phage surface include both covalent and non-covalent modifications, including some applied in conjunction with genetic modifications. Here, we survey viruses chemically augmented with capabilities limited only by imagination.
... In fact, several works have described the M13 phage as a multipurpose platform for performing organic synthesis and combinatorial chemistry on the nanoscale. 35,36 Thus, the thiolated phage can be successfully employed to produce mesophases decorated with metal NPs. Au and Pt NPs were located partially within the silica wall, leaving the pore free and becoming a rather promising catalyst. ...
By taking advantage of the physical and chemical properties of the M13 bacteriophage, we have used this virus to synthesize mesoporous silica structures. Major coat protein p8 was chemically modified by attaching thiol groups. As we show, the resulting thiolated phage can be used as a biotemplate able to direct the formation of mesoporous silica materials. Simultaneously, this thiol functionality acts as an anchor for binding metal ions, such as Au(3+) and Pt(4+), forming reactive M13-metal ionic complexes which evolve into metal nanoparticles (NPs) trapped in the mesoporous network. Interestingly, Au(3+) ions are reduced to Au(0) NPs by the protein residues without requiring an external reducing agent. Likewise, silica mesostructures decorated with Au and Pt NPs are prepared in a one-pot synthesis and characterized using different techniques. The obtained results allow us to propose a mechanism of formation. In addition, gold-containing mesoporous structures are tested for the reduction of 4-nitrophenol (4-NP) and methylene blue (MB) in the presence of NaBH4. Although all of the gold-containing catalysts exhibit catalytic activity, those obtained with thiolated phages present a better performance than that obtained with M13 alone. This behavior is ascribed to the position of the Au NPs, which are partially embedded in the wall of the final mesostructures.
Phage-nanomaterial conjugates are functional bio-nanofibers with various applications. While phage display can select for phages with desired genetically encoded functions and properties, nanomaterials can endow the phages with additional features at nanoscale dimensions. Therefore, combining phages with nanotechnology can construct bioconjugates with unique characteristics. One strategy for filamentous phages is to adsorb nanoparticles onto the side wall, composed of pVIII subunits, through electrostatic interactions. However, a noncovalent approach may cause offloading if the environment changes, potentially causing side effects especially for in vivo applications. Therefore, building stable phage-bioconjugates is an important need. We previously reported the construction of chimeric M13 phage conjugated with gold nanorods, named “phanorods,” without weakening the binding affinity to the bacterial host cells. Herein, we give a detailed protocol for preparing the chimeric M13 phage and covalently conjugating gold nanorods to the phage.
Viruses have recently emerged as promising nanomaterials for biotechnological applications. One of the most important applications of viruses is phage display, which has already been employed to identify a broad range of potential therapeutic peptides and antibodies, as well as other biotechnologically relevant polypeptides (including protease inhibitors, minimizing proteins, and cell/organ targeting peptides). Additionally, their high stability, easily modifiable surface, and enormous diversity in shape and size, distinguish viruses from synthetic nanocarriers used for drug delivery. Indeed, several plant and bacterial viruses (e.g., phages) have been investigated and applied as drug carriers. The ability to remove the genetic material within the capsids of some plant viruses and phages produces empty viral-like particles that are replication-deficient and can be loaded with therapeutic agents. This review summarizes the current applications of plant viruses and phages in drug discovery and as drug delivery systems and includes a discussion of the present status of virus-based materials in clinical research, alongside the observed challenges and opportunities.
Both direct and non‐competitive two‐site (sandwich) immunoassays are reported for 3‐phenoxybenzoic acid (3‐PBA) utilizing signal transduction by electrochemical impedance spectroscopy (EIS), and the sandwich immunoassay reduces the detection limit for this small molecule analyte by ∼70×. The direct EIS immunoassay utilizes a polyclonal antibody to 3PBA for biomolecular recognition, while the sandwich EIS immunoassay utilizes in addition a previously reported antiimmunocomplex M13 bacteriophage clone. For both immunoassays, the polyclonal antibody film is immobilized atop an Au electrode by amide bond formation. The direct EIS immunoassay exhibits a 3‐PBA sensitivity of 5.4×10⁴ kΩ cm² M¹, and a detection limit of 2.5x10⁻⁴ M (54 μg/ml), while the sandwich EIS immunoassay exhibits a 3PBA sensitivity of 4.2×10⁶ kΩ cm² M⁻¹, and a detection limit of 3.4×10⁻⁶ M (0.74 μg/ml). For the sandwich EIS immunoassay, a constant excess (80×10⁹ PFU/ml) of bacteriophage is maintained during all experiments. This is the first report of a bacteriophage‐assisted sandwich EIS assay.
La grande majorité des bactériophages connus ont un virion équipé d'une queue permettant la reconnaissance de l'hôte, la perforation de l'enveloppe bactérienne et l'éjection du matériel génétique viral directement dans le cytoplasme de la bactérie. La famille des Siphoviridae représente 60% des phages caudés et est caractérisée par une queue longue et non-contractile. Le tube de la queue est formé par un empilement de protéine majeur de tube (TTP) polymérisé autour de la protéine vernier (TMP). L'extrémité distale de la queue est équipé d'un complexe de protéine dans lequel se trouve les protéines de liaison au récepteur (RBP). La séquence d'évènement permettant l'éjection de l'ADN et l'infection est encore mal décrite.Au cours de cette thèse, la structure de pb6, la TTP du phage T5 qui s'assemble de façon non-canonique en trimères, a été résolue par cristallographie à une résolution de 2,2 Å. L'analyse de cette structure confirme cependant une homologie structurale de pb6 avec les autres TTPs et avec des protéines bactériennes du système de sécrétion de type VI et des pyocines R. Une étude RMN comparant pb6 dans ses états de monomère et de tube polymérisé est en cours et permettra à terme une description très fine de cet assemblage.De plus, les structures du complexe distal de queue et du tube de la queue (tube de pb6) ont été résolue des résolutions intermédiaires avant et après interaction avec le récepteur bactérien. Ces structures obtenues par cryo-microscopie électronique révèle une absence de changements structuraux au niveau du tube, en contradiction avec le modèle jusque là proposé que la TTP transmettait l'information de fixation du récepteur à la capside.Bien qu'à un stade préliminaire, les reconstructions du complexe distal sont très informatives sur les rôles de protéines pb2 et pb4.L'ensemble de ses données ainsi que des expériences biochimique et la comparaison avec d'autres systèmes bien décrits dans la littérature permet de proposer un nouveau modèle pour les premières étapes de l'infection des Siphoviridae. Ce modèle a également un intérêt pour l'étude du mécanisme d'autres familles de virus (Myoviridae). Les différences, similarité et parenté d'éléments de la queue du phage T5 avec d'autres systèmes de perforation de membrane sont discutés.
We report on the construction of multiarm colloidal molecules by tip-linking filamentous bacteriophages, functionalized either by biological engineering or chemical conjugation. The affinity for streptavidin of a genetically modified vector phage displaying Strep-tags fused to one end of the viral particle, is measured by determining the dissociation constant, Kd. In order to both improve the colloidal stability and the efficiency of the self-assembly process, a biotinylation protocol having a chemical yield higher than 90% is presented to regio-selectively functionalize the cystein residues located at one end of the bacteriophages. For both viral systems, a theoretical comparison is performed by developing a quantitative model of the self-assembly and interaction of the modified viruses with streptavidin compounds, which accurately accounts for our experimental results. Multiarm colloidal structures of different valencies are then produced by conjugation of these tip-functionalized viruses with streptavidin activated nanoparticles. We succeed to form stable virus based colloidal molecules, whose number of arms, called valency, is solely controlled by tuning the molar excess. Thanks to a fluorescent labeling of the viral arms, the dynamics of such systems is also presented in real time by fluorescence microscopy.
Analysis of molecular events in T4-infected Escherichia coli has revealed some of the most important principles of biology, including relationships between structures of genes and their products, virus-induced acquisition of metabolic function, and morphogenesis of complex structures through sequential gene product interaction rather than sequential gene activation. T4 bacteriophages and related strains were applied in the first formulations of many fundamental biological concepts. These include the unambiguous recognition of nucleic acids as the genetic material, the definition of the gene by fine-structure mutation, recombinational and functional analyses, the demonstration that the genetic code is triplet, the discovery of mRNA, the importance of recombination and DNA replications, light-dependent and light-independent DNA repair mechanisms, restriction and modification of DNA, self-splicing of intron/exon arrangement in prokaryotes, translation bypassing and others. Bacteriophage T4 possesses unique features that make it a good tool for a multicomponent vaccine platform. Hoc/Soc-fused antigens can be assembled on the T4 capsid in vitro and in vivo. T4-based phage display combined with affinity chromatography can be applied as a new method for bacteriophage purification. The T4 phage display system can also be used as an attractive approach for cancer therapy. The data show the efficient display of both single and multiple HIV antigens on the phage T4 capsid and offer insights for designing novel particulate HIV or other vaccines that have not been demonstrated by other vector systems.
M13 filamentous bacteriophage has been used in displaying disease-specific antibodies, biomarkers, and peptides. One of the major drawbacks of using phage in diagnostic assays is the aspecific adsorption of proteins leading to a high background signal and decreasing sensitivity. To deal with this, we developed a genetically pure, exchangeable dual-display phage system in which biomarkers and streptavidin-binding protein (SBP) are displayed at opposite ends of the phage. This approach allows for sample purification, using streptavidin-coated magnetic beads resulting in a higher sensitivity of signal detection assays. Our dual-display cassette system approach also allows for easy exchange of both the anchor protein (SBP) and the displayed biomarker. The presented principle is applied for the detection of antibody reactivity against UH-RA.21 which is a good candidate biomarker for rheumatoid arthritis (RA). The applicability of dual-display phage preparation using a helper plasmid system is demonstrated, and its increased sensitivity in phage ELISA assays using patient serum samples is shown.
Bacteriophage (phage) Lambda (λ) has played a key historic role in driving our understanding of molecular genetics. The lytic nature of λ and the conformation of its major capsid protein gpD in capsid assembly offer several advantages as a phage display candidate. The unique formation of the λ capsid and the potential to exploit gpD in the design of controlled phage decoration will benefit future applications of λ display where steric hindrance and avidity are of great concern. Here, we review the recent developments in phage display technologies with phage λ and explore some key applications of this technology including vaccine delivery, gene transfer, bio-detection, and bio-control.
Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.
Adeno-associated virus (AAV) is a member of the family Parvoviridae that has been widely used as a vector for gene therapy because of its safety profile, its ability to transduce both dividing and non-dividing cells, and its low immunogenicity. AAV has been detected in many different tissues of several animal species but has not been associated with any disease. As a result of natural infections, antibodies to AAV can be found in many animals including humans. It has been shown that pre-existing AAV antibodies can modulate the safety and efficacy of AAV vector-mediated gene therapy by blocking vector transduction or by redirecting distribution of AAV vectors to tissues other than the target organ. This review will summarize antibody responses against natural AAV infections, as well as AAV gene therapy vectors and their impact in the clinical development of AAV vectors for gene therapy. We will also review and discuss the various methods used for AAV antibody detection and strategies to overcome neutralizing antibodies in AAV-mediated gene therapy.
Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA.
In the present work we describe an original method for dual display of large proteins on the surface of lambda particles. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins.
Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.
Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.
Phage display screening allows the study of functional protein-protein interactions at the cell surface, but investigating intracellular organelles remains a challenge. Here we introduce internalizing-phage libraries to identify clones that enter mammalian cells through a receptor-independent mechanism and target-specific organelles as a tool to select ligand peptides and identify their intracellular receptors. We demonstrate that penetratin, an antennapedia-derived peptide, can be displayed on the phage envelope and mediate receptor-independent uptake of internalizing phage into cells. We also show that an internalizing-phage construct displaying an established mitochondria-specific localization signal targets mitochondria, and that an internalizing-phage random peptide library selects for peptide motifs that localize to different intracellular compartments. As a proof-of-concept, we demonstrate that one such peptide, if chemically fused to penetratin, is internalized receptor-independently, localizes to mitochondria, and promotes cell death. This combinatorial platform technology has potential applications in cell biology and drug development.
While the resistance of bacteria to traditional antibiotics is a major public health concern, the use of extremely potent antibacterial agents is limited by their lack of selectivity. As in cancer therapy, antibacterial targeted therapy could provide an opportunity to reintroduce toxic substances to the antibacterial arsenal. A desirable targeted antibacterial agent should combine binding specificity, a large drug payload per binding event, and a programmed drug release mechanism. Recently, we presented a novel application of filamentous bacteriophages as targeted drug carriers that could partially inhibit the growth of Staphylococcus aureus bacteria. This partial success was due to limitations of drug-loading capacity that resulted from the hydrophobicity of the drug. Here we present a novel drug conjugation chemistry which is based on connecting hydrophobic drugs to the phage via aminoglycoside antibiotics that serve as solubility-enhancing branched linkers. This new formulation allowed a significantly larger drug-carrying capacity of the phages, resulting in a drastic improvement in their performance as targeted drug-carrying nanoparticles. As an example for a potential systemic use for potent agents that are limited for topical use, we present antibody-targeted phage nanoparticles that carry a large payload of the hemolytic antibiotic chloramphenicol connected through the aminoglycoside neomycin. We demonstrate complete growth inhibition toward the pathogens Staphylococcus aureus, Streptococcus pyogenes, and Escherichia coli with an improvement in potency by a factor of approximately 20,000 compared to the free drug.
There is considerable interest in the use of bacteriophage vectors for mammalian cell gene transfer applications, due to their
stability, excellent safety profile and inexpensive mass production. However, to date, phage vectors have been plagued by
mediocre performance as gene transfer agents. This may reflect the complexity of the viral infection process in mammalian
cells and the need to refine each step of this process in order to arrive at an optimal, phage-based gene transfer system.
Therefore, a flexible system was designed that alowed for the introduction of multiple modifications on the surface of bacteriophage
lambda. Using this novel method, multiple peptides were displayed simultaneously from both the phage head and tail. Surface
head display of an ubiquitinylation motif greatly increased the efficiency of phage-mediated gene transfer in a murine macrophage
cell line. Gene transfer was further increased when this peptide was displayed in combination with a tail-displayed CD40-binding
motif. Overall, this work provides a novel system that can be used to rationally improve bacteriophage gene transfer vectors
and shows it may be possible to enhance the efficiency of phage-mediated gene transfer by targeting and optimizing multiple
steps within the viral infection pathway.
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.
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, HIS(6) 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.
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.
Phage display is a powerful technique that enables easy identification of targets for any type of ligand. Targets are displayed at the phage surface as a fusion protein to one of the phage coat proteins. By means of a repeated process of affinity selection on a ligand, specific enrichment of displayed targets will occur. In our studies using C-terminal display of cDNA fragments to phage coat protein p6, we noticed the occasional enrichment of targets that do not contain an open reading frame. This event has previously been described in other phage display studies using N-terminal display of targets to phage coat proteins and was due to uncommon translational events like frameshifting. The aim of this study was to examine if C-terminal display of targets to p6 is also subjected to frameshifting. To this end, an enriched target not containing an open reading frame was selected and an E-tag was coupled at the C-terminus in order to measure target display at the surface of the phage. The tagged construct was subsequently expressed in 3 different reading frames and display of both target and E-tag measured to detect the occurrence of frameshifting. As a result, we were able to demonstrate display of the target both in the 0 and in the +1 reading frame indicating that frameshifting can also take place when C-terminal fusion to minor coat protein p6 is applied.
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.
Viral Battery
In developing materials for batteries, there is a trade-off between charge capacity, conductivity, and chemical stability. Nanostructured materials improve the conductivity for some resistive materials, but fabricating stable materials at nanometer-length scales is difficult. Harnessing their knowledge of viruses as toolkits for materials fabrication, Lee et al. (p. 1051; published online 2 April) modified two genes in the filamentous bacteriophage M13 to produce a virus with an affinity for nucleating amorphous iron phosphate along its length and for attaching carbon nanotubes at one of the ends. In nanostructured form, the amorphous iron phosphate produced a useful cathode material, while the carbon nanotubes formed a percolating network that significantly enhanced conductivity.
Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.
History Bacteriophages T2,T4, and T6 were among seven Escherichia coli phages selected by Max Delbrück to study fundamentals of viral replication in a limited number of model viruses. These studies led to the first formulation of many concepts that are now accepted foundations of molecular biology: the fundamental differences between growth of viruses and cells (figure 18-1) (109); the demonstration that nucleic acids of virus particles suffice to establish infection and to direct synthesis of complete virions (163); the concept of the gene, including distinctions between units of recombination, mutation, and function (30); genetic recombination as exchange between DNA molecules involving ‘‘heterozygous’’ overlaps (91, 164, 165); the demonstration of messenger RNA (mRNA) (55, 414) and the non-overlapping triplet code (83, 390) with nonsense triplets as termination signals (31); the repair of DNA damage in the light (103) and in the dark (149); restriction and modification of DNA (247); the presence of spliced and nonspliced introns in prokaryotes (25, 171); the definition of pathways leading to the assembly of complex macromolecular structures (105); and the importance of protein complexes (machines), which change composition during various DNA transactions (9, 275).
Extensive research has been directed toward the development of multipurpose lambda vectors for cloning ever since the potential of using coliphage lambda as a cloning vector was recognized in the late 1970s. An understanding of the intrinsic molecular organization and of the genetic events which determine lysis or lysogeny in lambda has allowed investigators to modify it to suit the specific requirements of gene manipulations. Unwanted restriction sites have been altered and arranged together into suitable polylinkers. The development of a highly efficient in vitro packaging system has permitted the introduction of chimeric molecules into hosts. Biological containment of recombinants has been achieved by introducing amber mutations into the lambda genome and by using specific amber suppressor hosts. Taking advantage of the limited range of genome size (78 to 105% of the wild-type size) for its efficient packaging, an array of vectors has been devised to accommodate inserts of a wide size range, the limit being 24 kbp in Charon 40. The central dispensable fragment of the lambda genome can be replaced by a fragment of heterologous DNA, leading to the construction of replacement vectors such as Charon and EMBL. Alternatively, small DNA fragments can be inserted without removing the dispensable region of the lambda genome, as in lambda gt10 and lambda gt11 vectors. In addition, the introduction of many other desirable properties, such as NotI and SfiI sites in polylinkers (e.g., lambda gt22), T7 and T3 promoters for the in vitro transcription (e.g., lambda DASH), and the mechanism for in vivo excision of the intact insert (e.g., lambda ZAP), has facilitated both cloning and subsequent analysis. In most cases, the recombinants can be differentiated from the parental phages by their altered phenotype. Libraries constructed in lambda vectors are screened easily with antibody or nucleic acid probes since several thousand clones can be plated on a single petri dish. Besides the availability of a wide range of lambda vectors, many related techniques such as rapid isolation of lambda DNA, a high efficiency of commercially available in vitro packaging extracts, and in vitro amplification of DNA via the polymerase chain reaction have collectively contributed to lambda's becoming one of the most powerful and popular tools for molecular cloning.
A family of homomultimeric outer-membrane proteins termed secretins mediates the secretion of large macromolecules such as enzymes and filamentous bacteriophages across bacterial outer membranes to the extracellular milieu. The secretin encoded by filamentous phage f1 was purified. Mass determination of individual molecules by scanning transmission electron microscopy revealed two forms, a unit multimer composed of about 14 subunits and a multimer dimer. The secretin is roughly cylindrical and has an internal diameter of about 80 angstroms, which is large enough to accommodate filamentous phage (diameter of 65 angstroms).
M13 bacteriophage display presents polypeptides as fusions to phage coat proteins. Such phage-displayed ligands offer useful reagents for biosensors. Here, we report a modified phage propagation protocol for the consistent and robust display of two different, genetically encoded ligands on the major coat protein, P8. The results demonstrate that the phage surface reaches a saturation point for maximum peptide display.
Filamentous bacteriophages are interesting paradigms in structural molecular biology, in part because of the unusual mechanism of filamentous phage assembly. During assembly, several thousand copies of an intracellular DNA-binding protein bind to each copy of the replicating phage DNA, and are then displaced by membrane-spanning phage coat proteins as the nascent phage is extruded through the bacterial plasma membrane. This complicated process takes place without killing the host bacterium. The bacteriophage is a semi-flexible worm-like nucleoprotein filament. The virion comprises a tube of several thousand identical major coat protein subunits around a core of single-stranded circular DNA. Each protein subunit is a polymer of about 50 amino-acid residues, largely arranged in an α-helix. The subunits assemble into a helical sheath, with each subunit oriented at a small angle to the virion axis and interdigitated with neighbouring subunits. A few copies of "minor" phage proteins necessary for infection and/or extrusion of the virion are located at each end of the completed virion. Here we review both the structure of the virion and aspects of its function, such as the way the virion enters the host, multiplies, and exits to prey on further hosts. In particular we focus on our understanding of the way the components of the virion come together during assembly at the membrane. We try to follow a basic rule of empirical science, that one should chose the simplest theoretical explanation for experiments, but be prepared to modify or even abandon this explanation as new experiments add more detail.
M13 bacteriophage has been used as a scaffold to organize materials for various applications. Building more complex multiphage devices requires precise control of interactions between the M13 capsid proteins. Toward this end, we engineered a loop structure onto the pIII capsid protein of M13 bacteriophage to enable sortase-mediated labeling reactions for C-terminal display. Combining this with N-terminal sortase-mediated labeling, we thus created a phage scaffold that can be labeled orthogonally on three capsid proteins: the body and both ends. We show that covalent attachment of different DNA oligonucleotides at the ends of the new phage structure enables formation of multiphage particles oriented in a specific order. These have potential as nanoscale scaffolds for multi-material devices.
M13 bacteriophage is a well-characterized platform for peptide display. The utility of the M13 display platform is derived from the ability to encode phage protein fusions with display peptides at the genomic level. However, the genome of the phage is complicated by overlaps of key genetic elements. These overlaps directly couple the coding sequence of one gene to the coding or regulatory sequence of another, making it difficult to alter one gene without disrupting the other. Specifically, overlap of the end of gene VII and the beginning of gene IX has prevented the functional genomic modification of the N-terminus of p9. By redesigning the M13 genome to physically separate these overlapping genetic elements, a process known as "refactoring," we enabled independent manipulation of gene VII and gene IX and the construction of the first N-terminal genomic modification of p9 for peptide display. We demonstrate the utility of this refactored genome by developing an M13 bacteriophage-based platform for targeted imaging of and drug delivery to prostate cancer cells in vitro. This successful use of refactoring principles to re-engineer a natural biological system strengthens the suggestion that natural genomes can be rationally designed for a number of applications.
The sensitive detection of cancer biomarkers in urine could revolutionize cancer diagnosis and treatment. Such detectors must be inexpensive, easy to interpret, and sensitive. This report describes a bioaffinity matrix of viruses integrated into PEDOT films for electrochemical sensing of prostate specific membrane antigen (PSMA), a prostate cancer biomarker. High sensitivity to PSMA resulted from synergistic action by two different ligands to PSMA on the same phage particle. One ligand was chemically synthesized and wrapped around the phage, and the second was genetically encoded. The dual ligands result in a bidentate binder with high copy, dense ligand display for enhanced PSMA detection through a chelate-based, avidity effect. Biosensing with virus-PEDOT films provides a 100 pM limit of detection for PSMA in synthetic urine without requiring enzymatic or other amplification.
Application of nano-particles to diagnostic fields has attracted much attention. Biotechnology can contribute to produce useful nano-materials by engineering bacteriophage nano-particles, which are easily prepared by infecting phages to bacterial host cells. In this study, establishment of nano-bioprobes was demonstrated, based on the T7 phage display system, by constructing phage particles displaying a ligand polypeptide S-tag and a green fluorescent protein (GFP) at the same time on the surface of phage head. To achieve this purpose, two types of phage particles were tested: One displayed S-tag and GFP as a single polypeptide (tandem display), and another displayed these molecules as two different polypeptides (parallel display). Only the parallelly displayed phage could be detected with ligand blotting using S-protein and with immunoblotting using an anti-GFP antibody. S-protein-coated magnetic beads and nano-particles were successively labeled with fluorescence using the parallelly displayed phage but could not be labeled with the tandemly displayed phage. Thus, the parallel display of a ligand molecule and fluorescent protein on the head surface of bacteriophage T7 could provide a new scheme of producing fluorescent nano-bioprobes for diagnostic applications.
PhoPhabs have been created by incorporating alkaline phosphatase and antibody Fab's from combinatorial libraries on a filamentous phage framework. These PhoPhabs are antigen specific and can replace antibodies and eliminate the need for immunizations in ELISA (enzyme linked immunoabsorbent) methods.
One-dimensional ring structures from M13 viruses were constructed by two genetic modifications encoding binding peptides and synthesis of a heterobifunctional linker molecule. The bifunctional viruses displayed an anti-streptavidin peptide and hexahistidine peptide at opposite ends of the virus as pIII and pIX fusions. Stoichiometric addition of the streptavidin-NiNTA linker molecule led to the reversible formation of virus-based nanorings with circumferences corresponding to lengths of the packagable DNAs. These virus-based ring structures will be further engineered to nucleate inorganic materials and form metallic, magnetic, or semiconductor nanorings using trifunctionalized viruses.
Molecular imaging allows clinicians to visualize the progression of tumours and obtain relevant information for patient diagnosis and treatment. Owing to their intrinsic optical, electrical and magnetic properties, nanoparticles are promising contrast agents for imaging dynamic molecular and cellular processes such as protein-protein interactions, enzyme activity or gene expression. Until now, nanoparticles have been engineered with targeting ligands such as antibodies and peptides to improve tumour specificity and uptake. However, excessive loading of ligands can reduce the targeting capabilities of the ligand and reduce the ability of the nanoparticle to bind to a finite number of receptors on cells. Increasing the number of nanoparticles delivered to cells by each targeting molecule would lead to higher signal-to-noise ratios and would improve image contrast. Here, we show that M13 filamentous bacteriophage can be used as a scaffold to display targeting ligands and multiple nanoparticles for magnetic resonance imaging of cancer cells and tumours in mice. Monodisperse iron oxide magnetic nanoparticles assemble along the M13 coat, and its distal end is engineered to display a peptide that targets SPARC glycoprotein, which is overexpressed in various cancers. Compared with nanoparticles that are directly functionalized with targeting peptides, our approach improves contrast because each SPARC-targeting molecule delivers a large number of nanoparticles into the cells. Moreover, the targeting ligand and nanoparticles could be easily exchanged for others, making this platform attractive for in vivo high-throughput screening and molecular detection.
Phage display is a powerful technique in medical and health biotechnology. This technology has led to formation of antibody libraries and has provided techniques for fast and efficient search of these libraries.
The phage display technique has been used in studying the protein-protein or protein-ligand interactions, constructing of the antibody and antibody fragments and improving the affinity of proteins to receptors.
Recently phage display has been widely used to study immunization process, develop novel vaccines and investigate allergen-antibody interactions. This technology can provide new tools for protection against viral, fungal and bacterial infections. It may become a valuable tool in cancer therapies, abuse and allergies treatment. This review presents the recent advancements in diagnostic and therapeutic applications of phage display. In particular the applicability of this technology to study the immunization process, construction of new vaccines and development of safer and more efficient delivery strategies has been described.
We report a convenient new technique for the labeling of filamentous phage capsid proteins. Previous reports have shown that phage coat protein residues can be modified, but the lack of chemically distinct amino acids in the coat protein sequences makes it difficult to attach high levels of synthetic molecules without altering the binding capabilities of the phage. To modify the phage with polymer chains, imaging groups, and other molecules, we have developed chemistry to convert the N-terminal amines of the ~4200 coat proteins into ketone groups. These sites can then serve as chemospecific handles for the attachment of alkoxyamine groups through oxime formation. Specifically, we demonstrate the attachment of fluorophores and up to 3000 molecules of 2 kDa poly(ethylene glycol) (PEG2k) to each of the phage capsids without significantly affecting the binding of phage-displayed antibody fragments to EGFR and HER2 (two important epidermal growth factor receptors). We also demonstrate the utility of the modified phage for the characterization of breast cancer cells using multicolor fluorescence microscopy. Due to the widespread use of filamentous phage as display platforms for peptide and protein evolution, we envision that the ability to attach large numbers of synthetic functional groups to their coat proteins will be of significant value to the biological and materials communities.
This review summarizes the electron microscopical descriptions of prokaryote viruses. Since 1959, nearly 6300 prokaryote viruses have been described morphologically, including 6196 bacterial and 88 archaeal viruses. As in previous counts, the vast majority (96.3 %) are tailed, and only 230 (3.7 %) are polyhedral, filamentous, or pleomorphic. The family Siphoviridae, whose members are characterized by long, noncontractile tails, is by far the largest family (over 3600 descriptions, or 57.3 %). Prokaryote viruses are found in members of 12 bacterial and archaeal phyla. Archaeal viruses belong to 15 families or groups of family level and infect members of 16 archaeal genera, nearly exclusively hyperthermophiles or extreme halophiles. Tailed archaeal viruses are found in the Euryarchaeota only, whereas most filamentous and pleomorphic archaeal viruses occur in the Crenarchaeota. Bacterial viruses belong to 10 families and infect members of 179 bacterial genera, mostly members of the Firmicutes and γ-proteobacteria.
Although the natural hosts for bacteriophages are bacteria, a growing body of data shows that phages can also interact with some populations of mammalian cells, especially with cells of the immune system. In general, these interactions include two main aspects. The first is the phage immunogenicity, that is, the capacity of phages to induce specific immune responses, in particular the generation of specific antibodies against phage antigens. The other aspect includes the immunomodulatory activity of phages, that is, the nonspecific effects of phages on different functions of major populations of immune cells involved in both innate and adaptive immune responses. These functions include, among others, phagocytosis and the respiratory burst of phagocytic cells, the production of cytokines, and the generation of antibodies against nonphage antigens. The aim of this chapter is to discuss the interactions between phages and cells of the immune system, along with their implications for phage therapy. These topics are presented based on the results of experimental studies and unique data on immunomodulatory effects found in patients with bacterial infections treated with phage preparations.
Second near-infrared (NIR) window light (950-1400 nm) is attractive for in vivo fluorescence imaging due to its deep penetration depth in tissues and low tissue autofluorescence. Here we show genetically engineered multifunctional M13 phage can assemble fluorescent single-walled carbon nanotubes (SWNTs) and ligands for targeted fluorescence imaging of tumors. M13-SWNT probe is detectable in deep tissues even at a low dosage of 2 μg/mL and up to 2.5 cm in tissue-like phantoms. Moreover, targeted probes show specific and up to 4-fold improved uptake in prostate specific membrane antigen positive prostate tumors compared to control nontargeted probes. This M13 phage-based second NIR window fluorescence imaging probe has great potential for specific detection and therapy monitoring of hard-to-detect areas.
The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical pore-forming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes.
The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA(257-264) antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation.
The M13 bacteriophage has been demonstrated to be a robust scaffold for bionanomaterial development. In this paper, we report on the chemical modifications of three kinds of reactive groups, i.e., the amino groups of lysine residues or N-terminal, the carboxylic acid groups of aspartic acid or glutamic acid residues, and the phenol group of tyrosine residues, on M13 surface. The reactivity of each group was identified through conjugation with small fluorescent molecules. Furthermore, the regioselectivity of each reaction was investigated by HPLC-MS-MS. By optimizing the reaction condition, hundreds of fluorescent moieties could be attached to create a highly fluorescent M13 bacteriophage. In addition, cancer cell targeting motifs such as folic acid could also be conjugated onto the M13 surface. Therefore, dual-modified M13 particles with folic acid and fluorescent molecules were synthesized via the selective modification of two kinds of reactive groups. Such dual-modified M13 particles showed very good binding affinity to human KB cancer cells, which demonstrated the potential applications of M13 bacteriophage in bioimaging and drug delivery.
Combinatorial chemistry and phage display technologies provide robust means for the rapid discovery of tumor antigen-avid peptides. Combinatorial affinity maturation experiments have been performed on a small number of these peptides, including P30, in an attempt to improve affinity and in vivo binding. Phage display has also been performed using cultured human carcinoma cells or in situ with laser captured micro-dissected cancer cells resulting in many new peptides as potential imaging probes. Phage display-selected peptides been used in cancer imaging, but they have also been used in vivo to reduce tumor growth. The ability of unlabeled peptides to functionally modulate tumor growth and spread should positively impact future studies aimed at developing peptide-based cancer therapeutics. Thus, phage display technology has developed into a rapid, economical, and efficacious approach to the development of agents for the molecular imaging and diagnosis of cancer.
Phage display, the presentation of (poly)peptides as fusions to capsid proteins on the surface of bacterial viruses, celebrates its 25th birthday in 2010. The technique, coupled with in vitro selection, enables rapid identification and optimization of proteins based on their structural or functional properties. In the last two decades, it has advanced tremendously and has become widely accepted by the scientific community. This by no means exhaustive review aims to inform the reader of the key modifications in phage display. Novel display formats, innovative library designs and screening strategies are discussed. I will also briefly review some recent uses of the technology to illustrate its incredible versatility.
Two-step and three-step pretargeting systems utilizing biotinylated prostate tumor-homing bacteriophage (phage) and (111)In-radiolabeled streptavidin or biotin were developed for use in cancer radioimaging. The in vivo selected prostate carcinoma-specific phage (G1) displaying up to five copies of the peptide IAGLATPGWSHWLAL was the focus of the present study.
The ability of G1 phage to extravasate and target prostate tumor cells was investigated using immunohistochemistry. G1 phages were biotinylated, streptavidin was conjugated to diethylenetriaminepentaacetic acid (DTPA) and biotin was conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). Biodistribution studies and single-photon emission computed tomography (SPECT)/CT imaging of xenografted PC-3 tumors via two-step pretargeted (111)In-labeled streptavidin and three-step pretargeted (111)In-labeled biotin were performed in SCID mice to determine the optimal pretargeting method.
The ability of G1 phage to extravasate the vasculature and bind directly to human PC-3 prostate carcinoma tumor cells in vivo was demonstrated via immunocytochemical analysis. Comparative biodistribution studies of the two-step and three-step pretargeting strategies indicated increased PC-3 human prostate carcinoma tumor uptake in SCID mice of 4.34+/-0.26 %ID g(-1) at 0.5 h postinjection of (111)In-radiolabeled biotin (utilized in a three-step protocol) compared to 0.67+/-0.06 %ID g(-1) at 24 h postinjection of (111)In radiolabeled streptavidin (employed in a two-step protocol). In vivo SPECT/CT imaging of xenografted PC-3 tumors in SCID mice with the three-step pretargeting method was superior to that of the two-step pretargeting method, and, importantly, blocking studies demonstrated specificity of tumor uptake of (111)In-labeled biotin in the three-step pretargeting scheme.
This study demonstrates the use of multivalent bifunctional phage in a three-step pretargeting system for prostate cancer radioimaging.
Mutation in the tubby gene causes adult-onset obesity, progressive retinal, and cochlear degeneration with unknown mechanism. In contrast, mutations in tubby-like protein 1 (Tulp1), whose C-terminus is highly homologous to tubby, only lead to retinal degeneration. We speculate that their diverse N-terminus may define their distinct disease profile. To elucidate the binding partners of tubby, we used tubby N-terminus (tubby-N) as bait to identify unknown binding proteins with open-reading-frame (ORF) phage display. T7 phage display was engineered with three improvements: high-quality ORF phage display cDNA library, specific phage elution by protease cleavage, and dual phage display for sensitive high throughput screening. The new system is capable of identifying unknown bait-binding proteins in as fast as approximately 4-7 days. While phage display with conventional cDNA libraries identifies high percentage of out-of-frame unnatural short peptides, all 28 tubby-N-binding clones identified by ORF phage display were ORFs. They encode 16 proteins, including 8 nuclear proteins. Fourteen proteins were analyzed by yeast two-hybrid assay and protein pull-down assay with ten of them independently verified. Comparative binding analyses revealed several proteins binding to both tubby and Tulp1 as well as one tubby-specific binding protein. These data suggest that tubby-N is capable of interacting with multiple nuclear and cytoplasmic protein binding partners. These results demonstrated that the newly-engineered ORF phage display is a powerful technology to identify unknown protein-protein interactions.
A phage display-based bifunctional display system was developed for simple and sensitive immunoassay. The resulting bifunctional phage could simultaneously display a few single-chain variable fragment (ScFv) and many copies of the gold-binding peptide on its surface, thereby mediating antigen recognition and signal amplification. As a demonstration study, it was possible for bifunctional phage-based immunoassay to identify Bacillus anthracis spores from other Bacillus strains with detection sensitivity 10-fold higher than that of conventional phage enzyme-linked immunosorbent assay (ELISA). This protocol may be applied to build other bifunctional phage clones for broad applications (e.g., immunoassay kits, affinity biosensors, biorecognition assays).
An Achilles heel inherent to all molecular display formats, background binding between target and display system introduces false positives into screens and selections. For example, the negatively charged surfaces of phage, mRNA, and ribosome display systems bind with unacceptably high nonspecificity to positively charged target molecules, which represent an estimated 35% of proteins in the human proteome. Here we report the first systematic attempt to understand why a broad class of molecular display selections fail, and then solve the underlying problem for both phage and RNA display. Firstly, a genetic strategy was used to introduce a short, charge-neutralizing peptide into the solvent-exposed, negatively charged phage coat. The modified phage (KO7(+)) reduced or eliminated nonspecific binding to the problematic high-pI proteins. In the second, chemical approach, nonspecific interactions were blocked by oligolysine wrappers in the cases of phage and total RNA. For phage display applications, the peptides Lys(n) (where n=16 to 24) emerged as optimal for wrapping the phage. Lys(8), however, provided effective wrappers for RNA binding in assays against the RNA binding protein HIV-1 Vif. The oligolysine peptides blocked nonspecific binding to allow successful selections, screens, and assays with five previously unworkable protein targets.
Fluorogenic imaging agents emitting in the near-infrared are becoming important research tools for disease investigation in vivo. Often pathophysiological states such as cancer and cystic fibrosis are associated with disruptions in acid/base homeostasis. The development of optical sensors for pH imaging would facilitate the investigation of these diseased conditions. In this report, the design and synthesis of a ratiometric near-infrared emitting probe for pH quantification is detailed. The pH-responsive probe is prepared by covalent attachment of pH-sensitive and pH-insensitive fluorophores to a bacteriophage particle scaffold. The pH-responsive cyanine dye, HCyC-646, used to construct the probe, has a fluorogenic pKa of 6.2, which is optimized for visualization of acidic pH often associated with tumor hypoxia and other diseased states. Incorporation of pH-insensitive reference dyes enables the ratiometric determination of pH independent of the probe concentration. With the pH-responsive construct, measurement of intracellular pH and accurate determination of pH through optically diffuse biological tissue is demonstrated.
Tens of millions of short peptides can be easily surveyed for tight binding to an antibody, receptor or other binding protein using an "epitope library." The library is a vast mixture of filamentous phage clones, each displaying one peptide sequence on the virion surface. The survey is accomplished by using the binding protein to affinity-purify phage that display tight-binding peptides and propagating the purified phage in Escherichia coli. The amino acid sequences of the peptides displayed on the phage are then determined by sequencing the corresponding coding region in the viral DNA's. Potential applications of the epitope library include investigation of the specificity of antibodies and discovery of mimetic drug candidates.
Foreign DNA fragments can be inserted into filamentous phage gene III to create a fusion protein with the foreign sequence in the middle. The fusion protein is incorporated into the virion, which retains infectivity and displays the foreign amino acids in immunologically accessible form. These "fusion phage" can be enriched more than 1000-fold over ordinary phage by affinity for antibody directed against the foreign sequence. Fusion phage may provide a simple way of cloning a gene when an antibody against the product of that gene is available.