Engineering the HK97 virus-like particle as a nanoplatform for biotechnology applications.

The research described here looks at the development of virus-like particles (VLPs) derived from bacteriophage HK97 as versatile scaffolds for bionanomaterials construction. Based on molecular models, the Prohead I HK97 VLP was engineered to allow attachment of small molecules to the interior by introducing a reactive cysteine into the genetic sequence of the HK97 GP5 protein that self assembles to form the VLP structure. In addition, methods for entrapping large protein macromolecules were evaluated and found to produce high encapsulation numbers of green fluorescent proteins (GFP) in the internal space of the HK97 VLP. A method for modular modification of the external surface was engineered by constructing a plasmid allowing the addition of peptide sequences to the C-terminus of the GP5 protein, which was validated by appending the sortase recognition peptide sequence, LPETG, to the C-terminus of GP5 and showing the attachment of a polyglycine-GFP to the HK97 VLP through sortase mediated ligation. To demonstrate the potential for advanced applications, an HK97 VLP covalently labeled on the interior surface with fluorescein and containing an externally displayed integrin binding peptide sequence (RGD) was evaluated and found to be preferentially localized at C2C12 cells relative to the HK97 VLP lacking the RGD peptide. Together, these results support the potential of the HK97 VLP as a versatile nanoparticle platform that can be modified internally and externally in a modular fashion for the purpose of programming the VLP for desired applications.

Delayed In Vivo Encapsulation of Enzymes Alters the Catalytic Activity of Virus-Like Particle Nanoreactors.

Encapsulation of enzymes inside protein cage structures, mimicking protein-based organelle structures found in nature, has great potential for the development of new catalytic materials with enhanced properties. In vitro and in vivo methodologies have been developed for the encapsulation of enzymes within protein cage structures of several types, particularly virus-like particles (VLPs), with the ability to retain the activity of the encapsulated enzymes. Here, we examine the in vivo encapsulation of enzymes within the bacteriophage P22 derived VLP and show that some enzymes may require a delay in encapsulation to allow proper folding and maturation before they can be encapsulated inside P22 as fully active enzymes. Using a sequential expression strategy, where enzyme cargoes are first expressed, allowed to fold, and later encapsulated by the expression of the P22 coat protein, altered enzymatic activities are obtained in comparison to enzymes encapsulated in P22 VLPs using a simultaneous coexpression strategy. The strategy and results discussed here highlight important considerations for researchers investigating the encapsulation of enzymes inside confined reaction environments via in vivo routes and provide a potential solution for those that have been unable to produce active enzymes upon encapsulation.

Encapsulation of Pseudomonas aeruginosa elastase inside the P22 virus-like particle for controlling enzyme-substrate interactions.

Controlling interactions between enzymes and interaction partners, such as substrates, is important for applications in cellular biology and molecular biochemistry. A strategy for controlling enzyme access with substrate interaction partners is to exploit encapsulation of enzymes inside nanoparticles to limit the accessibility of the enzymes to large macromolecules, but allow free exchange of small-molecule substrates. The research here evaluates the encapsulation of Pseudomonas aeruginosa elastase inside the bacteriophage P22 virus-like particle (VLP) to examine the ability to allow free soluble substrates access to the enzyme while blocking large macromolecular substrate interactions. The results show that the active elastase protease can be encapsulated inside the P22 VLP, which blocks its ability to disrupt cell monolayers, but allows soluble substrates to be catalytically cleaved, supporting the viability of this approach for future investigations.

Engineering soluble artificial epidermal growth factor receptor mimics capable of spontaneous in vitro dimerization.

Epidermal growth factor receptor (EGFR) is a clinically validated target for a multitude of human cancers. The receptor is activated upon ligand binding through a critical dimerization step. Dimerization can be replicated in vitro by locally concentrating the receptor kinase domains on the surface of lipid-based vesicles. In this study we investigated the use of coiled coils to induce spontaneous receptor kinase domain dimerization in vitro to form non-membrane-bound artificial receptor mimics in solution. Two engineered forms of EGFR kinase domain fused to coiled coil complementary peptides were designed to self-associate upon mixing. Two fusion protein species (P3-EGFR and P4-EGFR) independently showed the same activity and polymerization profile known to exist with EGFR kinase domains. Upon mixing the two species, coiled coil heterodimers were formed that induced EGFR association to form dimers of the kinase domains. This was accompanied by 11.5-fold increase in the phosphorylation rate indicative of kinase domain activation equivalent to the levels achieved using vesicle localization and mimicking in vivo ligand-induced activation. This study presents a soluble tyrosine kinase receptor mimic capable of spontaneous in vitro activation that can facilitate functional and drug discovery studies for this clinically important receptor class.

Engineering DNA recognition and allosteric response properties of TetR family proteins by using a module-swapping strategy.

The development of synthetic biological systems requires modular biomolecular components to flexibly alter response pathways. In previous studies, we have established a module-swapping design principle to engineer allosteric response and DNA recognition properties among regulators in the LacI family, in which the engineered regulators served as effective components for implementing new cellular behavior. Here we introduced this protein engineering strategy to two regulators in the TetR family: TetR (UniProt Accession ID: P04483) and MphR (Q9EVJ6). The TetR DNA-binding module and the MphR ligand-binding module were used to create the TetR-MphR. This resulting hybrid regulator possesses DNA-binding properties of TetR and ligand response properties of MphR, which is able to control gene expression in response to a molecular signal in cells. Furthermore, we studied molecular interactions between the TetR DNA-binding module and MphR ligand-binding module by using mutant analysis. Together, we demonstrated that TetR family regulators contain discrete and functional modules that can be used to build biological components with novel properties. This work highlights the utility of rational design as a means of creating modular parts for cell engineering and introduces new possibilities in rewiring cellular response pathways.

Encapsulation of Active Enzymes within Bacteriophage P22 Virus-Like Particles.

Virus-like particles (VLPs) are nonpathogenic protein cage structures derived from viral coat proteins that have found utility in the area of biomaterials and nanotechnology. VLPs have been exploited as containers for the sequestration and encapsulation of a wide range of guest molecules in their hollow interiors. The robust nature of VLPs lend them as versatile scaffolds that can be exploited to provide protection to encapsulated guest molecules, such as enzymes which are often susceptible to inactivation and degradation, and for organization and construction of new nanomaterials incorporating the chemical properties of the guest molecules. In this chapter a background and methodology for the encapsulation of enzymes on the interior of the bacteriophage P22 derived VLP is described.

Modular Self-Assembly of Protein Cage Lattices for Multistep Catalysis.

The assembly of individual molecules into hierarchical structures is a promising strategy for developing three-dimensional materials with properties arising from interaction between the individual building blocks. Virus capsids are elegant examples of biomolecular nanostructures, which are themselves hierarchically assembled from a limited number of protein subunits. Here, we demonstrate the bio-inspired modular construction of materials with two levels of hierarchy: the formation of catalytically active individual virus-like particles (VLPs) through directed self-assembly of capsid subunits with enzyme encapsulation, and the assembly of these VLP building blocks into three-dimensional arrays. The structure of the assembled arrays was successfully altered from an amorphous aggregate to an ordered structure, with a face-centered cubic lattice, by modifying the exterior surface of the VLP without changing its overall morphology, to modulate interparticle interactions. The assembly behavior and resultant lattice structure was a consequence of interparticle interaction between exterior surfaces of individual particles and thus independent of the enzyme cargos encapsulated within the VLPs. These superlattice materials, composed of two populations of enzyme-packaged VLP modules, retained the coupled catalytic activity in a two-step reaction for isobutanol synthesis. This study demonstrates a significant step toward the bottom-up fabrication of functional superlattice materials using a self-assembly process across multiple length scales and exhibits properties and function that arise from the interaction between individual building blocks.

A Superior Synthesis of Longitudinally Twisted Acenes.

Seven longitudinally twisted acenes (an anthracene, two tetracenes, three pentacenes, and a hexacene) have been synthesized by the addition of aryllithium reagents to the appropriate quinone precursors, followed by SnCl2 -mediated reduction of their diol intermediates, and several of these acenes have been crystallographically characterized. The new syntheses of the three previously reported twisted acenes, decaphenylanthracene (1), 9,10,11,20,21,22-hexaphenyltetrabenzo[a,c,l,n]pentacene (2), and 9,10,11,12,13,14,15,16-octaphenyldibenzo[a,c]tetracene (14), resulted in a reduction of the number of synthetic steps. As a consequence their overall yields were increased by factors of 50-, 24-, and 66-fold, respectively. All of the twisted acene syntheses reported here are suitable for the synthesis of at least gram quantities of these remarkable hydrocarbon materials.

Sortase-Mediated Ligation as a Modular Approach for the Covalent Attachment of Proteins to the Exterior of the Bacteriophage P22 Virus-like Particle.

Virus-like particles are unique platforms well suited for the construction of nanomaterials with broad-range applications. The research presented here describes the development of a modular approach for the covalent attachment of protein domains to the exterior of the versatile bacteriophage P22 virus-like particle (VLP) via a sortase-mediated ligation strategy. The bacteriophage P22 coat protein was genetically engineered to incorporate an LPETG amino acid sequence on the C-terminus, providing the peptide recognition sequence utilized by the sortase enzyme to catalyze peptide bond formation between the LPETG-tagged protein and a protein containing a polyglycine sequence on the N-terminus. Here we evaluate attachment of green fluorescent protein (GFP) and the head domain of the influenza hemagglutinin (HA) protein by genetically producing polyglycine tagged proteins. Attachment of both proteins to the exterior of the P22 VLP was found to be highly efficient as judged by SDS-PAGE densitometry. These results enlarge the tool kit for modifying the P22 VLP system and provide new insights for other VLPs that have an externally displayed C-terminus that can use the described strategy for the modular modification of their external surface for various applications.

Two-Dimensional Crystallization of P22 Virus-Like Particles.

Virus-like particles (VLPs) are well established platforms for constructing functional biomimetic materials. The VLP from the bacteriophage P22 can be used as a nanocontainer to sequester active enzymes, at high concentration, within its cavity through a process of directed self-assembly. Construction of ordered 2D assemblies of these catalytic VLPs can be envisioned as a functional membrane. To achieve this, it is important to establish methods to fabricate densely packed monolayers of VLPs. Highly ordered assemblies of P22 can also be utilized as a two-dimensional (2D) crystal for electron crystallography to get precise structural information on the VLP. Here we report 2D crystallization of different P22 morphologies: P22 procapsid (PC), enzyme encapsulated PC (ß-glycosidase and enhanced green fluorescent protein), empty shell (PC without scaffold proteins, ES), the expanded form of P22 (EX), and enzyme encapsulated EX (NADH oxidase). The 2D crystals of P22 VLPs were formed on a positively charged lipid monolayer at the water-air interface with a subphase containing 1% trehalose. A P22 solution, injected underneath the lipid monolayer, floated to the surface because of the density difference between the subphase and protein solution. The lipid monolayer, with adsorbed P22, was transferred to a holey carbon grid and was examined by electron microscopy. 2D crystals were obtained from a subphase containing 100 mM NaCl, 10 mM MES (pH 5.0), and 1% trehalose. The diffraction spots from the transferred film extended to the sixth order in negatively stained samples and the 10th order in cryo-electron microscopy samples.

Self-assembling biomolecular catalysts for hydrogen production.

The chemistry of highly evolved protein-based compartments has inspired the design of new catalytically active materials that self-assemble from biological components. A frontier of this biodesign is the potential to contribute new catalytic systems for the production of sustainable fuels, such as hydrogen. Here, we show the encapsulation and protection of an active hydrogen-producing and oxygen-tolerant [NiFe]-hydrogenase, sequestered within the capsid of the bacteriophage P22 through directed self-assembly. We co-opted Escherichia coli for biomolecular synthesis and assembly of this nanomaterial by expressing and maturing the EcHyd-1 hydrogenase prior to expression of the P22 coat protein, which subsequently self assembles. By probing the infrared spectroscopic signatures and catalytic activity of the engineered material, we demonstrate that the capsid provides stability and protection to the hydrogenase cargo. These results illustrate how combining biological function with directed supramolecular self-assembly can be used to create new materials for sustainable catalysis.

Viruslike Particles Encapsidating Respiratory Syncytial Virus M and M2 Proteins Induce Robust T Cell Responses.

Subunit vaccines provide a safe, focused alternative to conventional vaccines. However, these vaccines often require significant adjuvants and are particularly hard to target toward cytotoxic T lymphocyte (CTL) immunity. Viruslike particles (VLPs) provide biomaterial scaffolds with pathogen-like polyvalent structures making them useful platforms for biomimetic antigen delivery to the immune system. Encapsidation of antigens within VLPs has been shown to enhance antigen availability for CD8 T cell responses. Here, we examine the potential to generate complex responses to multiple subunit antigens localized within the same VLP particle. Two proteins of respiratory syncytial virus (RSV) with well-characterized CD8 T cell responses, the matrix (M) and matrix 2 (M2) proteins, were successfully coencapsidated within the P22 VLP. Upon intranasal administration in mice, the particles stimulated CD8 T cell memory responses against both antigens. In addition, vaccination elicited tissue-resident T cell populations. Upon subsequent RSV challenge, P22-M/M2-treated mice displayed significantly reduced lung viral titers. This demonstrates the utility of the P22 VLP in directing immune responses to multiple encapsidated viral antigens, demonstrating the potential of this technology to facilitate immunity to multiple targets simultaneously.

Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery.

BACKGROUND: The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. RESULTS: We have designed a nano-bioreactor based on the encapsulation of a cytochrome P450 (CYP) inside the capsid derived from the bacteriophage P22. An enhanced peroxigenase, CYPBM3, was selected as a model enzyme because of its potential in enzyme prodrug therapy. A total of 109 enzymes per capsid were encapsulated with a 70 % retention of activity for cytochromes with the correct incorporation of the heme cofactor. Upon encapsulation, the stability of the enzyme towards protease degradation and acidic pH was increased. Cytochrome P450 activity was delivered into Human cervix carcinoma cells via transfecting P22-CYP nanoparticles with lipofectamine. CONCLUSION: This work provides a clear demonstration of the potential of biocatalytic virus-like particles as medical relevant enzymatic delivery vehicles for clinical applications.

Large area synthesis of a nanoporous two-dimensional polymer at the air/water interface.

We present the synthesis of a two-dimensional polymer at the air/water interface and its nm-resolution imaging. Trigonal star, amphiphilic monomers bearing three anthraceno groups on a central triptycene core are confined at the air/water interface. Compression followed by photopolymerization on the interface provides the two-dimensional polymer. Analysis by scanning tunneling microscopy suggests that the polymer is periodic with ultrahigh pore density.

Virus-like particles as antigenic nanomaterials for inducing protective immune responses in the lung.

The lung is a major entry point for many of the most detrimental pathogens to human health. The onslaught of pathogens encountered by the lung is counteracted by protective immune responses that are generated locally, which can be stimulated through vaccine strategies to prevent pathogen infections. Here, we discuss the use of virus-like particles (VLPs), nonpathogen derivatives of viruses or protein cage structures, to construct new vaccines exploiting the lung as a site for immunostimulation. VLPs are unique in their ability to be engineered with near molecular level detail and knowledge of their composition and structure. A summary of research in developing VLP-based vaccines for the lung is presented that suggests promising results for future vaccine development.

Encapsulation of an enzyme cascade within the bacteriophage P22 virus-like particle.

Developing methods for investigating coupled enzyme systems under conditions that mimic the cellular environment remains a significant challenge. Here we describe a biomimetic approach for constructing densely packed and confined multienzyme systems through the co-encapsulation of 2 and 3 enzymes within a virus-like particle (VLP) that perform a coupled cascade of reactions, creating a synthetic metabolon. Enzymes are efficiently encapsulated in vivo with known stoichiometries, and the kinetic parameters of the individual and coupled activities are characterized. From the results we develop and validate a mathematical model for predicting the expected kinetics for coupled reactions under co-localized conditions.

Characterization of a highly flexible self-assembling protein system designed to form nanocages.

The design of proteins that self-assemble into well-defined, higher order structures is an important goal that has potential applications in synthetic biology, materials science, and medicine. We previously designed a two-component protein system, designated A-(+) and A-(-), in which self-assembly is mediated by complementary electrostatic interactions between two coiled-coil sequences appended to the C-terminus of a homotrimeric enzyme with C3 symmetry. The coiled-coil sequences are attached through a short, flexible spacer sequence providing the system with a high degree of conformational flexibility. Thus, the primary constraint guiding which structures the system may assemble into is the symmetry of the protein building block. We have now characterized the properties of the self-assembling system as a whole using native gel electrophoresis and analytical ultracentrifugation (AUC) and the properties of individual assemblies using cryo-electron microscopy (EM). We show that upon mixing, A-(+) and A-(-) form only six different complexes in significant concentrations. The three predominant complexes have hydrodynamic properties consistent with the formation of heterodimeric, tetrahedral, and octahedral protein cages. Cryo-EM of size-fractionated material shows that A-(+) and A-(-) form spherical particles with diameters appropriate for tetrahedral or octahedral protein cages. The particles varied in diameter in an almost continuous manner suggesting that their structures are extremely flexible.

Constructing catalytic antimicrobial nanoparticles by encapsulation of hydrogen peroxide producing enzyme inside the P22 VLP.

Here we examine a self-assembling virus like particle to construct catalytically active nanoparticles that can inhibit bacterial growth. The results suggest that encapsulation of enzymes inside VLPs can be exploited to develop new bionanomaterials with useful functionalities.

Rescuing recombinant proteins by sequestration into the P22 VLP.

Here we report the use of a self-assembling protein cage to sequester and solubilize recombinant proteins which are usually trafficked to insoluble inclusion bodies. Our results suggest that protein cages can be used as novel vehicles to rescue and produce soluble proteins that are otherwise difficult to obtain using conventional methods.

Biomimetic antigenic nanoparticles elicit controlled protective immune response to influenza.

Here we present a biomimetic strategy toward nanoparticle design for controlled immune response through encapsulation of conserved internal influenza proteins on the interior of virus-like particles (VLPs) to direct CD8+ cytotoxic T cell protection. Programmed encapsulation and sequestration of the conserved nucleoprotein (NP) from influenza on the interior of a VLP, derived from the bacteriophage P22, results in a vaccine that provides multistrain protection against 100 times lethal doses of influenza in an NP specific CD8+ T cell-dependent manner. VLP assembly and encapsulation of the immunogenic NP cargo protein is the result of a genetically programmed self-assembly making this strategy amendable to the quick production of vaccines to rapidly emerging pathogens. Addition of adjuvants or targeting molecules were not required for eliciting the protective response.