Dustin P. Patterson

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.

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.

Virus-like particle nanoreactors: Programmed encapsulation of the thermostable CelB glycosidase inside the P22 capsid

Self-assembling biological systems hold great potential for the synthetic construction of new active soft nanomaterials. Here we demonstrate the hierarchical bottom-up assembly of bacteriophage P22 virus-like particles (VLPs) that encapsulate the thermostable CelB glycosidase creating catalytically active nanoreactors. The in vivo assembly and encapsulation produces P22 VLPs with a high packaging density of the tetrameric CelB, but without loss of enzyme activity or the ability of the P22 VLP to undergo unique morphological transitions that modify the VLPs internal volume and shell porosity. The P22 VLPs encapsulating CelB are also shown to retain a high percentage of the enzyme activity upon being embedded and immobilized in a polymeric matrix.

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.

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.

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.

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.

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.

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.

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 (β-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.