Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle.

Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly.

A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8.

Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes.

The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli.

Synthetic biology design to display an 18 kDa rotavirus large antigen on a modular virus-like particle.

Virus-like particles are an established class of commercial vaccine possessing excellent function and proven stability. Exciting developments made possible by modern tools of synthetic biology has stimulated emergence of modular VLPs, whereby parts of one pathogen are by design integrated into a less harmful VLP which has preferential physical and manufacturing character. This strategy allows the immunologically protective parts of a pathogen to be displayed on the most-suitable VLP.

Positive selection of digestive Cys proteases in herbivorous Coleoptera.

Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences.

Assembly and Purification of Polyomavirus-Like Particles from Plants.

Polyomaviruses are small DNA viruses that have a history of use in biotechnology. The capsids of a number of species have been developed into experimental prophylactic and therapeutic virus-like particle (VLP) vaccines. In order to explore plants as a host for the expression and purification of polyomavirus-like particles, we have transiently expressed the major capsid protein, VP1, in Nicotiana benthamiana leaves.

Beyond disease, how biomedical engineering can improve global health.

Biomedical engineering tools can be harnessed to address some of the world's most challenging nondisease-focused problems.

Computational study of elements of stability of a four-helix bundle protein biosurfactant.

Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants.

Protective efficacy of a bacterially produced modular capsomere presenting M2e from influenza: extending the potential of broadly cross-protecting epitopes.

Influenza A viruses drift and shift, emerging as antigenically distinct strains that lead to epidemics and pandemics of varying severity. Even epitopes associated with broad cross-protection against different strains, such as the ectodomain of matrix protein 2 (M2e), mutate unpredictably. Vaccine protective efficacy is only ensured when the emerging virus lies within the vaccine's cross-protective domain, which is poorly defined in most situations.

Transient expressions of synthetic biology in plants.

Recent developments in transient expression methods have enabled the efficient delivery and expression of multiple genes within the same plant cell over a timescale of days. In some cases, the vectors deployed can be fine-tuned to allow differential expression of the various genes. This has opened the way to the deployment of transient expression for such applications as the production of macromolecular complexes and the analysis and manipulation of metabolic pathways.

Microbially synthesized modular virus-like particles and capsomeres displaying group A streptococcus hypervariable antigenic determinants.

Effective and low-cost vaccines are essential to control severe group A streptococcus (GAS) infections prevalent in low-income nations and the Australian aboriginal communities. Highly diverse and endemic circulating GAS strains mandate broad-coverage and customized vaccines. This study describes an approach to deliver cross-reactive antigens from endemic GAS strains using modular virus-like particle (VLP) and capsomere systems.

Magnetic mesoporous silica nanoparticles end-capped with hydroxyapatite for pH-responsive drug release.

Mesoporous silica nanoparticles (MSNs) have emerged as one of the most promising vehicles for potential application in drug delivery. In this paper, we report a novel multifunctional nanocomposite composed of a magnetite nanocrystal core and a mesoporous silica shell (FeO@mSiO), end-capped with pH-stimuli-responsive hydroxyapatite (HAp) nanovalves for pH-responsive drug release. Iron oxide core and mesoporous silica shell nanoparticles were synthesized using a microemulsion method, and were then employed as templates for surface coating of hydroxyapatite.

Sensitivity of immune response quality to influenza helix 190 antigen structure displayed on a modular virus-like particle.

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen.

Multiphase flow microfluidics for the production of single or multiple emulsions for drug delivery.

Considerable effort has been directed towards developing novel drug delivery systems. Microfluidics, capable of generating monodisperse single and multiple emulsion droplets, executing precise control and operations on these droplets, is a powerful tool for fabricating complex systems (microparticles, microcapsules, microgels) with uniform size, narrow size distribution and desired properties, which have great potential in drug delivery applications. This review presents an overview of the state-of-the-art multiphase flow microfluidics for the production of single emulsions or multiple emulsions for drug delivery.

Effects of pre-existing anti-carrier immunity and antigenic element multiplicity on efficacy of a modular virus-like particle vaccine.

Modularization of a peptide antigen for presentation on a microbially synthesized murine polyomavirus (MuPyV) virus-like particle (VLP) offers a new alternative for rapid and low-cost vaccine delivery at a global scale. In this approach, heterologous modules containing peptide antigenic elements are fused to and displayed on the VLP carrier, allowing enhancement of peptide immunogenicity via ordered and densely repeated presentation of the modules. This study addresses two key engineering questions pertaining to this platform, exploring the effects of (i) pre-existing carrier-specific immunity on modular VLP vaccine effectiveness and (ii) increase in the antigenic element number per VLP on peptide-specific immune response.

Stimuli-responsive peptide nanostructures at the fluid-fluid interface.

The self-organization of peptide-based nanostructures at a confined fluid-fluid interface, for example, the air-water or oil-water interface, is important in the context of stabilizing macroscopic soft-matter foams and emulsions. The unique ability to design interfacial nanostructures by controlling the subtle cooperativity that drives peptide self-assembly, and the ability to switch molecular cooperativity by facile triggers such as pH, opens new vistas for controlling macroscopic soft matter in industries as diverse as healthcare and industrial processing. Here we describe research aimed at developing new understanding into soft-matter formation and control, through variation of peptide sequence and bulk conditions.

Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS).

Group A streptococcus (GAS) causes a wide range of diseases, some of them related to autoimmune diseases triggered by repeated GAS infections. Despite the fact that GAS primarily colonizes the mucosal epithelium of the pharynx, the main mechanism of action of most vaccine candidates is based on development of systemic antibodies that do not cross-react with host tissues, neglecting the induction of mucosal immunity that could potentially block disease transmission. Peptide antigens from GAS M-surface protein can confer protection against infection; however, translation of such peptides into immunogenic mucosal vaccines that can be easily manufactured remains a challenge.

Insights into the role of protein molecule size and structure on interfacial properties using designed sequences.

Mixtures of a large, structured protein with a smaller, unstructured component are inherently complex and hard to characterize at interfaces, leading to difficulties in understanding their interfacial behaviours and, therefore, formulation optimization. Here, we investigated interfacial properties of such a mixed system. Simplicity was achieved using designed sequences in which chemical differences had been eliminated to isolate the effect of molecular size and structure, namely a short unstructured peptide (DAMP1) and its longer structured protein concatamer (DAMP4).

Dispersive mixing and intraparticle partitioning of protein in size-exclusion chromatographic refolding.

Refolding enables bioprocesses predicated on proteins expressed as inclusion bodies in Escherichia coli. Optimization of size-exclusion chromatography (SEC) refolding is a significant challenge because a wide range of factors, including the choice of gel media, the column dimensions and configuration, affect the final yield in a protein-specific manner. In this study, we investigated these factors by relating them to dispersive mixing and partitioning of refolding molecules within the SEC pore structure.

Comparison of positional surfactant isomers for displacement of rubisco protein from the air-water interface.

Protein-surfactant interaction, which is a function of the protein and surfactant characteristics, is a common phenomenon in a wide range of industrial applications. In this work, we used rubisco, the most abundant protein in nature, as a model protein and sodium dodecylbenzenesulfonate (SDOBS), one of the most widely used commercial surfactants, with two positional isomers (SDOBS-2 and SDOBS-6), as a model surfactant. We first examined the surface tension and the mechanical properties of interfacial mixed rubisco-SDOBS films adsorbed at the air-water interface.

Analysis of monoPEGylated human galectin-2 by small-angle X-ray and neutron scattering: concentration dependence of PEG conformation in the conjugate.

Protein conjugation with polyethylene glycol (PEG) is a valuable means for improving stability, solubility, and bioavailability of pharmaceutical proteins. Using human galectin-2 (hGal-2) and 5 kDa PEG as a model system we first produced a PEG-hGal-2 conjugate exclusively at the Cys75 residue, resulting in two monosubstituted subunits per hGal-2 homodimer. Small angle X-ray and neutron scattering (SAXS and SANS) were combined to provide complementary structural information about the PEG-hGal-2 conjugate, wherein signal generation in SAXS depends mainly on the protein while SANS data presents signals from both the protein and PEG moieties.

Microbial production of virus-like particle vaccine protein at gram-per-litre levels.

This study demonstrates the feasibility of large-scale production of murine polyomavirus VP1 protein in recombinant Escherichia coli as pentamers which are able to subsequently self-assemble in vitro into virus-like particles (VLPs). High-cell-density pH-stat fed-batch cultivation was employed to produce glutathione-S-transferase (GST)-VP1 fusion protein in soluble form. The expression of recombinant VP1 was induced with IPTG at different cell optical densities (OD at 600 nm of 20, 60 or 100).

The construction, fouling and enzymatic cleaning of a textile dye surface.

The enzymatic cleaning of a rubisco protein stain bound onto Surface Plasmon Resonance (SPR) biosensor chips having a dye-bound upper layer is investigated. This novel method allowed, for the first time, a detailed kinetic study of rubisco cleanability (defined as fraction of adsorbed protein removed from a surface) from dyed surfaces (mimicking fabrics) at different enzyme concentrations. Analysis of kinetic data using an established mathematical model able to decouple enzyme transfer and reaction processes [Onaizi, He, Middelberg, Chem.

Modeling the competition between aggregation and self-assembly during virus-like particle processing.

Understanding and controlling aggregation is an essential aspect in the development of pharmaceutical proteins to improve product yield, potency and quality consistency. Even a minute quantity of aggregates may be reactogenic and can render the final product unusable. Self-assembly processing of virus-like particles (VLPs) is an efficient method to quicken the delivery of safe and efficacious vaccines to the market at low cost.