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. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles.

Receptor-specific delivery of protein antigen to dendritic cells by a nanoemulsion formed using top-down non-covalent click self-assembly.

A new class of targeted and immune-evading nanocarrier made using only biological components and facile processes is assembled in a bottom-up fashion. Simple top-down sequential addition of immune-evading or receptor-specific antibody elements conjugated to biosurfactant protein DAMP4 promotes self-assembly at an interface previously formed in the presence of peptide surfactant AM1, leading to a functional display at the interface through non-covalent molecular self-assembly.

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

Microbial bio-production of a recombinant stimuli-responsive biosurfactant.

Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states.

Encapsulation of DNA and non-viral protein changes the structure of murine polyomavirus virus-like particles.

Asymmetrical-flow field flow fractionation with multiple-angle light scattering (AFFFF-MALS) was, for the first time, used to characterize the size of murine polyomavirus virus-like particles (MPV VLPs) packaged with either insect cell genomic DNA or non-viral protein. Encapsidation of both genomic DNA and non-viral protein were found to cause a contraction in VLP radii of gyration by approximately 1 nm. Non-viral protein packaged into VLPs consisted of a series of glutathione-S-transferase, His and S tags attached to the N-terminal end of the MPV structural protein VP2 (M(r) = 67108). Transmission electron microscopy analysis of MPV VLPs packaging non-viral protein suggested that VLPs grew in diameter by approximately 5 nm, highlighting the differences between this invasive technique and the relatively non-invasive AFFFF-MALS technique. Encapsulation of non-viral protein into MPV VLPs was found to prevent co-encapsidation of genomic DNA. Further investigation into why this occurred led to the discovery that encapsulation of non-viral protein alters the nuclear localization of MPV VLPs during in vivo assembly. VLPs were relocated away from the ring zone and the nuclear membrane towards the centre of the nucleus amongst the virogenic stroma. The change in nuclear localization away from the site where VLP assembly usually occurs is a likely reason why encapsidation of genomic DNA did not take place.

Expression and purification of a nanostructure-forming peptide.

Peptides have recently attracted interest as building blocks for the assembly of novel functional materials including switchable surfactants, nanocoatings, hydrogels and aqueous vesicles. We expressed a beta-sheet forming peptide that has been widely studied in self-assembly processing, P(11)-2, as a monomer, dimer, tetramer and nonamer fused to an insoluble expression partner, ketosteroid isomerase, using minimal media. Expression was followed by whole cell extraction and isolation of the fusion protein to greater than 90% purity via a single immobilised metal affinity chromatography (IMAC) step. Peptides were chemically cleaved from each other and from the fusion partner, followed by acetone precipitation of the contaminating protein fragments. Pure peptide was recovered by reversed-phase HPLC. The expression level of the fusion protein decreased as the peptide concatamer number increased, as did the efficiency of the chemical cleavage, making the single-peptide process the most efficient overall. Applying this laboratory process to the single-peptide fusion protein nevertheless resulted in a pure peptide yield of greater than 30% of the expressed peptide.

The interfacial structure and Young's modulus of peptide films having switchable mechanical properties.

We report the structure and Young's modulus of switchable films formed by peptide self-assembly at the air-water interface. Peptide surfactant AM1 forms an interfacial film that can be switched, reversibly, from a high- to low-elasticity state, with rapid loss of emulsion and foam stability. Using neutron reflectometry, we find that the AM1 film comprises a thin (approx. 15A) layer of ordered peptide in both states, confirming that it is possible to drastically alter the mechanical properties of an interfacial ensemble without significantly altering its concentration or macromolecular organization. We also report the first experimentally determined Young's modulus of a peptide film self-assembled at the air-water interface (E=80MPa for AM1, switching to E<20MPa). These findings suggest a fundamental link between E and the macroscopic stability of peptide-containing foam. Finally, we report studies of a designed peptide surfactant, Lac21E, which we find forms a stronger switchable film than AM1 (E=335MPa switching to E<4MPa). In contrast to AM1, Lac21E switching is caused by peptide dissociation from the interface (i.e. by self-disassembly). This research confirms that small changes in molecular design can lead to similar macroscopic behaviour via surprisingly different mechanisms.

The economics of inclusion body processing.

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

Quantification of solid cell material by detection of membrane-associated proteins and peptidoglycan.

Quantification of solid cell material (cell debris) is necessary for the optimisation of the efficiency of bioseparations. Cell debris can be quantified by detection of a component present in the cell wall that can act as a marker for cell debris. Membrane-associated proteins have previously been used as a marker for cell debris. This marker was quantified by SDS-PAGE with densiometry. In this paper cell debris quantification methods are presented that are faster and more accurate, i.e. membrane-associated protein quantification with the Protein 50 Labchip of Agilent Technologies, or that make use of peptidoglycan as marker for cell debris, i.e. a spectrophotometric muramic acid assay.

Quantitative magnetic resonance imaging of urea and lysozyme in protein chromatography.

Magnetic resonance imaging (MRI) techniques have been implemented to enable quantitative imaging of protein and urea within a 5 ml HiTrap size-exclusion chromatography desalting column, without introduction of contrast agents. One-, two- and three-dimensional images of urea injected at concentrations of 2, 4, 6 and 8 M were acquired. One-dimensional profiles of lysozyme at concentrations between 5 and 25 mg ml(-1) were also obtained. All data were accurate to within +/- 15% when compared to the known amount injected. Quantitative MRI elution profiles of both urea and lysozyme were then obtained in real-time during a desalting separation.

Dielectrophoretic manipulation of surface-bound DNA.

Dielectrophoretic manipulation enables the positioning and orientation of DNA molecules for nanometer-scale applications. However, the dependence of the dielectrophoretic force and torque on the electric field magnitude and frequency has to be well characterised to realise fully the potential of this technique. DNA in solution is attracted to the strongest electric field gradient (i.e. the electrode edge) as a result of the dielectrophoretic force, while the dielectrophoretic torque aligns the DNA with its longest axis parallel to the electric field. In this work, the authors attached -DNA fragments (48 and 25 kilobases) to an array of gold microelectrodes via a terminal thiol bond and characterised the orientation and elongation as a function of electric field magnitude (0.1-0.8 MVm) and frequency (0.08-1.1 MHz). Maximum elongation was observed between 200 and 500 kHz for the attached DNA. Dielectrophoresis is limited by thermal randomisation at electric fields below 0.1 MVm and by electrothermal effects above 0.7 MVm. The authors conclude that dielectrophoresis can be used to manipulate surface-immobilised DNA reproducibly.

The production of human papillomavirus type 16 L1 vaccine product from Escherichia coli inclusion bodies.

The major capsid protein L1 of the human papillomavirus type 16 (HPV16) has been previously expressed recombinantly in Escherichia coli cells as inclusion bodies (IBs). The HPV16 L1 protein offers potential as a vaccine candidate against cervical cancer, but the reported E. coli process is limited in its ability to economically produce significant quantities of material. In this study, a scaleable laboratory process for the purification of recombinant His-tagged L1 protein and its processing to give an immunogenic product is developed. The performances of ion-exchange chromatography (IEX) and immobilised metal affinity chromatography (IMAC) for the purification of L1 protein in the presence of concentrated denaturant are compared. IEX was found to be superior to IMAC when taking into account the complexity of operation, cost of adsorbent, selectivity and purity of the final product. Following purification, reduction of denaturant concentration was performed by dilution to yield a product suitable for formulation. The simplicity and ease of scale-up of dilution makes it an attractive option for process scale production and superior to the existing approach employing dialysis. It was found that direct dilution of denaturant into suitable buffer can give rise to products which have neutralising conformational epitopes identified by strong antibody-binding properties, as assessed by ELISA with a conformational monoclonal antibody. Analysis of the results showed negative main effects of protein concentration and PEG addition on antibody-binding yields, but positive main effects of the addition of detergent and L-arginine to the buffer. The diluted product had antigenic properties as assessed by ELISA and may be formulated easily for use by diafiltration and the addition of adjuvant. This work demonstrates the feasibility of producing viral vaccines using E. coli and scaleable unit operations.