Development and Application of Synthetic Affinity Ligands for the Purification of Ferritin-Based Influenza Antigens.

A recently developed novel recombinant influenza antigen vaccine has shown great success in preclinical studies in ferrets and mice. It provides broader protection, and is efficient to manufacture compared to the conventional trivalent influenza vaccines (TIV). Each strain of the recombinant antigen has a constant self-assembled bacterial ferritin core which, if used as a target for affinity chromatography, could lead to a universal purification method. Ferritin in silico models were used to explore potential target binding sites against ligands synthesized by the four-component Ugi reaction. Two ligands, SJ047 and SJ055, were synthesized in solution, characterized by 1H, 13C, and 2D NMR spectroscopy, and subsequently immobilized on the PEG-functionalized beads. Ligands SJ047 and SJ055 displayed apparent Kd values of 2.04 × 10-7 M and 1.91 × 10-8 M, respectively, against the ferritin. SJ047 and SJ055-functionalized resins were able to purify hemagglutinin (New Caledonia)-ferritin expressed in a crude Human Embryonic Kidney (HEK) cell supernatant in a single step to a purity of 85 ± 0.5% (97 ± 1% yield) and 87.5 ± 0.5% (95.5 ± 1.5% yield), respectively. Additionally, SJ047 and SJ055-functionalized resins purified the recombinant antigens when spiked at known concentrations into HEK supernatants. All three strains, hemagglutinin (New Caledonia)-ferritin, hemagglutinin (California)-ferritin, and hemagglutinin (Singapore)-ferritin were purified, thereby offering an ideal alternate platform for affinity chromatography. Following elution from the affinity adsorbents, absorbance at 350 nm showed that there was no aggregation of the recombinant antigens and dynamic light scattering studies further confirmed the structural integrity of the recombinant antigen. The use of Ugi ligands coupled to a PEG-spacer arm to target the ferritin core of the strain is entirely novel and provides an efficient purification of these recombinant antigens. This approach represents a potentially universal method to purify any ferritin-based vaccine.

A benzoboroxole-based affinity ligand for glycoprotein purification at physiological pH.

Developing ligands capable of carbohydrate recognition has become increasingly important as the essential roles of glycoproteins and glycolipids in a diverse array of cellular signaling, pathophysiology, and immune response mechanisms are elucidated. Effective ligands for the glycan portion of glycoproteins and glycolipids are needed for pre-enrichment proteomics strategies, as well as for the purification of individual glycoproteins from complex biological milieu encountered both in biochemistry research and bio-pharmaceutical development. In this work, we developed a carbohydrate specific affinity ligand for glycoprotein purification using a one-pot, multi-component synthesis reaction (Ugi synthesis) and an amine-functionalized benzoboroxole moiety immobilized on agarose beads. Benzoboroxoles are unique boronic acid derivatives that have recently been found to bind specifically to the cis-diol groups of carbohydrates at physiological pH, with superior affinity to any other Wulff-type boronic acid. The solid-phase affinity ligand developed herein specifically binds the carbohydrate moiety of the glycoprotein glucose oxidase, as well as a fluorescein isothiocyanate-dextran, as shown through deglycosylation binding studies. Additionally, the ligand is able to purify glucose oxidase from crude Escherichia coli lysate, at physiological pH, equitably to commercially available boronic acid-functionalized agarose beads that required alkaline pH conditions. Thus, this affinity ligand is a marked improvement on current, commercially available boronic acid-based glycoprotein enrichment matrices and has the potential to exhibit high individual glycoprotein specificity because of the additional functional groups available for variation on the Ugi scaffold.

Reusable, robust, and accurate laser-generated photonic nanosensor.

Developing noninvasive and accurate diagnostics that are easily manufactured, robust, and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment. We have developed a clinically relevant optical glucose nanosensor that can be reused at least 400 times without a compromise in accuracy. The use of a single 6 ns laser (λ = 532 nm, 200 mJ) pulse rapidly produced off-axis Bragg diffraction gratings consisting of ordered silver nanoparticles embedded within a phenylboronic acid-functionalized hydrogel. This sensor exhibited reversible large wavelength shifts and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 510-1100 nm. The experimental sensitivity of the sensor permits diagnosis of glucosuria in the urine samples of diabetic patients with an improved performance compared to commercial high-throughput urinalysis devices. The sensor response was achieved within 5 min, reset to baseline in ∼10 s. It is anticipated that this sensing platform will have implications for the development of reusable, equipment-free colorimetric point-of-care diagnostic devices for diabetes screening.

Spore germination mediated by Bacillus megaterium QM B1551 SleL and YpeB.

Previous work demonstrated that Bacillus megaterium QM B1551 spores that are null for the sleB and cwlJ genes, which encode cortex-lytic enzymes (CLEs), either of which is required for efficient cortex hydrolysis in Bacillus spores, could germinate efficiently when complemented with a plasmid-borne copy of ypeB plus the nonlytic portion of sleB encoding the N-terminal domain of SleB (sleB(N)). The current study demonstrates that the defective germination phenotype of B. megaterium sleB cwlJ spores can partially be restored when they are complemented with plasmid-borne ypeB alone. However, efficient germination in this genetic background requires the presence of sleL, which in this species was suggested previously to encode a nonlytic epimerase. Recombinant B. megaterium SleL showed little, or no, activity against purified spore sacculi, cortical fragments, or decoated spore substrates. However, analysis of muropeptides generated by the combined activities of recombinant SleB and SleL against spore sacculi revealed that B. megaterium SleL is actually an N-acetylglucosaminidase, albeit with apparent reduced activity compared to that of the homologous Bacillus cereus protein. Additionally, decoated spores were induced to release a significant proportion of dipicolinic acid (DPA) from the spore core when incubated with recombinant SleL plus YpeB, although optimal DPA release required the presence of endogenous CLEs. The physiological basis that underpins this newly identified dependency between SleL and YpeB is not clear, since pulldown assays indicated that the proteins do not interact physically in vitro.

A tailor-made "tag-receptor" affinity pair for the purification of fusion proteins.

A novel affinity "tag-receptor" pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×10(5) M(-1) ). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.

Investigating the functional hierarchy of Bacillus megaterium PV361 spore germinant receptors.

Spores of Bacillus megaterium QM B1551 germinate rapidly when exposed to a number of single-trigger germinant compounds, including glucose, proline, leucine, and certain inorganic salts. However, spores of strain PV361, a plasmidless QM B1551 derivative that lacks the GerU germinant receptor (GR) responsible for mediating germination in response to single-trigger compounds, can germinate efficiently when incubated in nutritionally rich media, presumably via activation of additional germinant receptors. In this work, we have identified five chromosomally encoded GRs and attempted to characterize, by mutational analysis, germinant recognition profiles associated with the respective receptors in strain PV361. Of strains engineered with single GR insertion-deletions, only GerK-null spores displayed significant defective germination phenotypes when incubated in 5% (wt/vol) beef extract or plated on rich solid medium. Cumulative decreases in viability were observed in GerK-null spores that also lacked GerA or GerA2, indicating that these GRs, which exerted little effect on spore germination when disrupted individually, have a degree of functionality. Unexpectedly, an efficient germination response to combinations of germinants was restored in GerA(+) spores, which lack all other functional GRs, providing evidence for negative cooperativity between some GRs within the spore. Tetrazolium-based germinative assays conducted with purified spores indicated that these newly characterized B. megaterium GRs are cognate for a wide and chemically diverse range of germinant molecules, but unlike GerU, can only be induced to trigger germination when stimulated by at least two different germinants.

A biomimetic Protein G affinity adsorbent: an Ugi ligand for immunoglobulins and Fab fragments based on the third IgG-binding domain of Protein G.

This work reports the development of a synthetic affinity adsorbent for immunoglobulins based on the Fab-binding domain of Streptococcal Protein G (SpG-domain III). The ligand (A2C7I1) was synthesized by the four-component Ugi reaction to generate a substituted peptoidal scaffold mimicking key amino acid residues of SpG. Computer-aided analysis suggests a putative binding site on the CH 1 domain of the Fab molecule. In silico studies, supported by affinity chromatography in comparison with immobilized SpG, as well as analytical characterization by liquid chromatography/electrospray ionization-mass spectrometry and (1) H nuclear magnetic resonance of the ligand synthesized in solution, indicated the authenticity and suitability of the designed ligand for the purification of immunoglobulins. The immobilized ligand displayed an apparent static binding capacity of ~17 mg IgG ml(-1) and a dissociation constant of 5.34 × 10(-5) M. Preparative chromatography demonstrated the ability of the immobilized ligand to purify IgG and Fab fragments from crude mammalian and yeast cell cultures, under near physiological ionic strength and pH, to yield proteins of 99% and 93% purity, respectively.

Design, synthesis, and assessment of a de novo affinity adsorbent for the purification of recombinant human erythropoietin.

This work describes the assessment of a de novo synthetic affinity ligand for recombinant human erythropoietin (rHuEPO), based on the multicomponent Ugi reaction. Four Ugi ligands were designed based on the X-ray crystallographic structure of the complex between human erythropoietin and site 1 of its cell-surface receptor (EPObp)2 ; screening of the ligands with pure rHuEPO samples identified a lead ligand (A9C10I8) immobilized on aldehyde-functionalized agarose beads, which was able to bind and elute erythropoietin, as determined by SDS-PAGE and Western blot analyses. Furthermore, small-scale affinity chromatography performed on the immobilized adsorbent showed its ability to isolate rHuEPO from a spiked mammalian cell supernatant with a purity of ∼80%, as estimated with gel densitometry. This approach could lead to the development of a cost-effective downstream process for rHuEPO, as an alternative to the current multi-step purification protocols.

An acoustic dielectric and mechanical spectrometer.

In this report, the dielectric constant of glycerol solutions (0-70% (w/w)) and the mechanical transitions of poly(2-hydroxylethyl methacrylate-co-methacrylic acid) films (600-800 nm, 1.5-10 mol% cross-linker) have been investigated by the magnetic acoustic resonance sensor (MARS), which is an electrode-free acoustic sensor and operates over a continuous frequency spectrum (6-200 MHz). When a glycerol solution was loaded, the response of the MARS decayed exponentially as the operating frequency was increased. The decay rate against frequency as a function of the glycerol concentration reflects the change of the dielectric property of the glycerol solutions. In addition, mechanical relaxation of the poly(2-hydroxylethyl methacrylate-co-methacrylic acid) film has been observed on the MARS and the corresponding viscoelastic transition frequency has been estimated. The viscoelastic transition frequency increased as the polymer was more highly cross-linked. The MARS system behaved as a dielectric and mechanical spectrometer, monitoring the electrical and mechanical properties of viscoelastic materials or on the solid-liquid interfaces simultaneously, which has prospective application in studies of biomaterials, molecular interactions and drug deliveries.

Identification of a receptor subunit and putative ligand-binding residues involved in the Bacillus megaterium QM B1551 spore germination response to glucose.

The molecular basis for the recognition of glucose as a germinant molecule by spores of Bacillus megaterium QM B1551 has been examined. A chromosome-located locus (BMQ_1820, renamed gerWB) is shown to encode a receptor B-protein subunit that interacts with the GerUA and GerUC proteins to form a receptor that is cognate for both glucose and leucine. GerWB represents the third receptor B protein that binds to glucose in this strain. Site-directed mutagenesis (SDM) experiments conducted on charged proline and aromatic residues predicted to reside in the transmembrane domains of a previously characterized receptor B protein, GerVB, reveal the importance to receptor function of a cluster of residues predicted to reside in the middle of the transmembrane 6 (TM6) domain. Reductions in the region of 70- to 165-fold in the apparent affinity of receptors for glucose in which Glu196, Tyr191, and Phe192 are individually replaced by SDM indicate that some or all of these residues may be directly involved in the binding of glucose and perhaps other germinants to the germinant receptor.

Mutational analysis of Bacillus megaterium QM B1551 cortex-lytic enzymes.

Molecular-genetic and muropeptide analysis techniques have been applied to examine the function in vivo of the Bacillus megaterium QM B1551 SleB and SleL proteins. In common with Bacillus subtilis and Bacillus anthracis, the presence of anhydromuropeptides in B. megaterium germination exudates, which is indicative of lytic transglycosylase activity, is associated with an intact sleB structural gene. B. megaterium sleB cwlJ double mutant strains complemented with engineered SleB variants in which the predicted N- or C-terminal domain has been deleted (SleB-ΔN or SleB-ΔC) efficiently initiate and hydrolyze the cortex, generating anhydromuropeptides in the process. Additionally, sleB cwlJ strains complemented with SleB-ΔN or SleB-ΔC, in which glutamate and aspartate residues have individually been changed to alanine, all retain the ability to hydrolyze the cortex to various degrees during germination, with concomitant release of anhydromuropeptides to the surrounding medium. These data indicate that while the presence of either the N- or C-terminal domain of B. megaterium SleB is sufficient for initiation of cortex hydrolysis and the generation of anhydromuropeptides, the perceived lytic transglycosylase activity may be derived from an enzyme(s), perhaps exclusively or in addition to SleB, which has yet to be identified. B. megaterium SleL appears to be associated with the epimerase-type activity observed previously in B. subtilis, differing from the glucosaminidase function that is apparent in B. cereus/B. anthracis.

Effect of enzymatic deimination on the conformation of recombinant prion protein.

Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.

Holographic enzyme inhibition assays for drug discovery.

Optical sensors are widely utilized in drug discovery to analyze biomolecular interactions in vitro. Aside from additional time and cost demands, other issues associated with labeled screening methods include signal interference that can arise from the label per se and/or the screened compounds. This report describes an enzyme inhibition-based holographic sensor as a potential label-free detection system, using acetylcholinesterase (acetylcholine acetylhydrolase; EC 3.1.1.7; abbreviated herein AChE) as the model enzyme. pH-responsive reflection holograms, incorporated into "smart" hydrogel films bearing ionizable monomers, were used to monitor the pH change resulting from acetic acid produced by the hydrolysis of the substrate acetylcholine. The enzyme was immobilized on the sensor by an entrapment and in situ cross-linking method; no chemical modification and/or prelabeling of the enzyme (or the substrate) was required. The fully reversible sensor exhibited good operational and storage stability, allowing relatively short assay times and repeated use of a single sensor. Apparent inhibition parameters for several drug inhibitors of the enzyme were determined. The feasibility of adapting these sensors into an array format for prospective high-throughput screening, without compromising their intrinsic optical and functional properties, was also demonstrated.

One-step polymeric phononic crystal manufacture.

A versatile system to construct bulk polymeric phononic crystals by using acoustic waves is described. In order to fabricate this material, a customised cavity device fitted with a ∼2 MHz acoustic transducer and an acoustic reflector is employed for the acoustic standing wave creation in the device chamber. The polymer crystal is formed when the standing waves are created during the polymerisation process. The resulting crystals are reproduced into the shape of the tunable device cavity with a unique periodic feature. The separation is related to the applied acoustic wave frequency during the fabrication process and each unit cell composition was found to be made up to two material phases. To assess the acoustic properties of the polymer crystals their average acoustic velocity is measured relative to monomer solutions of different concentrations. It is demonstrated that one of the signature characteristics of phononic crystal, the slow wave effect, was expressed by this polymer. Furthermore the thickness of a unit cell is analysed from images obtained with microscope. By knowing the thickness the average acoustic velocity is calculated to be 1538 m/s when the monomer/cross-linker concentration is 1.5 M. This numerical calculation closely agrees with the predicted value for this monomer/cross-linker concentration of 1536 m/s. This work provides a methodology for rapid accessing a new type of adaptable phononic crystal based on flexible polymers.

Miniaturized pH Holographic Sensors for the Monitoring of Lactobacillus casei Shirota Growth in a Microfluidic Chip.

Bioreactors have been used both to develop new, and to improve bioprocess yields for, biopharmaceutical products. However, efforts to miniaturize bioreactors, in order to save costs and accelerate process development times, have been limited by the lack of on-site monitoring capabilities available at such scales. In this study, small volume (3 nL) nonconsumptive holographic sensors were integrated into a glass-PDMS microfluidic chip to monitor via a blue-shift in the resultant holographic replay wavelength, the change in pH during microbial growth of Lactobacillus casei ( L. casei) Shirota. Within the optimal growth pH range of L. casei, the accuracy of the miniaturized pH sensors was comparable to that of a conventional pH meter. Conceivably, this approach could be extrapolated to an array of miniaturized holographic sensors sensitive to different analytes, and thereby paving the way for reliable, real-time, noninvasive monitoring of microorganisms in a nanobioreactor.

Towards Closed-Loop Integration of Point-of-Care Technologies.

Point-of-care (PoC) diagnostics promises to yield test results accessible anytime and anywhere. Its application has expanded from providing healthcare necessities to the real-time monitoring of the ageing and health conscious population. Following the evolving consumer demand, there is a trend toward developing non- and minimally invasive PoC tests. Emerging PoC sensors have not only demonstrated multifunctional capabilities such as sweat stimulation but also can be connected to drug delivery units via a wireless network, enabling an active role of the technology in disease management. This review article summarises the latest developments in non- and minimally invasive PoC diagnostics and provides an overview on the progress towards closed-loop integration of complementary technologies for comprehensive and autonomous patient care.

Density Modulation of Embedded Nanoparticles via Spatial, Temporal, and Chemical Control Elements.

Nanoparticle polymer composites have enabled material multifunctionalities that are difficult to obtain otherwise. A simple modification to a commercially available resin system enables a universal methodology to embed nanoparticles in resins via spatial, temporal, thermal, concentration, and chemical control parameters. Changes in nanoparticle density distribution are exploited to demonstrate dynamic optical and electronic properties that can be processed on-demand, without the need for expensive equipment or cleanroom facilities. This strategy provides access to the control of optical (cooperative plasmonic effects), electronic (insulator to a conductor), and chemical parameters (multimetal patterning). Using the same composite resin system, the followings are fabricated: i) diffraction gratings with tuneable diffraction efficiencies (10-78% diffraction efficiencies), ii) organic electrochemical transistors with a low drive voltage, and iii) embedded electrodes in confined spaces for potential diagnostic applications.

Amino acid substitutions in transmembrane domains 9 and 10 of GerVB that affect the germination properties of Bacillus megaterium spores.

The molecular basis for differences in germinant recognition of Bacillus megaterium QM B1551 spores containing the GerVB and/or GerUB receptor proteins has been examined by site-directed mutagenesis and the construction of cross-homologue chimeras. Focusing on nonconserved residues predicted to reside in transmembrane domains 9 and 10, we demonstrate that GerVB residues Ser319 and Leu345 are of particular importance in defining the specificity and apparent affinity of the receptor for germinants. Kinetic analyses of mutants with different amino acid substitutions at these positions indicate that Ser319 and Leu345 are not involved directly in the binding of germinants, but probably reside in regions of the receptor where structural perturbations can affect the conformation of, or access to, germinant binding sites. Position 345 is also shown to be of importance in GerUB, where the F345A mutation severely impairs receptor function. Functionality is restored in the GerUB Ala345 background by substituting putative outer-loop residues adjacent to TM10 for the corresponding residues in GerVB, indicating that a degree of structural coordination between these regions is important to receptor function.