Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.

Background: Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula.

Selection and characterization of DNA aptamers for the rat major urinary protein 13 (MUP13) as selective biorecognition elements for sensitive detection of rat pests.

Among invasive mammalian predators, rats represent a major threat, endangering ecosystem functioning worldwide. After rat-control operations, detecting their continued presence or reinvasion requires more sensitive and lower cost detection technologies. Here, we develop a new sensing paradigm by using a specific rat urine biomarker (MUP13) to unambiguously signal the presence of rats.

Insect odorant receptor-based biosensors: Current status and prospects.

Whilst the senses of vision and hearing have been successfully automated and miniaturized in portable formats (e.g. smart phone), this is yet to be achieved with the sense of smell.

Synergistic improvement in the performance of insect odorant receptor based biosensors in the presence of Orco.

Insect odorant receptors (ORs) are believed to be a complex of an odorant binding subunit, OrX, and an ion channel forming subunit, Orco. In our previous study, we showed that the OrX subunit on its own in liposomes could detect volatile organic compounds (VOCs) ultrasensitively using Electrochemical Impedance Spectroscopy (EIS). In this study, we investigated the effect of the presence of Orco on the response of the OrX subunit to detect the VOCs.

Biosensing with Insect Odorant Receptor Nanodiscs and Carbon Nanotube Field-Effect Transistors.

Insect odorant receptors have been reconstituted into lipid nanodiscs and tethered to carbon nanotube field-effect transistors to function as a biosensor. Here, four different insect odorant receptors (ORs) from Drosophila melanogaster (DmelOR10a, DmelOR22a, DmelOR35a, and DmelOR71a) were expressed in Sf9 cells, purified, and reconstituted into lipid nanodiscs. We have demonstrated that each of these ORs produce a selective and highly sensitive electrical response to their respective positive ligands, methyl salicylate, methyl hexanoate, trans-2-hexen-1-al, and 4-ethylguaiacol, with limits of detection in the low femtomolar range.

Data on preparation and characterization of an insect odorant receptor based biosensor.

Insect Odorant receptors (OrXs) can be used as the recognition element in a biosensor as they demonstrate high levels of sensitivity and selectivity towards volatile organic compounds. Herein, we describe a method to express and purify insect odorant receptors and reconstitute them into artificial lipid bilayers (liposomes). These OrX/liposomes were covalently attached to a gold surface and characterized using quartz crystal microbalance with dissipation monitoring (QCM-D).

Common Variants of the Plant microRNA-168a Exhibit Differing Silencing Efficacy for Human Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1).

Background: The discovery that a plant microRNA (miRNAs) from rice (Oryza sativa miR168a) can modify post-transcriptional expression of the mammalian. Low-Density Lipoprotein Receptor Adaptor Protein 1 (LDLRAP1) gene highlights the potential for cross-kingdom miRNAmRNA interactions.

Objective: To investigate whether common variants of the conserved miR168a family have the capability for similar cross-kingdom regulatory functions, we selected sequences from three dietary plant sources: rice (Oryza sativa), tomato (Solanum lycopersicum), apple (Malus domestica) and compared their ability to regulate human LDLRAP1 expression.

An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants.

Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100-200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface.

Data on liquid gated CNT network FETs on flexible substrates.

This article presents the raw and analyzed data from a set of experiments performed to study the role of junctions on the electrostatic gating of carbon nanotube (CNT) network field effect transistor (FET) aptasensors. It consists of the raw data used for the calculation of junction and bundle densities and describes the calculation of metallic content of the bundles. In addition, the data set consists of the electrical measurement data in a liquid gated environment for 119 different devices with four different CNT densities and summarizes their electrical properties.

Metallic-semiconducting junctions create sensing hot-spots in carbon nanotube FET aptasensors near percolation.

Easily fabricated random network carbon nanotube field-effect transistors (CNT-FETs) have benefitted from improved separation techniques to deliver CNTs with current formulations providing at least 99% semiconducting tube content. Amongst the most promising applications of this device platform are electronic biosensors, where the network conduction is affected through tethered probes such as aptamers which act as molecular scale electrostatic gates. However, the prevailing assumption that these biosensor devices would be optimized if metallic tubes were entirely eliminated has not been examined.

Improving odorant chemical class prediction with multi-layer perceptrons using temporal odorant spike responses from drosophila melanogaster olfactory receptor neurons.

In this work, we examine the possibility of improving the prediction performance of an olfactory biosensor through the use of temporal spiking data. We present an Artificial Neural Network (ANN), in the form of an optimal hybrid Multi-Layer Perceptron (MLP) system for the classification of chemical odorants from olfactory receptor neuron spike responses of the Drosophila melanogaster fruit fly (DmOrs). The data used in this study contains the responses to 34 odorants from 6 individual DmOrs, of which we exploit the temporal spiking responses of a 500ms odorant stimulus window.

Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

Background: The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts.

Functional implications of large backbone amplitude motions of the glycoprotein 130-binding epitope of interleukin-6.

Human interleukin (IL)-6 plays a pivotal role in the immune response, hematopoiesis, the acute-phase response, and inflammation. IL-6 has three distinct receptor epitopes, termed sites I, II, and III, that facilitate the formation of a signaling complex. IL-6 signals via a homodimer of glycoprotein 130 (gp130) after initially forming a heterodimer with the nonsignaling α-receptor [IL-6 α-receptor (IL-6R)] via site I.

A cell-free expression screen to identify fusion tags for improved protein expression.

Cell-free protein synthesis can now be routinely used for the rapid screening of protein expression at the microliter level using PCR-amplified templates. However, identification of the optimal expression construct for a target protein can still be a problem. A rapid cell-free procedure is described here for the systematic assessment of a range of diverse fusion tags on the expression and solubility of any given target protein.

Polymorphism of FtsZ filaments on lipid surfaces: role of monomer orientation.

FtsZ is a bacterial cytoskeletal protein involved in cell division. It forms a ringlike structure that attaches to the membrane to complete bacterial division. It binds and hydrolyzes GTP, assembling into polymers in a GTP-dependent manner.

Insights into subunit interactions within the insect olfactory receptor complex using FRET.

Insect olfactory receptors (ORs) are a novel family of ligand-gated cation channels that can respond to volatile organic compounds at low concentrations. They are involved in the detection of odorants associated with mate recognition, food localisation and predator avoidance. These receptors form a complex that is currently thought to contain at least two subunit members: the non-canonical Orco ion channel subunit and a ligand-binding receptor subunit.

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags.

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days.

Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase.

Cell-free synthesis and combinatorial selective 15N-labeling of the cytotoxic protein amoebapore A from Entamoeba histolytica.

Amoebapore A is a pore-forming protein produced by the pathogenic parasite Entamoeba histolytica, which causes human amoebic dysentery. The pore-forming activity of amoebapore A is regulated by pH-dependent dimerization, a prerequisite for membrane insertion and pore formation. Understanding of these important processes has been hampered by the cytotoxicity of amoebapore A, which prevents the production of this protein in cell-based expression systems.

Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants.

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture.

Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins.

Olfaction in Drosophila is mediated by a large family of membrane-bound odorant receptor proteins (Ors). In heterologous cells, we investigated whether the structural features and signalling mechanisms of ligand-binding Drosophila Ors are consistent with them being G protein-coupled receptors (GPCRs). The detailed membrane topology of Or22a was determined by inserting epitope tags into the termini and predicted loop regions.

Functional analysis of a Drosophila melanogaster olfactory receptor expressed in Sf9 cells.

Olfactory receptors (ORs) are seven transmembrane proteins that are responsible for the transduction of volatiles into neuronal signals. Their low sequence homology means that the prediction of ligands for ORs based on extrapolation from empirical data of other ORs is difficult, so an experimental approach must be used. Here, we report a functional assay for insect ORs using calcium-imaging in Sf9 cells.

Kinetic and crystallographic analysis of mutant Escherichia coli aminopeptidase P: insights into substrate recognition and the mechanism of catalysis.

Aminopeptidase P (APPro) is a manganese-dependent enzyme that cleaves the N-terminal amino acid from polypeptides where the second residue is proline. APPro shares a similar fold, substrate specificity, and catalytic mechanism with methionine aminopeptidase and prolidase. To investigate the roles of conserved residues at the active site, seven mutant forms of APPro were characterized kinetically and structurally.