Antibody mimetic receptor proteins for label-free biosensors.

The development of high sensitivity biosensors, for example for clinical diagnostics, requires the identification of suitable receptor molecules which offer high stability, specificity and affinity, even when embedded into solid-state biosensor transducers. Here, we present an electrochemical biosensor employing small synthetic receptor proteins (Mw < 15 kDa) which emulate antibodies but with improved stability, sensitivity and molecular recognition properties, in particular when immobilized on a solid sensor surface. The synthetic receptor protein is a non-antibody-based protein scaffold with variable peptide regions inserted to provide the specific binding, and was designed to bind anti-myc tag antibody (Mw ∼ 150 kDa), as a proof-of-principle exemplar. Both the scaffold and the selected receptor protein were found to have high thermostability with melting temperatures of 101 °C and 85 °C, respectively. Furthermore, the secondary structures of the receptor protein were found to be very similar to that of the original native scaffold, despite the insertion of variable peptide loops that create the binding sites. A label-free electrochemical sensor was fabricated by functionalising a microfabricated gold electrode with the receptor protein. A change in the phase of the electrochemical impedance was observed when the biosensor was subjected to anti-myc tag antibodies at concentrations between 6.7 pM and 6.7 nM. These findings demonstrate that these non-antibody receptor proteins are excellent candidates for recognition molecules in label-free biosensors.

Visualization of dioxygen bound to copper during enzyme catalysis.

X-ray crystal structures of three species related to the oxidative half of the reaction of the copper-containing quinoprotein amine oxidase from Escherichia coli have been determined. Crystals were freeze-trapped either anaerobically or aerobically after exposure to substrate, and structures were determined to resolutions between 2.1 and 2.4 angstroms. The oxidation state of the quinone cofactor was investigated by single-crystal spectrophotometry. The structures reveal the site of bound dioxygen and the proton transfer pathways involved in oxygen reduction. The quinone cofactor is regenerated from the iminoquinone intermediate by hydrolysis involving Asp383, the catalytic base in the reductive half-reaction. Product aldehyde inhibits the hydrolysis, making release of product the rate-determining step of the reaction in the crystal.

Alternative reagents to antibodies in imaging applications.

Antibodies have been indispensable tools in molecular biology, biochemistry and medical research. However, a number of issues surrounding validation, specificity and batch variation of commercially available antibodies have prompted research groups to develop novel non-antibody binding reagents. The ability to select highly specific monoclonal non-antibody binding proteins without the need for animals, the ease of production and the ability to site-directly label has enabled a wide variety of applications to be tested, including imaging. In this review, we discuss the success of a number of non-antibody reagents in imaging applications, including the recently reported Affimer.

Crystal structure of a quinoenzyme: copper amine oxidase of Escherichia coli at 2 A resolution.

BACKGROUND: Copper amine oxidases are a ubiquitous and novel group of quinoenzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes, with concomitant reduction of molecular oxygen to hydrogen peroxide. The enzymes are dimers of identical 70-90 kDa subunits, each of which contains a single copper ion and a covalently bound cofactor formed by the post-translational modification of a tyrosine side chain to 2,4,5-trihydroxyphenylalanine quinone (TPQ). RESULTS: The crystal structure of amine oxidase from Escherichia coli has been determined in both an active and an inactive form. The only structural differences are in the active site, where differences in copper coordination geometry and in the position and interactions of the redox cofactor, TPQ, are observed. Each subunit of the mushroom-shaped dimer comprises four domains: a 440 amino acid C-terminal beta sandwich domain, which contains the active site and provides the dimer interface, and three smaller peripheral alpha/beta domains (D1-D3), each of about 100 amino acids. D2 and D3 show remarkable structural and sequence similarity to each other and are conserved throughout the quinoenzyme family. In contrast, D1 is absent from some amine oxidases. The active sites are well buried from solvent and lie some 35 A apart, connected by a pair of beta hairpin arms. CONCLUSIONS: The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

Label-free electrochemical impedance biosensor to detect human interleukin-8 in serum with sub-pg/ml sensitivity.

Biosensors with high sensitivity and short time-to-result that are capable of detecting biomarkers in body fluids such as serum are an important prerequisite for early diagnostics in modern healthcare provision. Here, we report the development of an electrochemical impedance-based sensor for the detection in serum of human interleukin-8 (IL-8), a pro-angiogenic chemokine implicated in a wide range of inflammatory diseases. The sensor employs a small and robust synthetic non-antibody capture protein based on a cystatin scaffold that displays high affinity for human IL-8 with a KD of 35 ± 10 nM and excellent ligand specificity. The change in the phase of the electrochemical impedance from the serum baseline, ∆θ(ƒ), measured at 0.1 Hz, was used as the measure for quantifying IL-8 concentration in the fluid. Optimal sensor signal was observed after 15 min incubation, and the sensor exhibited a linear response versus logarithm of IL-8 concentration from 900 fg/ml to 900 ng/ml. A detection limit of around 90 fg/ml, which is significantly lower than the basal clinical levels of 5-10 pg/ml, was observed. Our results are significant for the development of point-of-care and early diagnostics where high sensitivity and short time-to-results are essential.

Crystallization of the NADP(+)-dependent glutamate dehydrogenase from Escherichia coli.

The NADP(+)-dependent hexameric glutamate dehydrogenase from Escherichia coli has been crystallized as the apo-enzyme and also in the presence of its substrates 2-oxoglutarate, glutamate or NADP+, using either pulsed equilibrium microdialysis, or the hanging drop method of vapour diffusion. Three non-isomorphous, but related, crystal forms have been obtained, all of which belong to the orthorhombic system and are most likely to be in space group P2(1)2(1)2(1). One crystal form is grown from ammonium sulphate, includes the apoenzyme and the binary complexes with 2-oxoglutarate or NADP+, and has cell dimensions a = 157.5 A, b = 212.5 A, c = 101.0 A with a hexamer in the asymmetric unit. Crystallizations using glutamate as the precipitant produced two further crystal forms, which show significant changes in the b and c cell dimensions with respect to the apo-enzyme crystals, with parameters a = 160.0 A, b = 217.5 A c = 92.4 A and a = 160.0 A, b = 223.0 A c = 92.4 A, respectively. X-ray diffraction photographs taken with synchrotron radiation show measurable reflections to beyond 3.0 A resolution.

Identification, cloning, and sequence analysis of the nitrogen regulation gene ntrC of Agrobacterium tumefaciens C58.

We describe the cloning of an ntrC gene of Agrobacterium tumefaciens C58 by interspecific complementation of an Escherichia coli ntrC mutant. Restriction mapping and Southern blot analysis of the complementing clone identified a 1.7-kb EcoRI-PvuII DNA fragment whose sequence was determined. Analysis of this sequence revealed coding regions corresponding to a complete ntrC gene and the C-terminal region of an ntrB gene. Amino acid sequence comparisons of A. tumefaciens NTRC protein with NTRC sequences from Rhizobium meliloti, Bradyrhizobium sp. (Parasponia), Klebsiella pneumoniae, E. coli, and Salmonella typhimurium show strong sequence conservation supporting DNA hybridization data, demonstrating strong evolutionary homology among ntrC genes of Rhizobiaceae. The C58 NTRC protein has been identified, by 35S-labeling, in a T7 RNA polymerase (pT7-7) expression vector system.

Continual green-fluorescent protein monitoring of cauliflower mosaic virus 35S promoter activity in nematode-induced feeding cells in Arabidopsis thaliana.

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressive from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.

Molecular analysis of the recA gene of Agrobacterium tumefaciens C58.

The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined. A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation. However, unlike its Escherichia coli equivalent, A. tumefaciens recA lacks the upstream 'SOS box', suggesting a different mechanism of regulation for this gene.

Alginate lyase from Klebsiella pneumoniae, subsp. aerogenes: gene cloning, sequence analysis and high-level production in Escherichia coli.

The alyA gene, encoding a secreted guluronate-specific alginate lyase (Aly) from Klebsiella pneumoniae subsp. aerogenes type 25, has been cloned. DNA sequence analysis reveals two possible translation start sites for the precursor form of Aly and a long open reading frame (ORF) predicted to encode a 287-amino-acid (aa) mature form of Aly, in agreement with N-terminal aa sequence analysis of the protein. Aly has a calculated molecular mass of 31.4 kDa, in good agreement with SDS-PAGE analysis, and a calculated pI of 9.39. Comparison of the deduced aa sequence with a mannuronate-specific lyase from a marine bacterium reveals 19.3% identity and 28.8% similarity with a 9-aa conserved region close to the C terminus, probably of functional or structural significance. There is no obvious sequence similarity with pectate lyases which also catalyse a beta-elimination reaction. Heterologous expression of K. pneumoniae alyA in Escherichia coli yields 10 mg of Aly per litre of culture supernatant, apparently due to non-specific release from the periplasm.

Respiratory nitrate reductase of Escherichia coli. Sequence identification of the large subunit gene.

The nucleotide sequence is presented of part of the transcription initiation end of the nar operon of Escherichia coli K12, which encodes the respiratory nitrate reductase complex. The first coding sequence transcribed is the narG gene, encoding the large catalytic molybdoprotein of the complex. This sequence was assigned unambiguously by automated N-terminal amino acid sequencing of the purified large subunit. The deduced partial amino acid sequence of this polypeptide is hydrophilic and rich in basic residues. Membrane insertion does not involve N-terminal proteolytic processing of this subunit.

Structural and kinetic studies of a series of mutants of galactose oxidase identified by directed evolution.

Galactose oxidase (GO; E.C. 1.1.3.9) is a copper- containing enzyme that oxidizes a range of primary alcohols to aldehydes. This broad substrate specificity is reflected in a high K(M) for substrates. Directed evolution has previously been used to select variants of GO that exhibit enhanced expression and kinetic properties. In assays using unpurified enzyme samples, the variant C383S displayed a 5-fold lower K(M) than wild-type GO. In the present study, we have constructed, expressed, purified and characterized a number of single, double and triple mutants at residues Cys383, Tyr436 and Val494, identified in one of the directed evolution studies, to examine their relative contributions to improved catalytic activity of GO. We report kinetic studies on the various mutant enzymes. In addition, we have determined the three-dimensional structure of the C383S variant. As with many mutations identified in directed evolution experiments, the availability of structural information does not provide a definitive answer to the reason for the improved K(M) in the C383S variant protein.

Protease inhibitors and directed evolution: enhancing plant resistance to nematodes.

Plant nematodes are agricultural pests, the control of which relies on chemical nematicides and fumigants that are among the most toxic and environmentally damaging of all agrochemicals. New approaches to control, based on transgenic resistance, would provide important health and environmental benefits. In this chapter we consider briefly some targets for engineering nematode resistance and discuss the use of plant protease inhibitors as anti-feedants. This approach has provided plants that display good levels of resistance against a range of nematode species. To enhance this defence strategy further we are investigating the value of directed evolution to improve the characteristics of protease inhibitors. We describe the approaches of DNA shuffling and phage display that are being used to create and screen variant libraries in the search for inhibitors with improved features.

Characterization of cDNAs encoding serine proteinases from the soybean cyst nematode Heterodera glycines.

Three cDNAs encoding serine proteinases (HGSPI-III) were isolated from a cDNA library constructed from feeding females of Heterodera glycines. The library was screened with three separate serine proteinase gene fragments amplified from cDNA of H. glycines using consensus oligonucleotide primers. Each predicted protein contains a secretion signal sequence, a propeptide and a mature protein of 226-296 amino acids. One of the predicted enzymes, HGSP-II has 41% identity to a chymotrypsin-like enzyme from the mollusc, Haliotis rufescens, and analysis of key residues involved in substrate binding also suggests a chymotrypsin-like specificity. HGSP-I and HGSP-III show greatest homology to kallikreins but sequence analysis does not allow prediction of their substrate preferences. Southern blot analysis suggests that HGSP-II and HGSP-III are encoded by single-copy genes in contrast to HGSP-I which may have two or more homologues. The regions encoding the mature proteinases were cloned into an expression vector and recombinant protein produced in Escherichia coli. Both HGSP-I and HGSP-II were shown, after refolding, to cleave the synthetic peptide N-CBZ-Phe-Arg-7-amido-4-methylcoumarin, and this activity could be inhibited by the cowpea trypsin inhibitor, CpTI. HGSP-III showed no activity against the synthetic substrates tested. The information gained from these studies indicates that serine proteinases are an important group of enzymes in H. glycines and further characterization will aid the development of a proteinase inhibitor-based approach for transgenic plant resistance to plant parasitic nematodes.

Detection of a homologue to an E. coli glutamate synthase gene in a cyanobacterium.

The gltBY locus of E. coli, which codes for the two subunits of pyridine nucleotide dependent glutamate synthase, was used as a probe to detect homologues in genomic DNA of Synechococcus PCC6301, a unicellular cyanobacterium. Non-overlapping probes from gltB detected a single homologue with extensive homology, however gltY probes do not detect a strong homologue. The possibility that the gltB homologue encodes a ferredoxin-dependent glutamate synthase of the type found in cyanobacteria, algae and plants is discussed.

General practice -- the state of the art: part 2.

Most New Zealand general practitioners believe their patients should be given a full explanation about the aetiology of their illness and rationale of treatment. They also believe that patients can be trained not to make unnecessary or unreasonable demands, and that a general practitioner has to earn the respect of his patients. A high proportion of doctors believed in the desirability of regular cervical smears. Almost two-thirds of general practitioners had some access to hospital beds, mostly for obstetrics. Slightly less than one-quarter had hospital appointments, and a further one-quarter would welcome such an appointment. Young doctors were somewhat more likely to welcome such involvement than their more senior colleagues. New Zealand general practitioners do more practical procedures than their British counterparts. Less than one-half of respondents were in solo practice and the likelihood of entering solo practice appears to be related to age, with younger graduates tending to opt for partnerships or associations. Most New Zealand doctors use an appointment system and most have some ancillary help in the practice. Practice nurses were used in almost two-thirds of practices. Somewhat less than half of the doctors felt that their practice load was ideal and one-third considered that they cared for a larger practice population that they felt was desirable.

Image Analysis of the Growth of Globodera pallida and Meloidogyne incognita on Transgenic Tomato Roots Expressing Cystatins.

An approach based on image analysis that enables rapid collection and analysis of nematode size and shape during growth is reported. This technique has been applied to assess Meloidogyne incognita and Globodera pallida during their development over 35 and 42 days, respectively, on transgenic tomato roots expressing the wild-type rice cystatin Oc-I or an engineered variant, Oc-IAD86. Morphometric values were established that subdivided enlarged saccate females from other life stages. Analysis of this data subset indicates that the size of females and the frequency with which they parasitize roots expressing a cystatin are reduced. Results also demonstrate that cystatins can influence the growth of G. pallida prior to the adult stage. Similar image analysis procedures should be generally applicable to the study of host status or erivironmental factors that influence growth rates of plant-parasitic nematodes.

Major Sperm Protein Genes from Globodera rostochiensis.

Three genes in the major sperm protein (MSP) gene family from the potato cyst nematode Globodera rostochiensis were cloned and sequenced. In contrast to the absence of introns in Caenorhabditis elegans MSP genes, these genes in G. rostochiensis contained a 57 nucleotide intron, with normal exon-intron boundaries, in the same relative location as the intron in Onchocerca volvulus. The MSP genes of G. rostochiensis had putative CAAT, TATA, and polyadenylation signals. The predicted G. rostochiensis MSP gene product is 126 amino acids long, one residue shorter than the products in the other species. The comparison of MSP amino acid sequences from four diverse nematode species suggests that O. volvulus, Ascaris suum, and C. elegans may be more closely related to each other than they are to G. rostochiensis.