A microtiter plate-based assay for phosphoenolpyruvate carboxylase.

A sensitive, quantitative assay for phosphenolpyruvate carboxylase which utilizes microtiter plates is described. The assay depends upon the production of a colored compound in the reaction between oxaloacetate, the product of the phosphoenolpyruvate reaction, and the dye Fast Violet B. The method is particularly appropriate for monitoring chromatographic eluates and its utility for this purpose is demonstrated by the detection of phosphoenolpyruvate carboxylase in fractions of crude maize extract separated by size-exclusion chromatography.     

Development of a robust microtiter plate-based assay method for assessment of bioactivity

A microtiter plate-based assay was developed for the quantitative monitoring of bioactive compound production in Streptomyces hygroscopicus fermentation samples. The method reported demonstrates the successful application of the theories of disk diffusion based methods of bioactivity assessment, to a microtiter assay for high throughput analysis. The assay method facilitates the generation of the dose-response curve of test organisms (Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae) to a bioactive compound. Using this dose-response curve, the method facilitates definition of three distinct Minimum Inhibitory Concentration (MIC) values for use in the characterisation of the bioactive attributes of a sample. The assay uses established standard procedures to facilitate adaptation of the assay for use with a wider range of test microorganisms. Errors due to the assumption of a linear relationship between turbidity and biomass concentration are also reduced, due to incorporation of a step to convert turbidity to biomass concentration, for use in the calculation of bioactivity.     

High-risk genotype for type 1 diabetes: a new simple microtiter plate-based ELOSA assay

The main contribution to genetic susceptibility for type 1 diabetes (T1D) is conferred by the HLA class II genes, with a major involvement of the DQB1*02 and 0302 alleles. The aim of our study was to develop a simple and rapid method suitable for identifying individuals with an HLA-associated T1D risk using whole blood as a source of DNA and reverse hybridization on microtiter plates (ELOSA). DNA was extracted from whole blood using various extraction methods. The PCR-amplified second exon of the DQB1 gene was hybridized at 37 degrees C for 1 hr to a set of 11 capture probes immobilized on a microtiter plate (eight-well strip per test) and corresponding to T1D susceptibility (S), protection (P), or neutral (N) alleles. Colorimetric analysis was then performed using specific oligonucleotides coupled to horseradish peroxidase and OrthoPhenyl Peroxidase (OPD) substrate. DNA samples corresponding to French (Rh?ne-Alpes area) T1D patients (n = 128) have been genotyped with the HLA-T1D prototype. A strong correlation is observed between susceptible genotypes and the disease, because 92.2% of the T1D individuals screened have at least one susceptible allele (DQB1*02 or *0302), thereby strengthening interest in analyzing DQB1 alleles as HLA-linked T1D markers in our Rh?ne-Alpes area population. Interestingly, clear T1D-associated genotyping results have been observed when using DNA samples extracted from dried blood spots, making it possible to envisage such genotyping in geographically dispersed affected families, for large-scale newborn screening, and for the inclusion of high-risk patients in clinical trials aimed at preventing the disease.     

A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins

The Gram-negative bacterium Escherichia coli is an important host for the (heterologous) production of recombinant proteins. The development and optimization of a protocol to overproduce a desired protein in E. coli is often tedious. A novel high-throughput screening method based on the Luminex xMAP bead technology was developed allowing a rapid evaluation of a certain expression strategy. A variant of green fluorescent protein (GFPuv) from Aequorea victoria was used as a reporter to establish the methodology. The N-terminus and the C-terminus of GFPuv were engineered to contain a His(6)- and an HA-tag (YPYDVPDYA), respectively. The double-tagged protein was loaded onto Luminex-microspheres via its His(6)-tag, the presence of the HA-tag was verified using an anti-HA antibody. High-throughput detection of full-length proteins (containing both tags) on the beads was performed using an automated Luminex 100IS analyzer. The results were compared to results obtained by classical Western blot analysis. Comparison of the two methods revealed that the Luminex-based method is faster and more economical in detecting full-length (intact) soluble recombinant protein, allowing one to routinely screen a high number of parameters in gene expression experiments. As proof of concept, different protocols to overproduce double-tagged model eucaryotic proteins (human protein S6 kinase 1 and human tankyrase) in E. coli were monitored using the new approach. Relevant parameters for optimizing gene expression of the corresponding genes were rapidly identified using the novel high-throughput method.     

A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30 min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z′ score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.     

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.     

Variable specific activity of Escherichia coli beta-galactosidase in bacterial cells

Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.     

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.     

A microtiter plate assay for aspartate transcarbamylase

A discontinuous, colorimetric method for the assay of aspartate transcarbamylase has been adapted for use with 96-well microtiter plates. The method is based on that of L.M. Prescott and M.E. Jones (1969 Anal. Biochem. 32, 408-419) for the detection of ureido compounds, using monoxime and antipyrine. The enzymatic reaction is carried out in a volume of 150 microliters and is stopped by the addition of 100 microliters of a color mix. After development, the absorbance at 460 nm is directly proportional to the quantity of N-carbamyl-L-aspartate up to at least 0.125 mumol and to the quantity of Escherichia coli aspartate transcarbamylase up to about 7 ng. Kinetic parameters obtained from saturation curves for L-aspartate in 50 mM Tris-acetate, pH 8.0, are indistinguishable from those previously obtained: Vmax = 26,225 mumol h-1 mg-1; S0.5 = 14.7 mmol liter-1; hill constant = 2.5.     

A microtiter plate assay for inorganic phosphate

A microtiter assay for the detection of picomolar quantities of inorganic phosphate has been described. The assay, linear between 50 and 1000 pmol of inorganic phosphate, is simple and rapid, with results obtainable in several minutes. Results from 5'-nucleotidase and (Ca2+ + Mg2+)ATPase assays using this method were compared with conventional phosphate assays and showed a high degree of correlation. The high sensitivity of this assay and the small sample size needed allows its widespread use in biochemical studies involving the generation of inorganic phosphate.     

Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin

Production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633 is regulated in a quorum sensing-like mechanism with subtilin acting as autoinducer and signal transduction via the subtilin-specific two-component regulation system SpaRK. Here, we report the construction and application of a subtilin reporter strain in which subtilin induced lacZ gene expression in a B. subtilis ATCC 6633 spaS gene deletion mutant is monitored and visualized by the beta-galactosidase in a chromogenic plate assay. A quantitative microtiter plate subtilin bioassay was developed and optimized. Maximal sensitivity of the system was achieved after 6 h of incubation of the reporter strain together with subtilin in a medium containing 300 mM NaCl. This sensitive and unsusceptible method was applied to identify subtilin producing B. subtilis wild type strains from both, culture collections and soil samples. The B. subtilis lantibiotic ericin S with four amino acid exchanges compared to subtilin induces the subtilin reporter strain, in contrast to the structurally closely related Lactococcus lactis lantibiotic nisin. These observations suggest a certain substrate specificity of the histidine kinase SpaK, which however, also would allow the identification of subtilin-isoform producing microorganisms.