High sensitivity immunoassays using particulate fluorescent labels.

The use of polystyrene fluorescent microspheres as sensitive labels in direct-detection (not enzymatically amplified) heterogeneous equilibrium "sandwich" immunoassays in 96-well plates is described. With mouse IgG as a model antigen, a fluorescent particulate label is more sensitive than a corresponding soluble reporter. The limit of detection of mouse IgG in the multiparametrically optimized assay was 0.2 ng/ml (7.6 x 10(8) antigens/ml) for the particulate reporter and 50 ng/ml (1.9 x 10(11) antigens/ml) for the soluble reporter. The sensitivities of assays using the particulate label were dependent on the surface densities of the capture and reporter antibodies and the concentration of reporter beads. Sensitivity was improved by adding the preformed reporter antibody/fluorescent microsphere complex to trapped antigen on the well surfaces instead of sequentially adding the reporter antibody and then the fluorescent microspheres. Maximal (equilibrium) binding of the particulate reporter to captured antigen occurred after 20 h with a concentration of 1.4 x 10(9) reporter beads/ml. Thus, particulate fluorescent labels provide high sensitivity in direct-detection immunoassays.     

A novel in vivo assay for the analysis of protein-protein interaction.

The Ras Recruitment System (RRS) is a method for identification and isolation of protein-protein interaction. The method is based on translocation of cytoplasmic mammalian Ras protein to the inner leaflet of the plasma membrane through protein-protein interaction. The system is studied in a temperature-sensitive yeast strain where the yeast Ras guanyl nucleotide exchange factor is inactive at 36 degrees C. Protein-protein interaction results in cell growth at the restrictive temperature. We developed a gene reporter assay for the analysis of protein-protein interaction in mammalian cells. Ras activation in mammalian cells induces the mitogen-activated kinase cascade (MAPK), which can be monitored using Ras-dependent reporter genes. This greatly extends the usefulness of the system and provides a novel assay for protein-protein interaction in mammalian cells.     

Dual luciferase assay system for rapid assessment of gene expression in Saccharomyces cerevisiae

A new reporter system has been developed for quantifying gene expression in the yeast Saccharomyces cerevisiae. The system relies on two different reporter genes, Renilla and firefly luciferase, to evaluate regulated gene expression. The gene encoding Renilla luciferase is fused to a constitutive promoter (PGK1 or SPT15) and integrated into the yeast genome at the CAN1 locus as a control for normalizing the assay. The firefly luciferase gene is fused to the test promoter and integrated into the yeast genome at the ura3 or leu2 locus. The dual luciferase assay is performed by sequentially measuring the firefly and Renilla luciferase activities of the same sample, with the results expressed as the ratio of firefly to Renilla luciferase activity (Fluc/Rluc). The yeast dual luciferase reporter (DLR) was characterized and shown to be very efficient, requiring approximately 1 minute to complete each assay, and has proven to yield data that accurately and reproducibly reflect promoter activity. A series of integrating plasmids were generated that contain either the firefly or Renilla luciferase gene preceded by a multi-cloning region in two different orientations and the three reading frames to make possible the generation of translational fusions. Additionally, each set of plasmids contains either the URA3 or LEU2 marker for genetic selection in yeast. A series of S288C-based yeast strains, including a two-hybrid strain, were developed to facilitate the use of the yeast DLR assay. This assay can be readily adapted to a high-throughput platform for studies requiring numerous measurements.     

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.     

Solid-phase contact assay that uses a lux-marked Nitrosomonas europaea reporter strain to estimate toxicity of bioavailable linear alkylbenzene sulfonate in soil

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain of Nitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain of lux-marked N. europaea was slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.     

Solid-Phase Contact Assay That Uses a lux-Marked Nitrosomonas europaea Reporter Strain To Estimate Toxicity of Bioavailable Linear Alkylbenzene Sulfonate in Soil

Information about in situ toxicity of the bioavailable pools of adsorptive soil pollutants is a prerequisite for proper ecological risk assessment in contaminated soils. Such toxicity data may be obtained by assays allowing for direct exposure of introduced test microorganisms to the toxicants, as they appear in solid solution equilibria in the natural soil. We describe a novel sensitive solid-phase contact assay for in situ toxicity testing of soil pollutants based on a recombinant bioluminescent reporter strain of Nitrosomonas europaea. A slurry of the reporter strain and soil sample was shaken for 1 h, after which bioluminescence was measured either directly (soil slurry protocol) or in the supernatant obtained after centrifugation (soil extract protocol). The assay was validated for both protocols by using linear alkylbenzene sulfonate (LAS) as a toxic and adsorptive model compound in the soil samples. Interestingly, LAS showed the same toxicity to the reporter strain with either soil incubation (both protocols) or pure culture, suggesting that adsorbed LAS pools contributed to the observed toxicity. The solid-phase contact assay that used the reporter strain of lux-marked N. europaea was slightly more sensitive for the detection of LAS toxicity in soil than activity-based assays targeting indigenous nitrifiers and much more sensitive than assays targeting indigenous heterotrophic microbes. We conclude that the new solid-phase contact assay, which is based on direct interaction of the test microorganisms with bioavailable pools of the toxicants in soil, provides a most sensitive and relevant method for evaluating the in situ toxicity and assessing the risks of soil contaminants.     

Structure‐guided design of a reversible fluorogenic reporter of protein‐protein interactions

A reversible green fluorogenic protein‐fragment complementation assay was developed based on the crystal structure of UnaG, a recently discovered fluorescent protein. In living mammalian cells, the nonfluorescent fragments complemented and rapidly became fluorescent upon rapamycin‐induced FKBP and Frb protein interaction, and lost fluorescence when the protein interaction was inhibited. This reversible fluorogenic reporter, named uPPI [UnaG‐based protein‐protein interaction (PPI) reporter], uses bilirubin (BR) as the chromophore and requires no exogenous cofactor. BR is an endogenous molecule in mammalian cells and is not fluorescent by itself. uPPI may have many potential applications in visualizing spatiotemporal dynamics of PPIs.     

Selective extraction of Cyclopia for enhanced in vitro phytoestrogenicity and benchmarking against commercial phytoestrogen extracts

Previous work established the phytoestrogenicity of "unfermented"Cyclopia (honeybush) extracts. The current study investigated the phytoestrogenicity of four Cyclopia harvestings (M6-9) for preparation of extracts with enhanced phytoestrogenicity for benchmarking against commercial preparations. Two extracts, from M6 (C. subternata) and M7 (C. genistoides), were identified as most phytoestrogenic using estrogen receptor binding, an estrogen receptor response element containing promoter reporter assay, alkaline phosphatase activity, and E-screen. M6 and M7 were sequentially and non-sequentially extracted with five solvents of differing polarities. Additionally, two extracts were prepared in the traditional way of preparing a cup of honeybush tea. The resultant 22 extracts were evaluated for estrogenicity. Select extracts were analysed by high-pressure liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). The sequentially extracted M6 methanol extract (SM6Met) had the highest potency and the sequentially extracted M6 ethyl acetate extract (SM6EAc) had the highest efficacy of all the extracts. The HPLC results suggested enrichment of luteolin in SM6EAc and enrichment of an unidentified polyphenol in SM6Met. Benchmarking against four commercial phytoestrogenic preparations suggest that in terms of the assays used, Cyclopia extracts have comparable potency and efficacy to the commercial extracts and thus have potential as marketable phytoestrogenic nutraceuticals.     

SOX11结合p53并上调其转录活性

目的:研究SOX11对p53转录活性的影响,并检测二者的体外相互作用。方法:在H1299(p53缺失)和H460(含野生型p53)2种细胞中分别过表达SOX11和p53,用双萤光素酶方法测定p53的转录活性;用大肠杆菌DH5α表达GST和GST-p53融合蛋白并将其纯化,用GST pull-down实验检测SOX11与p53在体外是否存在相互作用。结果:萤光素酶实验结果表明,在H1299和H460细胞中,过表达SOX11分别能促进外源p53和内源p53的转录活性;GST pull-down实验表明SOX11能在体外与p53发生相互作用。结论:SOX11能在体外与p53发生相互作用并促进p53的转录活性,为进一步研究p53的功能提供了新的线索。     

The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathione S-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.     

Integration of cell-free protein coexpression with an enzyme-linked immunosorbent assay enables rapid analysis of protein-protein interactions directly from DNA

Assays that integrate detection of binding with cell-free protein expression directly from DNA can dramatically increase the pace at which protein-protein interactions (PPIs) can be analyzed by mutagenesis. In this study, we present a method that combines in vitro protein production with an enzyme-linked immunosorbent assay (ELISA) to measure PPIs. This method uses readily available commodity instrumentation and generic antibody-affinity tag interactions. It is straightforward and rapid to execute, enabling many interactions to be assessed in parallel. In traditional ELISAs, reporter complexes are assembled stepwise with one layer at a time. In the method presented here, all the members of the reporter complex are present and assembled together. The signal strength is dependent on all the intercomponent interaction affinities and concentrations. Although this assay is straightforward to execute, establishing proper conditions and analysis of the results require a thorough understanding of the processes that determine the signal strength. The formation of the fully assembled reporter sandwich can be modeled as a competition between Langmuir adsorption isotherms for the immobilized components and binding equilibria of the solution components. We have shown that modeling this process provides semiquantitative understanding of the effects of affinity and concentration and can guide strategies for the development of experimental protocols. We tested the method experimentally using the interaction between a synthetic ankyrin repeat protein (Off7) and maltose-binding protein. Measurements obtained for a collection of alanine mutations in the interface between these two proteins demonstrate that a range of affinities can be analyzed.     

Kinetic determination of cellular LacZ expression

A kinetic assay for the expression of β-galactosidase in cells transfected with the LacZ gene was developed using a 96-well-plate format. The assay involves solubilization of the cells followed by measuring hydrolytic rates of o-nitrophenyl β-D-galactoside on a standard 96-well-plate reader without other manipulations. The protocol requires only that reagent be added sequentially to the wells at ambient temperatures, thus permitting a semiautomated or fully automated determination of reporter expression. The rates of chromophore development were found to be linear over a 6-log enzyme concentration range, from 0.001 to 100 mU. Additionally, the use of kinetic data avoids the complications of non-enzymatic, background optical density.     

The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum

Myostatin, also known as growth and differentiation factor 8, is a member of the transforming growth factor beta superfamily that negatively regulates skeletal muscle mass (1). Recent experiments have shown that myostatin activity is detected in serum by a reporter gene assay only after activation by acid, suggesting that native myostatin circulates as a latent complex (2). We have used a monoclonal myostatin antibody, JA16, to isolate the native myostatin complex from normal mouse and human serum. Analysis by mass spectrometry and Western blot shows that circulating myostatin is bound to at least two major proteins, the myostatin propeptide and the follistatin-related gene (FLRG). The myostatin propeptide is known to bind and inhibit myostatin in vitro (3). Here we show that this interaction is relevant in vivo, with a majority (>70%) of myostatin in serum bound to its propeptide. Studies with recombinant V5-His-tagged FLRG protein confirm a direct interaction between mature myostatin and FLRG. Functional studies show that FLRG inhibits myostatin activity in a reporter gene assay. These experiments suggest that the myostatin propeptide and FLRG are major negative regulators of myostatin in vivo.     

A single secreted luciferase-based gene reporter assay

Promoter analysis typically employs a reporter gene fused to a test promoter combined with a second reporter fused to a control promoter that is used for normalization purposes. However, this approach is not valid when experimental conditions affect the control promoter. We have developed and validated a single secreted luciferase reporter (SSLR) assay for promoter analysis that avoids the use of a control reporter. The approach uses an early level of expression of a secreted luciferase linked to a test promoter as an internal normalization control for subsequent analysis of the same promoter. Comparison of the SSLR assay with the dual luciferase reporter (DLR) assay using HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase) and LDLR (low-density lipoprotein receptor) promoter constructs, which are down-regulated by 25-hydroxycholesterol, show that both assays yield similar results. Comparison of the response of the HMGCR promoter in SSLR transient assays compared very favorably with the response of the same promoter in the stable cell line. Overall, the SSLR assay proved to be a valid alternative to the DLR assay for certain applications and had significant advantages in that measurement of only one luciferase is required and monitoring can be continuous because cell lysis is not necessary.     

Relative quantification of protein-protein interactions using a dual luciferase reporter pull-down assay system

The identification and quantitative analysis of protein-protein interactions are essential to the functional characterization of proteins in the post-proteomics era. The methods currently available are generally time-consuming, technically complicated, insensitive and/or semi-quantitative. The lack of simple, sensitive approaches to precisely quantify protein-protein interactions still prevents our understanding of the functions of many proteins. Here, we develop a novel dual luciferase reporter pull-down assay by combining a biotinylated Firefly luciferase pull-down assay with a dual luciferase reporter assay. The biotinylated Firefly luciferase-tagged protein enables rapid and efficient isolation of a putative Renilla luciferase-tagged binding protein from a relatively small amount of sample. Both of these proteins can be quantitatively detected using the dual luciferase reporter assay system. Protein-protein interactions, including Fos-Jun located in the nucleus; MAVS-TRAF3 in cytoplasm; inducible IRF3 dimerization; viral protein-regulated interactions, such as MAVS-MAVS and MAVS-TRAF3; IRF3 dimerization; and protein interaction domain mapping, are studied using this novel assay system. Herein, we demonstrate that this dual luciferase reporter pull-down assay enables the quantification of the relative amounts of interacting proteins that bind to streptavidin-coupled beads for protein purification. This study provides a simple, rapid, sensitive, and efficient approach to identify and quantify relative protein-protein interactions. Importantly, the dual luciferase reporter pull-down method will facilitate the functional determination of proteins.     

A method for linking yeast artificial chromosomes.

A method for linking any standard yeast artificial chromosomes (YAC) is described. YACs are introduced into the same cell and joined by mitotic recombination between the vector arms and the homologous sequence in a linking vector; several YACs can be recombined sequentially. The linking vectors also contain the beta-galactosidase gene as an expression reporter in mammalian cells.