Streptavidin-coated Scintillation Proximity Assay Beads Research Articles

Scintillation Proximity Assay of Arginine Methylation

Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein posttranslational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, 3H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.

An Association Between the Autoimmune Susceptibility Locus CTLA4 and Alterations in Proximal TCR Signaling in Healthy Human Volunteers

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector inEscherichia colias fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor–ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Homogeneous Proximity Tyrosine Kinase Assays: Scintillation Proximity Assay versus Homogeneous Time-Resolved Fluorescence

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous (“mix-and-measure”) nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipeting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.

A Scintillation Proximity Assay for the Raf/MEK/ERK Kinase Cascade: High-Throughput Screening and Identification of Selective Enzyme Inhibitors

We have developed a quantitative scintillation proximity assay (SPA) that reproduces the Raf/MEK/ERK signal transduction pathway. The components of this assay include human cRaf1, MEK1, and ERK2 and a biotinylated peptide substrate for ERK2. cRaf1 was expressed as a his-tagged protein in insect cells in an active form. MEK1 and ERK2 were expressed inEscherichia colias glutathioneS-transferase (GST)-fusion proteins in their inactive forms. ERK2 was removed from the GST portion of the fusion protein by cleavage with thrombin protease. When the purified components are incubated together, cRaf-1 phosphorylates and activates MEK1, MEK1 phosphorylates and activates ERK2, and ERK2 phosphorylates the peptide, biotin-AAATGPLSPGPFA. Phosphorylation of the peptide using [γ-33P]ATP is detected following binding to streptavidin-coated SPA beads. The assay detects inhibitors of cRaf1, MEK1, or ERK2, and has been used to screen large numbers of compounds. The specific target of inhibition was subsequently identified with secondary assays described herein.

Anin Vitro96-Well Plate Assay of the Mitogen-Activated Protein Kinase Cascade

Mitogen-activated protein (MAP) kinases of the extracellular signal-regulated kinase (ERK) family are activated in response to many growth and differentiation factors as well as some oncogenes. ERK activation follows phosphorylation by a class of specific upstream MAP kinase/ERK kinase (MEK) exemplified by MEK-1. Activated ERKs control many short- and long-term changes in cell function through phosphorylating a number of intracellular target substrates which include stathmin, a phosphoprotein regulating microtubule stability. We report here the development of a simple, 96-well plate, quantitativein vitroassay measuring purified ERK2 catalytic activation by a constitutive MEK-1 mutant (S218E S222E). Enzymatic activity was detected by33P phosphorylation of purified biotinylated stathmin captured on streptavidin-coated scintillation proximity assay beads which eliminates the need for wash steps. The assay was optimized and theK0.5value for ATP was found to be 0.9 μM and theKmfor stathmin was determined to be 16 μM. The assay was also used to determine IC50values for the protein kinase inhibitors PD98059 and staurosporine. This simple assay allows several hundred quantitative measurements of MEK1-dependent ERK2 activation to be performed in a day.

Development of a Scintillation Proximity Assay for Histone Deacetylase Using a Biotinylated Peptide Derived from Histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.

Characterization of [ 125I-Tyr 0]-corticotropin releasing factor (CRF) binding to the CRF binding protein using a scintillation proximity assay

We describe the characterization of high affinity [ 125I-Tyr 0]-human CRF binding to purified recombinant human CRF-binding protein (CRF-BP) using a scintillation proximity assay (SPA). For this stable nonseparation technique developed in 96 well microtiter plates, biotinylated CRF-BP is captured by streptavidin-coated SPA beads for the detection of bound [ 125I-Tyr 0]-CRF. Unbound [ 125I-Tyr 0]-CRF represented little or no signal in the assay. Total binding observed was greater than 5000 cpm with a nonspecific signal of <100 cpm determined in the presence of excess unlabeled human CRF. A comparison of the SPA method with a charcoal precipitation method confirmed that the biotinylation procedure did not adversely affect affinity of the CRF-BP for [ 125I-Tyr 0]-CRF. Saturation binding analysis yielded an apparent equilibrium dissociation constant ( K d) of 208±5.0 pM (±S.D., n=3). An inhibition constant ( K i ) for unlabeled CRF was calculated to be 0.22±0.03 nM (±S.D., n=8) and a pharmacological profile for eight CRF-related neuropeptides gave a rank potency similar to previously reported results. Finally, the assay variability was assessed with intra- and inter-plate coefficients of variation which were less than 5% each.

Development of a Scintillation Proximity Assay for Calcineurin Phosphatase Activity

We have developed a scintillation proximity assay (SPA) that allows the Ca2+/calmodulin (CaM)-dependent Serine/threoine (Ser/Thr) phosphoprotein phosphatase 2B (calcineurin) activity to be analyzed. A [33P] labeled and biotinylated peptide containing a partial sequence of the regulatory subunit (Rn) of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase was synthesized and used as a synthetic substrate for calcineurin. Following incubation of the peptide with calcineurin, which removes the [33P] label, streptavidin-coated SPA beads were added to capture the biotinylated peptide (the level of the signal detected is inversely proportional to that of the calcineurin activity). Sensitivity is increased in this system by settling or centrifuging the streptavidin-coated SPA beads after binding has occurred. This method allows calcineurin phosphatase assays to be carried out in a 96-well format that is amenable to screening large numbers of compounds.

The Utility of FK506-Binding Protein as a Fusion Partner in Scintillation Proximity Assays: Application to SH2 Domains

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector inEscherichia colias fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor–ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Scintillation Proximity Assay for Human DNA Topoisomerase I Using Recombinant Biotinyl-Fusion Protein Produced in Baculovirus-Infected Insect Cells

DNA topoisomerases are well-established targets of important therapeutic agents which include the antibacterial quinolones and anticancer camptothecins. Screens for new classes of topoisomerase inhibitors generally employ methods, such as gel electrophoresis, which are not readily amenable to a rapid high-throughput format. We describe here a high-throughput assay to screen for inhibitors of human DNA topoisomerase I based on the scintillation proximity assay. The assay employs recombinant biotinyl-topoisomerase I fusion protein, a hybrid protein which contains a domain that is biotinylated duringin vivoexpression. The hybrid topoisomerase I fusion protein is found to be biotinylated, active, and nuclear-localized when produced in insect cells using a baculovirus expression system. The biotinyl-topoisomerase I fusion protein can be captured from crude nuclear extracts by immobilization on streptavidin-coated scintillation proximity assay beads. The assay detects binding of3H-labeled DNA to the bead-immobilized enzyme by scintillation counting. The method is also able to detect stabilization of covalent protein–DNA complexes by camptothecin, an inhibitor previously shown to stabilize covalent intermediates that form during catalysis.

Development of a Scintillation Proximity Assay for Human Cytomegalovirus Protease Using33Phosphorous

A scintillation proximity assay (SPA) using33phosphorous is described for human cytomegalovirus (HCMV) UL80 protease. This is the first demonstration that33phosphorous is compatible with the SPA system. The peptide substrate used in the assay contains an HCMV protease cleavage site and is biotinylated at its amino terminus. The peptide also contains a site for protein kinase A, enabling radiolabeling at its carboxy terminus with [γ-33P]ATP. Peptide is incubated with protease, followed by binding to streptavidin-coated SPA beads via biotin. Cleavage of the peptide by the protease results in a decrease in the radioactive signal, which is prevented by the presence of a protease inhibitor. This methodology is applicable to other proteases whose cleavage site is known.