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Kinase and ubiquination assays

a kinase and ubiquitination technology, applied in the field of fluorescent molecule and luminescent metal complex assays, can solve the problems of limiting the sensitivity of luminescence-based assays, detection of false positives or false negatives in drug or compound screens, etc., to reduce interference, enhance/up-regulate or modulate an activity, and inhibit the effect of interferen

Inactive Publication Date: 2007-11-15
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0024] As in some embodiments of the present invention, preparing protease (e.g., DUB) substrates with a genetically encoded acceptor fluorophore, avoids difficult “orthogonal” labeling strategies to site-specifically incorporate two distinct fluorophores into a single protein. In the case of whole-protein kinase substrates, labeled proteins are typically prepared through a random labeling of surface-accessible amine groups. As in one embodiment of the present invention, preparing enzyme substrates as fluorescent protein fusions, leads to improved lot-to-lot consistency of the substrate, which is a consideration in developing reagents for high-throughput screening applications.
[0029] Some embodiments of the invention involve a set of generic TR-FRET ubiquitin reagents for both ubiquitination and deubiquitination. By selectively incorporating the TR-FRET donor (e.g., terbium) and acceptors (e.g., fluorescein or fluorescent proteins) onto ubiquitin, universal high throughput screening reagents were created that enable robust HTS assays with high Z′ values (>0.7) with either kinetic or end-point readout. In addition, the time resolved signal from the terbium donor reduces the amount of interference from color quenchers and autofluorescent compounds that are frequently encountered in compound libraries. In some embodiments of the invention, TR-FRET ubiquitin platforms are provided herein as a simple, flexible set of reagents to accelerate compound screening to identify specific inhibitors of ubiquitin conjugating and deubiquitinating enzymes.

Problems solved by technology

Background luminescence (e.g., fluorescence or luminescence from assay components) and non-specific interactions of assay components, however, can limit the sensitivity of luminescence-based assays, particularly when luminophores having short lifetimes are used, resulting in the detection of false positives or false negatives in a drug or compound screen.

Method used

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Examples

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example 1

Labeling of an Antibody with a Luminescent Metal Chelate

[0294] 1 mg purified PY72 (anti-phosphotyrosine) IgG antibody, an antibody that preferentially binds amino acid sequences containing phosphorylated tyrosines (e.g., sequences phosphorylated by protein tyrosine kinases (PTKs)) and was dialyzed for 1.5 hours in a 100 mM sodium bicarbonate buffer, pH 9.5, using a 12-14,000 MWCO dialysis membrane. (PY72 hybridoma cells were obtained from the Salk Institute; the immunogen was phosphotyrosine conjugated to KLH. Ascites were produced by Harlan Bioproducts for Science, Indianapolis Ind. Ascites were purified with a protein G column (Pierce). Purified antibody is also available from Covance, Berkeley Calif. (Part # MMS414P).) The antibody was then removed from the dialysis membrane and concentrated to 48.8 uM (7.3 mg / mL) using a Centricon YM50 (Millipore) concentrator. 100 uL of this antibody solution was diluted to 5 mg / ml (33.4 uM) into the labeling reaction which consisted of 10 mM ...

example 2

Binding Curve Experiment Between Protein Tyrosine Kinase Product Tracer (PTK Tracer) and Anti-PTK Product (PY72) Antibody

[0296] A direct binding curve (showing luminescent metal chelate-labeled PY72 antibody binding to fluorescent acceptor labeled tracer) was generated by incubating serial dilutions of the labeled antibody (10 nM to 9.8 pM in two fold dilutions) with 1 nM fluorescent acceptor-labeled tracer (PTK labeled tracer; sequence F-ADE(pY)LIPQQS, where F is fluorescein and pY is a phosphorylated tyrosine, SEQ ID NO:1; note that the tracer is a phosphorylated tyrosine derivative of a protein tyrosine kinase (PTK) substrate) in FP dilution buffer (part #P2839, Invitrogen, Carlsbad, Calif.). After a 30 minute incubation, the fluorescence polarization of each composition in the plate was read on a Tecan Ultra plate reader using a 485 nm excitation filter (20 nm bandpass) and 535 nm emission filters (25 nm bandpass). Data was collected using 10 flashes per well and a 40 μs integr...

example 3

Competition Curve between Labeled Kinase Product Tracer and Unlabeled Kinase Product

[0298] A competition curve to show that the disruption of the antibody-tracer interaction could be monitored by both fluorescence polarization and time-resolved RET from the same sample was performed by incubating serial dilutions (10 μM to 19.5 nM in two-fold dilutions) of an unlabeled phosphotyrosine-containing peptide competitor (ADE(pY)LIPQQS, where pY is a phosphorylated tyrosine, SEQ ID NO:3) in the presence of 10 nM Tb-chelate labeled PY72 antibody and 1 nM labeled PTK labeled tracer, as described above. After a 30 minute incubation, the plate was read on a Tecan Ultra plate reader. Fluorescence polarization was measured using a 485 nm excitation filter (20 nm bandpass) and 535 nm emission filters (25 nm bandpass). Time-resolved RET was measured using a 340 nm excitation filter (35 nm bandpass) and 495 nm (10 nm bandpass) and 520 nm (25 nm bandpass) filters using a 200 μs integration window a...

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Abstract

Compositions, including antibodies, polypeptides, and organic molecules, kits, and methods for probing molecular interactions (e.g., deubiquination, ubiquination and kinase activity), e.g., using resonance energy transfer (RET) are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 832,114, filed Jul. 21, 2006, U.S. Provisional Application No. 60 / 774,236, filed Feb. 17, 2006, U.S. Provisional Application No. 60 / 759,545, filed Jan. 18, 2006, U.S. Provisional Application No. 60 / 735,812, filed Nov. 14, 2005, and U.S. Provisional Application No. 60 / 731,310, filed Oct. 28, 2005, the disclosures of which are incorporated herein by reference in their entireties.TECHNICAL FIELD [0002] This invention relates to assays employing a fluorescent molecule and a luminescent metal complex and to methods for monitoring and measuring molecular interactions, such as competitive binding or enzymatic activity (e.g., kinase, de-ubiquinating or ubiquination activity). BACKGROUND [0003] Ubiquitination primarily serves as a targeting signal, and proteins carrying the most common type of poly-Ubiquitin chain are targeted for destruction by the ubiquitin proteasome p...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/48
CPCG01N33/542C12Q1/37
Inventor VOGEL, KURT WILLIAMRIDDLE, STEVEN MICHAELHORTON, ROBERT ARONROBERS, MATTHEW BRIANMICHAUD, GREGORY ALLENMACHLEIDT, THOMASVEDVIK, KEVINHUWILER, KRISTIN G.
Owner LIFE TECH CORP
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