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Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes

a technology of ubiquitination enzymes and chimeras, which is applied in the field of targeted protein silencing using chimeras between antibodies and ubiquitination enzymes, can solve the problems of curtailing the ubiquitination of both native and novel substrates, unable to provide phenotypic insight into cellular processes and disease etiology, and only able to study cellular characteristics by employing, etc., to achieve simple and efficient, simple implementation

Inactive Publication Date: 2014-04-24
CORNELL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention is about creating new types of molecules called "ubiquibodies" that can target and remove specific proteins in cells. This allows researchers to study the functions of proteins in cells and determine if they are valid targets for treating diseases. The technology is simple and easy to use, and can be applied in any laboratory.

Problems solved by technology

Previously, however, cellular characteristics could only be studied by employing, for example, genetic “knock-outs” or RNA interference (“RNAi”) technology.
Notwithstanding the benefits of such technology, systems that function at the genetic level fail to provide phenotypic insight into cellular processes and disease etiology.
Nevertheless, it was determined that the core SCF complex was encumbered by the overexpression of F-box chimeras, which therefore curtailed the ubiquitination of both native and novel substrates.

Method used

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  • Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes
  • Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes
  • Targeted protein silencing using chimeras between antibodies and ubiquitination enzymes

Examples

Experimental program
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Effect test

example 1

Plasmids

[0133]For prokaryotic expression of ubiquibodies, pET28a with C-terminal FLAG and His6 tags was used. CHIP and CHIPΔTPR were cloned from pET30a-hCHIP-His6. scFv13 and scFv13R4 were cloned from pET-scFv13(R4)-SecM17 vectors (see Contreras-Martinez, et al., “Intracellular Ribosome Display via SecM Translation Arrest as a Selection for Antibodies with Enhanced Cytosolic Stability.”J Mol Biol 372(2): 513-24 (2007)), which is hereby incorporated by reference in its entirety, while and scFvD10 was cloned from pHK49-gpD-specific antibody D10. See Koch et al., “Direct Selection of Antibodies from Complex Libraries with the Protein Fragment Complementation Assay.”J Mol Biol 357(2): 427-41 (2006), which is hereby incorporated by reference in its entirety. scFvs were cloned between NcoI and EcoRI sites, followed by a Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 1) linker that was inserted by PCR before CHIPΔTPR followed by a SalI site. The FLAG and His6 tags were created by primer dimerization and ...

example 2

Antibodies and Reagents

[0135]Mouse monoclonal [M2] to DDDDK tag (HRP) from Abcam (ab49763) was used to probe for ubiquibodies while rabbit anti-HA produced by Sigma (H6908) was used to probe the antigen gpD and rabbit polyclonal anti-β-gal from Abcam (ab616) was used to probe the antigen β-gal. Rabbit polyclonal anti-ubiquitin from Abcam (ab8134) and rabbit anti-ubiquitin Lys48-specific from Millipore (clone Apu2) were used to detect poly-ubiquitin chains. Mouse monoclonal anti-HSP70 from Enzo Life Sciences (C92F3A-5) and mouse monoclonal anti-GFP from Roche were used as controls for the eukaryotic expression. All of the above were used with secondary antibodies from Promega, anti-mouse-HRP and anti-rabbit-HRP.

example 3

In Vitro Reconstitution Assays

[0136]Purified ubiquitin and ubiquitin activating enzyme (E1) were purchased from Boston Biochem, ubiquitin conjugating enzyme (E2, UbCH5a) was purchased from Millipore, Hsp70 was purchased from Enzo Life Sciences and β-gal was purchased from Sigma. Moreover, in vitro ubiquitination assays were performed in the presence of 20 mM MOPS pH 7.2, 100 mM KCl, 5 mM MgCl2 1 mM DTT, 4 mM ATP, 50 μM ubiquitin, 0.1 μM E1, 2 μM E2, 3 μM E3 (ubiquibody or control protein) and 3 μM target protein (β-gal, gpD or Hsp70). Reactions were carried out at 37° C. and analyzed by SDS-PAGE and immunoblotting.

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Abstract

The present invention relates to an isolated chimeric molecule comprising a degradation domain including a eukaryotic U-box motif and a targeting domain capable of immuno specifically directing the degradation domain to a substrate where the targeting domain is heterologous to the degradation domain. A linker couples the degradation domain to the targeting domain. Also disclosed are compositions as well as methods of treating a disease, substrate silencing, screening agents for therapeutic efficacy against a disease, and methods of screening for disease biomarkers.

Description

[0001]This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 61 / 468,435, filed Mar. 28, 2011, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention relates to targeted protein silencing using chimeras between antibodies and ubiquitination enzymes.BACKGROUND OF THE INVENTION[0003]Following discovery of the ubiquitin-proteasome system (“UPS”) in the 1980's, researchers have exploited its capacity to function as a degradative regulator of protein profusion. The UPS ensures homeostatic concentrations of intracellular proteins via the proteasome, which recognizes and degrades ubiquitinated proteins. The process of ubiquitination involves a cascade of three enzymes, i.e., the ubiquitin activating enzyme (“E1”), the ubiquitin conjugating enzyme (“E2”), and the ubiquitin ligase (“E3”). In brief, target protein modification, through ubiquitination, occurs when ubiquitin is activated by E1, which then ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/00C07K16/18
CPCC07K16/18C12N9/93C07K16/00C12Y603/02019C07K2319/00C07K2319/50C07K2319/95A61K38/00
Inventor DELISA, MATTHEWVARNER, JEFFREYPORTNOFF, ALYSE
Owner CORNELL UNIVERSITY
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