US 2006/0040335 A1 · Diagnostic and screening methods and kits associated with proteolytic activity

Abstract

Methods and kits for assessing proteolytic enzyme activity and a modulator's effect thereof employ a Ubiquitin or Ubiquitin-like Protein and a signal producing structure. The corresponding fusion polynucleotide is employed for production of transgenic cells, plants and animals that may be produced by stably transfection, optionally transforming the cell, plant or animal with a Ubiquitin-, UBL- or their C-terminal binding functional fragment-Reporter fusion polynucleotide. A method of diagnosing a disease or condition, comprises contacting or administering a sample obtained from a subject suspected of being afflicted with the disease or condition with the cell, plant or animal, detecting any signal produced by the reporter in the presence of the sample and comparing the signal to controls for 0% and 100% signals.

Claims85

Dependent claims

A method for assessing proteolytic enzyme activity, comprising

providing a fusion polymer comprising a first polymer that comprises Ubiquitin or a Ubiquitin-like protein (UBL) or a functional C-terminal segment thereof and a second polymer comprising a free N-terminal amino acid required for detection;
wherein the first and second polymers are operatively linked to one another through the UBL C-terminus and the second polymer N-terminus;
contacting the fusion polymer with a proteolytic enzyme that cleaves at the UBL C-terminus;
detecting a signal associated with either amount or activity of cleaved polymer, or a sample suspected of comprising the enzyme;
and
establishing a correlation between the cleaved polymer's signal to the proteolytic enzyme's activity.

3 more elements

A method for screening compounds for their effect on proteolytic activity, comprising

obtaining a fusion polymer comprising a first polymer that comprises Ubiquitin or a ubiquitin-like protein (UBL) or a binding functional C-terminal segment thereof and a second polymer comprising a free N-terminus amino acid;
wherein the first and second polymers are operatively linked to one another through the N-C-termini;
contacting the fusion polymer with a UBL C-terminus cleaving proteolytic enzyme under conditions effective for cleavage to occur;
detecting a signal associated with an amount of cleavage to obtain a 100% cleavage signal;
repeating the contacting and detecting steps in the presence of a full inhibitor of proteolytic enzyme activity to obtain a 0% cleavage signal;
obtaining a set of compounds;
separately repeating the fusion polymer obtaining, contacting and detecting steps in the presence of each compound to obtain a cleavage signal;
normalizing each compound cleavage signal by reference to the 0% and 100% cleavage signal and assigning a proteolytic enzyme activity value to each compound.

5 more elements

A proteolytic enzyme activity modulator screening kit, comprising

a fusion polymer comprising a first polymer that comprises Ubiquitin or a ubiquitin-like protein (UBL) or a C-terminal segment thereof and a second polymer comprising a polypeptide requiring a free N-amino acid terminus for detection;
wherein the first and second polymers are operatively linked to one another through the Ubiquitin or UBL C-terminus and the second polymer N-terminus;
and
instruction for conducting the proteolytic enzyme assay, detecting a signal associated with the amount or activity of the first and/or second polymers, and establishing a correlation of the detected signal to the enzyme's proteolytic activity for a plurality of modulators and controls;
and
optionally a source of a proteolytic enzyme that cleaves at the Ubiquitin or UBL C-terminus.

3 more elements

Dependencies are inferred from the claim text.

Diagnostic and screening methods and kits associated with proteolytic activity

Life of this patent

Diagnostic and screening methods and kits associated with proteolytic activity

Life of this patent

Abstract

Claims85