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Devices and methods for profiling enzyme substrates

a technology of enzyme substrates and devices, applied in the field of apparatus and methods for separating and detecting enzyme substrates, can solve the problems of unknown substrate phosphorylation spectrum and unwanted side effects

Active Publication Date: 2008-04-17
UNIV OF MARYLAND BALTIMORE COUNTY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In a still further embodiment, the present invention provides methods for identifying an inhibitor of enzyme activity, comprising providing a first and a second separation gel, each of which comprises at least one inactive enzyme that is capable of modifying at least one protein, wherein the at least one enzyme is contained within each of the separation gels, and wherein the enzyme is capable of re-activation, loading a collection of macromolecules comprising at least one enzyme substrate into the first and the second protein separation gels to generate loaded separation gels, resolving the at least one enzyme substrate on the first and second loaded separation gels to generate resolved separation gels, washing the resolved separation gels, incubating the first and second resolved separation gels in a protein refolding buffer to re-activate the enzyme, incubating the first resolved separation gel in an enzyme reaction buffer, incubating the second resolved separation gel in an enzyme reaction buffer that further comprises at least one inhibitor of enzyme activity and detecting at least one enzyme substrate in the first resolved separation gel and the second resolved separation gel and comparing the first resolved separation gel and the second resolved separation gel, wherein a reduction in the detection of the at least one enzyme substrate in the second resolved separation gel indicates

Problems solved by technology

However, wholesale inhibition of activity of certain kinases may lead to unwanted side effects.
A major impediment to the development of substrate-specific inhibitors is that for most enzymes such as kinases, the spectrum of substrates that can be phosphorylated is not known.

Method used

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  • Devices and methods for profiling enzyme substrates
  • Devices and methods for profiling enzyme substrates
  • Devices and methods for profiling enzyme substrates

Examples

Experimental program
Comparison scheme
Effect test

example 1

In-Gel Kinase Assay of CK2

[0077]CK2 regulates the half-life of the prostate-specific tumor suppressor NKX3.1 (Li, X., et al., Mol Cell Biol, 26:3008-3017 (2006)). Only the free monomeric form of one catalytic subunit, CK2α′, is involved in regulating NKX3.1.

Purification of CK2 and Demonstration of Enzyme Activity

[0078]The protein coding regions of human CK2α gene was cloned into a protein expression vector to produce the protein in E. coli. A CK2α kinase activity-deficient mutant was also generated in parallel. CK2 α was expressed in E. coli and partially purified by nickel resin chromatography. To confirm that the recombinant CK2 α possessed kinase activity that could be manifested after electrophoresis in a denaturing polyacrylamide gel, an in-gel kinase assay was performed on the partially purified CK2 α with casein as a substrate in the gel. Standard conditions for SDS removal (i.e., alcohol-comprising buffer), protein refolding, and a kinase reaction were used. An exemplary pro...

example 2

Detection of CK2 Substrates in an LNCaP Cell Extract by ID RIKA and KESTREL

[0110]To compare the extent to which RIKA and Kinase Substrate Tracking and Elucidation System (KESTREL) are capable of detecting potential substrates, a whole cell lysate from LNCaP cells (50 mgs protein) will be fractionated by anion exchange chromatography and eluted with a NaCl step gradient into 40 fractions. Equivalent volumes of the load, flow and elution fractions will then be analyzed in the RIKA and KESTREL analyses. To perform 1D CK2 RIKAs, two aliquots of each fraction will be treated with λ phosphatase and electrophoresed through denaturing acrylamide gels containing either 10 μg / ml active or kinase-dead CK2α. After electrophoretic resolution of proteins on the RIKA gel, SDS will be removed by incubation in 20% isopropanol. The gels will then be carried through a series of buffer changes to refold the proteins present in the gel and to restore the catalytic activity of CK2α. A kinase reaction wil...

example 3

Validation the RIKA by Demonstrating Its Ability to Detect Known CK2 Substrates and to Discover Previously Unknown Substrates

Mass Spectrometric Identification of Potential CK2α Substrates Revealed by RIKA.

[0116]Since the total number of true CK2 targets in human cells is unknown, it is difficult to derive a statistical argument to determine how many identifications should be performed to achieve a given degree of confidence. It is anticipated that by characterizing 80-100, likely both known and unknown potential substrates will be encountered. To achieve this goal, a series of 2D CK2α RIKAs will be performed using fractionated LNCaP extracts as a source of potential CK2α substrates

[0117]Aliquots of anion exchange fractions of LNCaP whole cell lysates will be focused on 18 cm linear IPG strips with a pH range of 3-6, 5-8, 7-9, and 3-10. Preliminary studies using pH 3-10 IPG strips demonstrated that the majority of potential targets lie within the range of pH 3-5. It is anticipated th...

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Abstract

The present invention relates to apparatus and methods for separating and detecting enzyme substrates using separation gels. For example, the apparatus and methods can be used to separate and detect kinase substrates for further analysis. The apparatus and methods can also be used to detect enzyme inhibitors, such as kinase inhibitors.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Patent Application No. 60 / 687,919, filed Jun. 6, 2005, the disclosure of which is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]Portions of this invention may have been made with United States Government support under a grant from the United States Army Medical Research & Material Command, Grant No. DAMD-1703-1-0091. As such, the U.S. Government may have certain rights in this invention. Portions of the invention may also have been made with support from the Maryland Technology Development Corporation.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present invention relates to apparatus and methods for separating and detecting enzyme substrates using separation gels. For example, the apparatus and methods can be used to separate and detect kinase substrates for further analysis. The apparatus and methods can al...

Claims

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Application Information

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IPC IPC(8): C12Q1/48C12M1/34
CPCC12Q1/00G01N2500/02C12Q1/485
Inventor BIEBERICH, CHARLESLI, XIANG
Owner UNIV OF MARYLAND BALTIMORE COUNTY
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