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Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme

A technology for removing butyrylase and propionylase, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, measuring devices, etc., and can solve problems such as research and affecting biological functions.

Active Publication Date: 2015-07-08
JINGJIE PTM BIOLAB HANGZHOU CO LTD
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, little is known about the screening of specific lysine depropionylation and debutyrylation enzymes and the determination of enzyme activities, which directly affects the biological function research of these two modification methods

Method used

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  • Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme
  • Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme
  • Method for screening and testing activity of lysine propionylation removal enzyme and butyrylation removal enzyme

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Embodiment Construction

[0030] Plasmids, Antibodies and Other Reagents Plasmids encoding HDAC3, HDAC6 cDNA and their corresponding enzyme-inactive mutant plasmids HDAC3S424A and HDAC6H216 / 611A were kindly provided by Edward Seto, H. Lee Moffitt Cancer Center. See previous reports for plasmid information (21-22) . The anti-butyryl-lysine antibody and the anti-histone site-specific butyryl-lysine antibody were prepared by the inventors themselves. Other antibodies used were: anti-acetylated lysine antibody (ImmunoChem Pharmaceuticals, Burnaby, British Columbia, Canada); anti-histone H3 antibody, anti-histone H4 antibody (Abcam, Cambridge, UK); anti-HA antibody (Roche Diagnostics , Indianapolis, IN); anti-Flag antibody (Sigma-Aldrich, St. Louis, MO). Recombinant HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10 and HDAC11 were purchased from BPS Bioscience Inc. (BPS Bioscience Inc., San Diego, CA). Butyryl-CoA, acetyl-CoA, Leptomycin B and acetate were purchased from Sigma-Aldrich...

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Abstract

The invention relates to a method for screening and testing activity of a lysine propionylation removal enzyme and a butyrylation removal enzyme. The basis thought of the method is that: lysine propionylation or butyrylation modification degree of a substrate can be reflected by the sensitivity of the substrate to the catalysis removal enzyme. The method comprises the following steps of: a, lysine propionylation substrate polypeptide marked with releasable signal substances; b, the lysine propionylation removal enzyme; and c, a detection method for distinguishing the modification lysine propionylation polypeptide from corresponding non-modification polypeptide. The method can be used for screening modification removal enzymes and testing the activity of the enzymes, and also can screen substrates influencing the activity of the enzymes to be further used in quick diagnosis of several diseases.

Description

technical field [0001] The invention relates to a method for screening lysine depropionylase and debutyrylase and measuring the activities of lysine depropionylase and debutyrylase. Especially by measuring Boc-Lys(Prop)-AMC (tert-butyroxybutylated lysine-4-amino-7-methylcoumarin) and Boc-Lys(Buty)-AMC (tert-butyroxy Fluorescence intensity of propionylated lysine-4-amino-7-methylcoumarin) catalyzed by lysine depropionylase and debutyrylase, combined with mass spectrometry identification, biochemical analysis and Cytological assays confirmed specific lysine depropionylase and debutyrylase and assayed for enzyme activity. Background technique [0002] The nucleosome is the basic repeating unit of chromatin, consisting of an octameric core histone surrounded by approximately 147 base pairs of DNA. Nucleosomes are capable of further folding into higher order structures (1) . Dynamic changes in chromatin structure regulate DNA plasticity, which in turn affects DNA template-bas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/25G01N30/02G01N30/72G01N30/74G01N21/64
Inventor 赵英明
Owner JINGJIE PTM BIOLAB HANGZHOU CO LTD
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