Deubiquitination enzyme activity detection method based on fluorescence polarization

A technology of deubiquitinating enzymes and fluorescence polarization, applied in the field of biomedicine, can solve the problems of limited binding strength of solid-phase substrates, easy to fall off detection results, and affect probe degradation, etc. simple effect

Pending Publication Date: 2021-10-19
荆州市第一人民医院
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Problems solved by technology

This method has the following problems: 1) This method uses His-tag to immobilize the fusion protein, and the binding strength between the His-tag and the solid-phase matrix is ​​limited and it is easy to fall off, which will affect the detection results; 2) One end of the probe Immobilized on the medium, the steric hindra...

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  • Deubiquitination enzyme activity detection method based on fluorescence polarization
  • Deubiquitination enzyme activity detection method based on fluorescence polarization
  • Deubiquitination enzyme activity detection method based on fluorescence polarization

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[0030] YUH1 is a very typical Ub carbon-terminal hydrolase, which belongs to the UCHs family (Yu HA et al., Protein expression and purification, 2007, 56(1): 20-26). Taking deubiquitinating enzyme YUH1 as an example, Ub-linker-BODIPY probe was used to detect the activity of YUH1. The digestion buffer conditions of the deubiquitinating enzyme YUH1 are 20 mM Tris, 1mM DTT, 1mM EDTA, pH7.5, and the optimal digestion temperature is 25°C. It should be clearly pointed out that the probes designed in the present invention are also applicable to the detection of other deubiquitinating enzyme activities.

[0031] The method comprises the steps of:

[0032] 1. Preparation of deubiquitinase detection probes:

[0033] (1) Preparation of fusion protein Ub-linker-Cys: The gene sequence of Ub-linker-Cys was cloned into the pET11a vector (Novagen), the restriction sites were NdeI and BamHI, and BL21 star was used as the expression host for the fusion protein is expressed when OD 600 When ...

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Abstract

The invention provides a deubiquitination enzyme activity detection method based on fluorescence polarization. The method comprises the following steps: 1, preparing a deubiquitinating enzyme detection probe: preparing a fusion protein Ub-linker-Cys, connecting a BODIPY probe, and carrying out mass spectrometric detection; 2, detecting the activity of the deubiquitination enzyme: selecting the deubiquitination enzyme and detecting the activity of the deubiquitination enzyme in real time according to the influence of deubiquitination on the FP value of the probe. The detection method is based on detection of fluorescence polarization, the fluorescence polarization detects the proportional relation of the fluorescence intensity in the parallel direction and the fluorescence intensity in the vertical direction, the detection method is not easily interfered by the concentration of the fluorescence probe, the solution temperature, the pH value and other fluorescence signals, when the activity of the deubiquitination enzyme is measured, the steps are simple, the consumed time is short, and the usage amount of the probe and the deubiquitination enzyme is small. In addition, the probe prepared in the method can also be used for screening deubiquitination enzyme inhibitors, and is high in throughput.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for detecting deubiquitinase activity based on fluorescence polarization. Background technique [0002] Ubiquitin modification widely exists in eukaryotic cells, and the "ubiquitin code" formed by ubiquitin (Ub) can regulate almost all physiological processes. Ubiquitination modification is also reversible, and ubiquitin can be released from substrates or ubiquitin chains by deubiquitinase (DUB). Deubiquitinases play a regulatory role in the ubiquitin-proteasome pathway, autophagy pathway, etc. Many neurodegenerative diseases and tumors are also related to deubiquitinases, so deubiquitinases are very important drugs target. Detecting the activity of deubiquitinating enzymes is an important way to study their biological functions, and it is also a necessary means for drug screening. At present, the main method for detecting deubiquitinase activity is as follows: st...

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Application Information

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IPC IPC(8): G01N21/64C12N15/70C12N15/62C12P21/02C07K19/00C12Q1/37
CPCG01N21/6445C12N15/70C07K14/8139C12Q1/37G01N33/6848C07K2319/95
Inventor 易华伟张嫦丽王清清范文罗伟辛陈琦
Owner 荆州市第一人民医院
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