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Plant in-vitro ubiquitin protein degradation system and application thereof

A plant protein, ubiquitin protein technology, applied in the field of plant in vitro ubiquitin protein degradation system, can solve the problems of single, false positive, false negative and so on

Inactive Publication Date: 2014-12-31
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for the test protein encoded by the plant gene, the commonly used in vitro ubiquitination reaction system has the following disadvantages: (1) the ubiquitin activating enzyme and ubiquitin conjugating enzyme used (for ubiquitin ligase autoubiquitination activity detection) and ubiquitin proteins are mostly not plant-encoded proteins (such as common yeast ubiquitin activating enzymes, human ubiquitin conjugating enzymes UBCH5b or UBCH5c, and human ubiquitin proteins, etc.), due to the homologous genes of different species The functions may not be exactly the same, so the detection of the activity of plant gene products in such a reaction system cannot fully represent the real situation in the plant system, so false positive or false negative results are often produced; (2) the ubiquitin binding There is a single type of enzyme, but there are actually a large number of specific combinations of E2-E3 in the body, and the activity of all E3 proteins cannot be detected by using a single E2, so some false negative results are inevitable.

Method used

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  • Plant in-vitro ubiquitin protein degradation system and application thereof
  • Plant in-vitro ubiquitin protein degradation system and application thereof
  • Plant in-vitro ubiquitin protein degradation system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Example 1. Construction of recombinant expression vector

[0090] 1. Preparation of recombinant plasmid pET28a-UBA2

[0091] 1. Design primers to amplify the full CDS sequence of Arabidopsis ubiquitin activating enzyme UBA2 gene (GenBank: U40566.1, nucleotide sequence from 414 to 3647 at the 5'end, sequence 2) and separate at both ends Add EcoRI and SalI restriction sites.

[0092] 2. Use the primers of step 1 to clone the CDS full sequence of UBA2 gene by PCR, and cut with restriction enzymes EcoRI and SalI.

[0093] 3. Cut the pET28a vector with restriction enzymes EcoRI and SalI to obtain the vector backbone.

[0094] 4. Connect the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid pET28a-UBA2. Sequencing showed that the recombinant plasmid pET28a-UBA2 was inserted between the EcoRI and SalI sites of the backbone vector pET28a: GenBank: U40566.1, from the 5'end 414 to 3647 nucleotide sequence, that is, the sequence 2 in the seque...

Embodiment 2

[0149] Example 2. Preparation of protein

[0150] 1. Expression and purification of 6×His fusion protein and UBCH5b protein

[0151] 1. Preparation of E. coli Bl21 (DE3) competent cells.

[0152] 2. The recombinant plasmids prepared in step 1 and step 6 of Example 1 with pET28a as the vector backbone were respectively heat shocked into B121 (DE3) competent cells.

[0153] 3. Pick 2-3 single clones in 3-10 mL of LB liquid medium supplemented with corresponding antibiotics, and cultivate overnight at 37°C at 200 rpm.

[0154] 4. Add the cell culture after overnight to 200-500mL LB liquid medium containing the corresponding antibiotics and 0.2% (0.2g / 100mL) glucose to make the OD600 about 0.1.

[0155] 5. When shaking culture at 18°C ​​until the OD600 is 0.5-0.6, add the inducer isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.2mM; induce at 18°C ​​for 12-16h.

[0156] 6. Centrifuge for 10 minutes at 4°C, 4000 rpm, and collect the bacteria.

[0157] 7. Pre-cooled Lysis...

Embodiment 3

[0180] Example 3, Example 2 Purified Arabidopsis ubiquitin activating enzyme and Arabidopsis ubiquitin protein activity detection

[0181] In this example, the activity of the Arabidopsis ubiquitin activating enzyme and the Arabidopsis ubiquitin protein purified in Example 2 was tested according to the following method:

[0182] (1) Take two 1.5mL Eppendorf tubes and mark them as sample 1 and sample 2. Add 0.2μg of human ubiquitin conjugating enzyme (UBCH5b) obtained in Example 2, 3-5μg of Arabidopsis wild-type monomeric ubiquitin protein (His-Ub) and 1μL of 20× reaction buffer ( Formula: 1M Tris pH7.5, 100mMATP, 50mM MgCl 2 , 2mM DTT, solvent is water). In addition, 0.05μg of Arabidopsis ubiquitin activating enzyme UBA2 (His-AtE1) was added to sample 1, and the same amount of wheat ubiquitin activating enzyme (His-wE1) was added to sample 2. Add ddH to both samples 2 O to a total volume of 20 μL.

[0183] (2) After mixing the samples, take out 1 / 2 volume of the reaction solution,...

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Abstract

The invention discloses a plant in-vitro ubiquitin protein degradation system and an application thereof. The plant in-vitro ubiquitin protein degradation system provided by the invention is a composition consisting of one ubiquitin activating enzyme from arabidopsis thaliana, 14 ubiquitin-conjugating enzymes and wild-type K48R and K63R point mutation ubiquitin protein. Compared with the currently common used in-vitro ubiquitination reaction component, all components of the composition provided by the invention come from the model plant arabidopsis thaliana; when the composition is used for detecting whether the to-be-detected protein of plant origin has ubiquitination activity, the analysis result is more real and credible; the composition provided by the invention contains the component capable of representing most of the subfamily characteristics of the ubiquitin-conjugating enzyme of arabidopsis thaliana, and the coverage is wide; the activity of ubiquitin ligase can be detected and the specific binding condition of E2-E3 can be analyzed more comprehensively by use of the components, and more clues can be provided for studying the gene functions; and moreover, the type of polyubiquitin chain formed by the ubiquitination reaction can be analyzed by use of the point mutation ubiquitin protein.

Description

Technical field [0001] The invention relates to a plant in vitro ubiquitin protein degradation system and its application, in particular to a plant in vitro ubiquitin protein degradation system in which all active components are derived from Arabidopsis and its application. Background technique [0002] The ubiquitin / 26S proteasome system UPS in eukaryotes can not only degrade abnormal proteins, but also remove most of the regulatory proteins with short half-lives (such as transcription factors, cyclins, etc.), so It plays an important regulatory role in maintaining the normal life activities of cells. Existing studies have confirmed that the impact of this system in plants is more important, and its function is almost involved in the control of key steps in all physiological pathways in plants, and it is one of the finest regulatory systems in plants. Covalently attaching ubiquitin as a protein tag to the target protein molecule requires the ubiquitin activating enzyme (E1), ub...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63
Inventor 谢旗赵庆臻
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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