Plant in-vitro ubiquitin protein degradation system and application thereof

A technology of ubiquitin protein and protein, which is applied in the field of plant in vitro ubiquitin protein degradation system, which can solve the problems of single, false negative, and inability to detect E3 protein activity.

Inactive Publication Date: 2013-05-08
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • 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] Embodiment 1, the construction of recombinant expression vector

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

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

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

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

[0094] 4. Ligate 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 into GenBank: U40566.1 between the EcoRI and SalI sites of the backbone vector pET28a, from nucleotide sequence 414 to 3647 at the...

Embodiment 2

[0149] Embodiment 2, the preparation of albumen

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

[0151] 1. Prepare Escherichia coli Bl21(DE3) competent cells.

[0152] 2. The recombinant plasmids prepared in Step 1 and Step 6 of Example 1 and using pET28a as the vector backbone were transformed into Bl21(DE3) competent cells by heat shock method respectively.

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

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

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

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

[0...

Embodiment 3

[0180] Activity detection of the Arabidopsis thaliana ubiquitin activating enzyme and the Arabidopsis ubiquitin protein purified in Example 3 and Example 2

[0181] In this example, the activities of the Arabidopsis thaliana ubiquitin activating enzyme and the Arabidopsis ubiquitin protein purified in Example 2 were detected according to the following method:

[0182] (1) Take two 1.5mL Eppendorf tubes and record them as sample 1 and sample 2 respectively. Add 0.2 μg of human ubiquitin-conjugating enzyme (UBCH5b) obtained in Example 2, 3-5 μg of Arabidopsis wild-type monomeric ubiquitin protein (His-Ub) and 1 μL of 20× reaction buffer ( Formulation: 1M Tris pH7.5, 100mM ATP, 50mM MgCl 2 , 2mM DTT, the solvent is water). In addition, 0.05 μg of Arabidopsis thaliana 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....

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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 come from Arabidopsis thaliana and its application. Background technique [0002] The ubiquitin / 26S proteasome system (UPS) in eukaryotes can not only degrade abnormal proteins, but also remove most regulatory proteins with short half-lives (such as transcription factors, cell cycle proteins, etc.), so It plays an important regulatory role in maintaining the normal life activities of cells. Existing studies have confirmed that the influence of this system is more important in plants, and its function is almost involved in the control of key steps of all physiological pathways in plants, and it is one of the most delicate regulatory systems in plants. The covalent attachment of ubiquitin as a protein tag to target protein molecules requires the sequentia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68C12N15/63
Inventor 谢旗赵庆臻
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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