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Mutant xsumo and related products

A technology of mutants and mutation sites, applied in the field of mutant xSUMO and its related products, can solve the problem of inability to distinguish between E2-specific substrates and E3-specific substrates, and achieve the effect of deepening understanding

Active Publication Date: 2021-09-03
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a mutant xSUMO and its related products, which are used to construct an orthogonal SUMO pathway, so as to solve the problem that the specific substrate of E2 or E3 cannot be distinguished in the prior art. The question of the specificity of the substrate

Method used

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  • Mutant xsumo and related products
  • Mutant xsumo and related products
  • Mutant xsumo and related products

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 total RNA extraction and cDNA acquisition

[0048] After counting 293 cells (Cell Bank, Chinese Academy of Sciences), centrifuge at 800rpm for 3min, and 6 Add TRIzol (Thermo Scientific) to cells / ml TRIzol, and repeatedly pipette to dissolve the cells. Centrifuge at 12000g for ten minutes at 4°C (to remove insoluble matter such as adipose tissue), and transfer the supernatant to a clean centrifuge tube. The above supernatant was lysed at room temperature for 5 min. Add chloroform at a ratio of 0.2ml chloroform / ml TRIzol, cover the lid, shake vigorously by hand, and then let stand on ice for 10min. 4°C, 12000r / min, centrifuge for ten minutes, divide into three layers after centrifugation, pay attention to absorb the upper layer of water into a new centrifuge tube, be careful not to suck into the middle interface. Add isopropanol at a ratio of 500ul / ml TRIzol, mix well, and let stand on ice for 10min. 4°C, 12000r / min, centrifuge for ten minutes, discard the s...

Embodiment 2

[0051] Plasmid Construction of Example 2 wtSUMO, wtSAE and wtUbc9

[0052] (1) Use the extracted cDNA as a template for PCR amplification:

[0053] A small HA peptide was fused in front of the wtSUMO protein as an antibody tag for later western validation. Design the upstream and downstream primers of wtHA-SUMO.

[0054] Table 1 Target genes and their primers

[0055]

[0056] The 50ul reaction system is: 1ul wtSUMO, 1ul upstream primer (10uM), 1ul downstream primer (10uM), 25ulrTaq Mix, 22ul ddH20. After adding and mixing the samples of the PCR reaction system, put them into the PCR instrument. The program is as follows: pre-denaturation at 94°C for 2 minutes, denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 1 minute, and reaction for 35 seconds. cycle, with a final extension of 7 minutes at 72°C and temporary storage at 4°C.

[0057] (2) Sample gel electrophoresis:

[0058] All samples were loaded into electrophoresis (acco...

Embodiment 3

[0066] Example 3 Prokaryotic expression and purification of wtSUMO, wtSAE and wtUbc9

[0067]Take 4ul wtSUMO plasmid and 50ul BL21(DE3) competent cells (Shanghai Kanglang Biotechnology Co., Ltd.) and mix carefully, and perform chemical transformation in the same way as above. Finally, take 400ul resuscitation liquid and spread it on the Kana antibiotic plate, and incubate the bacteria at 37℃ Incubate overnight in the incubator. Pick a single clone into 10ml LB (Kana), shake overnight at 37°C, transfer to 1L LB (Kana), shake and culture at 37°C until the bacterial density OD600=0.6-0.8, add 1ml IPTG (1M), 16°C Induced overnight at low temperature for 16 hours.

[0068] Collect the bacteria the next day, centrifuge the bacterial liquid at 4°C, 7000rpm for 10 minutes, pour off the supernatant, add 10ml lysisbuffer to resuspend the bacterial pellet at the bottom of the centrifuge bottle, and then add 10ul MgCl 2 , 10ul CaCl 2 And 5mg lysozyme, and mix well. The bacterial cells...

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Abstract

The present invention provides mutant xSUMO and related products thereof. The mutant xSUMO contains at least the following mutation sites for wild-type SUMO: the 70th R is replaced by E, the 91st Y is replaced by A, and the 93rd E is replaced by R . The construction of mutant xSUMO promoted the construction of xSUMO-xE1 mutant pair in the orthogonal SUMO pathway, laid a good foundation for the completion of the OST pathway, and deepened the understanding of the interaction and structure of SUMO and E1.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant xSUMO and related products. Background technique [0002] SUMO (Small ubiquitin-like modifier) ​​is a very important small molecular weight protein (~12kDa) in eukaryotic cells. There are 5 different SUMO proteins in the human body, which are SUMO1-5( figure 1 ). SUMO-1, the most common SUMO protein, contains 101 amino acids, has a molecular weight of 11.6 kDa, and has about 50% sequence similarity with SUMO-2 / 3. Although the sequence similarity between SUMO-1 and ubiquitin (Ub) is only 18%, the two have similar three-dimensional spatial structures, and SUMO-1 only has a stretch of about 20 amino acids more flexible than ubiquitin at the N-terminus Extended structure ( figure 1 ). As a post-translational modification, SUMOylation is involved in many important cellular functions, including DNA damage and repair, cell cycle regulation, cell apoptosis and proliferation, tr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N15/12G01N27/62
CPCC07K14/4702G01N33/6848
Inventor 赵博叶琳李新宇刘金钊
Owner SHANGHAI JIAOTONG UNIV
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