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Method for simply synthesizing ubiquitin probe Ub-Rho110-Gly

A ubiquitin and probe technology, applied in the field of protein synthesis, can solve problems such as high toxicity and limitations, and achieve the effects of low synthesis cost, simple synthesis steps, and high reaction conversion efficiency.

Active Publication Date: 2019-05-31
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a common staining agent, rhodamine has a wide range of applications, and its derivatives are also many. Among them, rhodamine B is a typical representative, but its toxicity is high, so its use is greatly restricted.

Method used

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  • Method for simply synthesizing ubiquitin probe Ub-Rho110-Gly
  • Method for simply synthesizing ubiquitin probe Ub-Rho110-Gly
  • Method for simply synthesizing ubiquitin probe Ub-Rho110-Gly

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Pick the ubiquitin mutant Ub77C monoclonal colony into 40mL LB medium containing ampicillin resistance, and culture at 37°C, 220rpm for 14h.

[0036] Take the bacteria liquid cultivated in the above steps and enlarge it into 4L LB medium containing ampicillin resistance according to the volume ratio of 1:100, and continue to cultivate at 37°C and 220rpm. When the OD 600 absorbance value of the bacteria liquid reaches 0.8, add 1.0M IPTG The final concentration of IPTG in the stock solution was 0.4mM, and the bacterial solution was induced, and cultured at 37°C and 200rpm for 6h.

[0037] Collect the cultured bacterial solution by centrifugation (25°C, 8000rpm, 10min), discard the supernatant and resuspend the obtained bacterial cells with 80mL lysis buffer (50mM Tris-HCl, 150mMNaCl, pH=7.4), and lyse the bacterial cells with a sonicator body. Use a high-speed refrigerated centrifuge to centrifuge the cell disruption solution (13000rpm, 30min, 4-8°C) and collect the supe...

Embodiment 2

[0041] 50mg of Ub(1-75)-NHNH 2 Dissolve in 6mL of 6M guanidine hydrochloride and 0.2M disodium hydrogen phosphate PBS buffer solution with pH=3.0, add 60uL of 1.0M sodium nitrite aqueous solution, and react at -20°C for 20min; after the reaction, add 39mg of Set sodium, and adjust the pH to 5.0, and react at room temperature for 20 minutes; after the reaction is completed, use semi-preparative high-performance liquid chromatography to purify the above-mentioned reaction solution, collect the purified solution, and freeze-dry to obtain 40 mg of ubiquitin thioester Ub ( 1-75) - Mesna.

Embodiment 3

[0043] Weigh 50mg of ubiquitin thioester Ub(1-75)-Mesna protein dry powder and dissolve in 5mL of pure DMSO, add 2% (volume fraction) of thiophenol according to the volume of the solution, add 129mg of Gly-Rho110-Gly, and Add 96mL of DIEA, stir to dissolve, and react at room temperature for 2h. After the reaction is complete, the reaction solution is precipitated with 20mL of ether, purified by semi-preparative high-performance liquid chromatography, and the purified solution is collected. and lyophilized to obtain the target product ubiquitin probe Ub-Rho110-Gly.

[0044] In summary, the present invention provides a synthetic method for easily obtaining the ubiquitin probe Ub-Rho110-Gly.

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Abstract

The invention discloses a method for simply synthesizing ubiquitin probe Ub-Rho110-Gly.The method comprises the following steps: firstly, preparing ubiquitin hydrazideUb (1-75)-NHNH2 by using an N-S acyl group migration semi-synthesis strategy, further converting into ubiquitin thioesterUb (1-75)-Mesna based on a hydrazide method, and then obtaining the ubiquitin probe Ub-Rho110-Gly by direct ammonolysis.The method has the characteristics of high preparation yield, high synthetic purity (about Ub (1-75)-NHNH230-40mg can be obtained per 1L culture medium after freeze-drying), simple operation and mass preparation.

Description

technical field [0001] The invention relates to a method for simply synthesizing ubiquitin probe Ub-Rho110-Gly, which belongs to the technical field of protein synthesis. Background technique [0002] Ubiquitination is a common post-translational modification involved in the regulation of many fundamental physiological processes, such as protein degradation, transcription, and DNA damage repair. Ubiquitination is generally achieved through the synergistic action of three enzymes (E1, E2, E3), that is, the glycine at the C-terminal of ubiquitin is connected to the side chain amino group of lysine in the substrate protein through an isopeptide bond. The ubiquitin molecule itself can also be used as a substrate protein, using its N-terminal Met or seven lysines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63) to form different lengths, uniform or non-uniform multimeric Ubiquitin chains, and both were identified in cell lysates. In addition, Met 1 at the N-terminal of ubiqui...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/36C07K1/34C07K1/16
Inventor 李宜明叶银杉樊健
Owner HEFEI UNIV OF TECH
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