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A simple method for the synthesis of 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 limitation, and achieve the effects of low synthesis cost, simple synthesis steps and high preparation yield

Active Publication Date: 2022-07-26
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • A simple method for the synthesis of ubiquitin probe ub-rho110-gly
  • A simple method for the synthesis of ubiquitin probe ub-rho110-gly
  • A simple method for the synthesis of ubiquitin probe ub-rho110-gly

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Experimental program
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Effect test

Embodiment 1

[0035] The ubiquitin mutant Ub77C monoclonal colonies were picked and placed in 40 mL of LB medium containing ampicillin resistance, and cultured at 37° C. and 220 rpm for 14 h.

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

[0037] The cultured bacterial liquid was collected by centrifugation (25°C, 8000 rpm, 10 min), the supernatant was discarded, and the resulting bacterial cells were fully resuspended with 80 mL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH=7.4), and the bacteria were lysed using a sonicator. body. Use a high-speed refrigerated centrifuge to centrifuge th...

Embodiment 2

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

Embodiment 3

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

[0044] In conclusion, 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 simple method for synthesizing ubiquitin probe Ub-Rho110-Gly. First, ubiquitin hydrazide Ub(1-75)-NHNH is prepared by using N-S acyl transfer semi-synthetic strategy 2 , and it was further converted into the ubiquitin thioester Ub(1-75)-Mesna based on the hydrazide method, and then the ubiquitin probe Ub-Rho110-Gly was obtained by the direct aminolysis method. The method of the invention has the advantages of high preparation yield and high synthesis purity (about Ub(1-75)-NHNH can be obtained per 1 L of culture medium after freeze-drying 2 30 ~ 40mg), easy to operate, can be prepared in large quantities.

Description

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

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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