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.
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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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