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SUMO fluorescent probe and preparation method thereof

A fluorescent probe, fluorescein technology, applied in the field of protein synthesis, to achieve the effects of high reaction conversion efficiency, low cost and low synthesis cost

Pending Publication Date: 2019-10-01
合肥科生景肽生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the preparation process of SUMO fluorescent probes still needs to be further improved.

Method used

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  • SUMO fluorescent probe and preparation method thereof
  • SUMO fluorescent probe and preparation method thereof
  • SUMO fluorescent probe and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Preparation of SUMO Hydrazide SUMO2(2-92)-NHNH Using the Intein Method N-S Migration Strategy 2 . It is based on commercial Intein-containing strains, and with the help of vectors based on conventional techniques, connect SUMO2 protein to the N-terminus of Intein, connect chitin-binding domain to its C-terminus, and transform Escherichia coli to obtain strains capable of expressing Intein and SUMO2 proteins . Among them, the strain containing Intein is commercialized BL21, purchased from Quanshijin Company, the vector cloned in this strain is PTXB1, and the restriction sites are NdeI and SapI.

[0058] The strain capable of expressing Intein and SUMO2 is cultured, lysed to obtain a supernatant, and then the chitin beads are used for affinity chromatography, and the chitin beads bind to the chitin-binding protein in the supernatant, and the chitin The binding protein is connected to the intein, and the other end of the intein is connected to the SUMO2 target ...

Embodiment 2

[0070] Example 2 Preparation of SUMO thioester SUMO2(2-92)-Mesna by protein hydrazide method, including the following steps:

[0071] 50mg of SUMO2(2-92)-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 Sodium, and adjust the pH to 5.0, and react at room temperature for 20 minutes; use semi-preparative high-performance liquid chromatography after the reaction is completed, such as image 3 As shown, the above reaction solution was purified, the purified solution was collected, and about 6-8 mg of SUMO thioester SUMO2(2-92)-Mesna could be obtained per 1 L of LB medium after lyophilization.

[0072] image 3 It is the high performance liquid chromatogram of SUMO thioester SUMO2(2-92)-Mesna, the abscissa represents the time, and the ordinate represents the absorption value. At the same time, th...

Embodiment 3

[0074] Embodiment 3 provides a kind of method utilizing SUMO thioester SUMO2(2-92)-Mesna to generate SUMO probe SUMO2-Rho110-Gly, which comprises: first by Boc 2 O acid anhydride protects SUMO thioester SUMO2(2-92)-Mesna to form Boc-SUMO2(2-92)-Mesna, followed by direct aminolysis of Boc-SUMO2(2-92)-Mesna to obtain SUMO probe SUMO2-Rho110 -Gly.

[0075] Specifically include the following steps:

[0076] 3a. Weigh 43 mg of SUMO2(2-92)-Mesna protein dry powder, dissolve it in 1.5 mL DMSO (dimethyl sulfoxide), and add 18.5 μL of Boc 2 O acid anhydride (di-tert-butyl dicarbonate) and 8 μL of DIEA (N,N-diisopropylethylamine), stirred at room temperature for 1 h, precipitated the protein with glacial ether, and dried naturally to obtain Boc-SUMO2(2- 92) - Mesna;

[0077] 3b. Dissolve the precipitated protein in 3a in 2.5mL DMSO, add 10μL of thiophenol, and then add 11.5mg of Gly-Rho110-Gly. After the small molecule Gly-Rho110-Gly is completely dissolved, add 8.5μL DIEA, after re...

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Abstract

The invention provides a SUMO fluorescent probe and a preparation method thereof. The method comprises the steps that 1, on the basis of Interin N-S migration method, SUMO hydrazide SUMO2(2-92)-NHNH2is prepared; 2, on the basis of the SUMO hydrazide SUMO2(2-92)-NHNH2, by means of the hydrazide method, SUMO thioester SUMO2(2-92)-Mesna is obtained; 3, on the basis of a reaction of the SUMO thioester SUMO2(2-92)-Mesna and the fluorescein micromolecule rhodamine, the SUMO fluorescent probe with rhodamine can be obtained conveniently. The preparation method has the advantages of being high in preparation yield, high in synthesis purity, easy to implement and capable of achieving large-scale preparation, and suitable for application of industrial production of the UMO fluorescent probe.

Description

technical field [0001] The invention relates to the technical field of protein synthesis, in particular to a SUMO fluorescent probe and a preparation method thereof, in particular to a preparation method of the SUMO probe SUMO2-Rho110-Gly. Background technique [0002] Post-translational modifications (PTMs) are essential for many cellular processes, which are often reversible. And there are a large number of post-translational modifications (such as phosphorylation, methylation, acetylation and glycosylation) that occur in cells to regulate various life activities. Among them, small ubiquitin-like modifier protein (SUMO) modification is a highly dynamic process that can be reversed by SUMO-specific proteases, and plays an important role in various cellular processes, such as regulating nucleoplasmic transport, protein stabilization, and DNA damage. Repair, cell cycle regulation, gene transcription, differentiation, and cell localization, etc. SUMO molecules regulate the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C07K1/113C07K19/00C09K11/06G01N33/68G01N33/574G01N33/573
CPCC07K14/00C07K19/00C09K11/06G01N33/6848G01N33/573G01N33/574G01N2333/4704G01N2333/95
Inventor 周莉
Owner 合肥科生景肽生物科技有限公司
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