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Method for finding E2-E3 specifically mediating target protein ubiquitination reaction based on known E1

A target protein, specific technology, applied in the field of biochemistry and molecular biology, can solve the problems of terror and unclear ubiquitin modification mechanism

Inactive Publication Date: 2013-02-20
JIANGSU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Similar studies have also shown that p12 and another regulatory subunit of Pol δ, p68, can also be modified by ubiquitination and SUMO (Liu, G. &. Warbrick, E., Biochem Biophys Res Commun, 2006, 349: 360-6 ), but so far, the ubiquitin modification mechanism of p12 is degraded (down-regulated), and the key point is that a good method has not been established to determine the specific recognition and mediation of target proteins from numerous E3 Ligase E3 for ubiquitination modification, finding the ubiquitination modification pathway of the E2 / E3 system to regulate target proteins has become a "horrible" experiment

Method used

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  • Method for finding E2-E3 specifically mediating target protein ubiquitination reaction based on known E1
  • Method for finding E2-E3 specifically mediating target protein ubiquitination reaction based on known E1
  • Method for finding E2-E3 specifically mediating target protein ubiquitination reaction based on known E1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Establishment of in vitro reaction system for ubiquitination modification and determination of E2

[0036] A 15 μL reaction system was used for in vitro ubiquitination reaction, containing: 10 μg ubiquitin, 30 nM UBE1, 20 ng of Ubiquitin aldehyde, 1x ENS, 300 ng GST-p12 as substrate, HeLa S-100 fraction as E3 source, and 500 nM UbcH5c, UbcH2, UbcH3(Cdc34) and UbcH13 / Uev1a complex as E2 respectively; reaction buffer is 5 mM MgCl 2 , 40 mM Tris-HCl, pH 7.5, 2 mM DDT, and GST was used as a control experiment. at 30 0 React under C condition for 60 minutes. At the end of the reaction, add 800 μL of Pull-down buffer (1xPBS containing 0.5% NP-40) to terminate the reaction, then add 10 μL of Glutathione Sepharose-4B beads, and shake at room temperature for 60 minutes; Wash the beads 6 times with Pull-down buffer; add 1 x SDS-PAGE loading buffer, 95 0 C for 5 minutes, and the samples were electrophoresed on a 12% SDS-PAGE gel and then transferred to the membrane. ...

Embodiment 2

[0037] Example 2: Determination of the E3 activity of each fraction after the first-stage immunoaffinity chromatography

[0038] about 2×10 8 Hela cells cultured in vitro, lysed by ultrasound, 4 0 After high-speed centrifugation at C, take the supernatant as the cell lysis extract (the cell lysis buffer used is: 20 mM Tris-Cl, pH7.8, 0.5 mM EGTA, 1 mM EDTA, 1 mM MgCl, 50 mM NaCl , 10% Glycerol, Protease Inhibitor). After such figure 2 The schematic diagram of the technical route shown, through the combination of four chromatographic columns to identify unknown E3. Detect the E3 activity of the eluted fractions in each step of chromatography, and then combine the E3 activity peaks and pass through the next chromatography column. After the last step of chromatography, conduct mass spectrometry identification on the fraction peaks containing E3 activity to determine the specific recognition target E3 of protein p12.

[0039] Taking UBE1 as E1 and the UbcH13 / Uev1a complex de...

Embodiment 3

[0040] Example 3: Determination of the E3 activity of each fraction after separation by the second-stage Phenyl Sepharose hydrophobic chromatography

[0041]The Ft in Example 2 was dialyzed in the dialysate TGEED (TGEE+1 mM DDT) / 500 mM ammonium sulfate, and the dialyzed Ft was separated by Phenyl Sepharose hydrophobic chromatography, and TGEED containing 500 mM to 0 mM ammonium sulfate for gradient elution, and then detect the E3 activity in each eluted fraction. Such as Figure 4 As shown, the E3 activity using GST-p12 as substrate mainly exists in fraction No. 50. Fractions 40-60 were pooled and dialyzed against TGEED dialysate to remove ammonium sulfate.

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Abstract

The invention relates to the fields of biochemistry and molecular biology, and specifically relates to a method forfinding an ubiquitin ligase E3 specifically identifying a target protein and a E2 / E3 system for regulating target protein ubiquitination reaction from a number of E3 of human cells. According to the invention, an in-vitro ubiquitination analysis system is established; a known ubiquitin activating enzyme E1 and HelaS100 fraction are adopted as E3 sources, and a ligase E2 mediating the target protein ubiquitination reaction is screened; and a multiple chromatographic column combination is combined with mass spectrometric analysis, such that the ubiquitin ligase E3 specifically identifying the target protein and the E2 / E3 system regulating target protein ubiquitination modification are searched from human cells. With the method, the target-protein-identifying substrate recognition factor which is ubiquitin ligase E3 can be conveniently and accurately found, and a E2 / E3 system mediating target protein ubiquitination modification can be established.

Description

technical field [0001] The invention relates to the fields of biochemistry and molecular biology. Specifically, it refers to a method for finding the E2 / E3 system that specifically recognizes target protein ubiquitin ligase E3 and regulates target protein ubiquitination reaction from numerous E3 in human cells. technical background [0002] Among various forms of protein post-translational modification, ubiquitination has attracted more and more attention in recent years. Like the phosphorylation modification process, the ubiquitination modification process is also a reversible covalent modification process, which is involved in many processes of cell life activities by regulating the stability, functional activity state and intracellular location of the modified protein. play a vital role. [0003] The target protein modified by polyubiquitination (Poly-ubiquitination) is generally recognized by the proteasome and then degraded. This process mainly involves three key enzy...

Claims

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Application Information

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IPC IPC(8): C12Q1/25G01N33/68
Inventor 张倩周亚竟陈慧卿陈克平麦维军刘晓勇何远清李国辉张磊刘留樊晓婷宋惠芳
Owner JIANGSU UNIV
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