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

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

[0036] The in vitro ubiquitination reaction used a 15 μL reaction system 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 was 5 mM MgCl 2 , 40 mM Tris-HCl, pH 7.5, 2 mM DDT, while GST was used as a control experiment. at 30 0 The reaction was carried out under the condition of C for 60 minutes. At the end of the reaction, 800 μL of Pull-down buffer (containing 0.5% NP-40 in 1xPBS) was added to stop the reaction, and then 10 μL of Glutathione Sepharose-4B beads were added, and shaken at room temperature for 60 minutes; Wash beads 6 times with Pull-down buffer; add loading buffer for 1 x SDS-PAGE, 95 0 C was incubated for 5 minutes, and the samples were electrophoresed on a 12% SDS-PAGE gel and then transferred to the membrane. Bead-adsorbed p12 polyubiquitin conjugates detected with an anti-ubiquitin polyclonal antibody such as figure 1 As shown in B, in known E1 (UBE1), using HeLa S-100 as the E3 source, the UbcH13/Uev1a complex (represented by UbcH13 in the figure) mediates the strongest polyubiquitin response signal of p12.

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

[0038] about 2×10 8 HeLa cells cultured in vitro, lysed by sonication, 4 0 After high-speed centrifugation at C, the supernatant was taken as the cell lysis extract (the cell lysis buffer used was: 20 mM Tris-Cl, pH 7.8, 0.5 mM EGTA, 1 mM EDTA, 1 mM MgCl, 50 mM NaCl) , 10% Glycerol, protease inhibitor). through such as figure 2 Schematic diagram of the technical route shown, identification of the unknown E3 by a combination of 4 chromatographic columns. Detect the E3 activity of the eluted fractions of each step of chromatography, then combine the E3 activity peaks and pass through the next chromatography column. After the last step of chromatography, perform mass spectrometry identification on the peaks of the fractions containing E3 activity to determine the specific recognition target. E3 of protein p12.

[0039] Taking UBE1 as E1 and the UbcH13/Uev1a complex determined in Example 1 as E2, the E3 activity of each fraction after separation by first-stage immunoaffinity chromatography was detected by the established ubiquitin reaction in vitro analysis system. . like image 3 As shown in A, the DNA Pol δ four-subunit protein complex was eluted from fraction 12 to fraction 30 by immunoaffinity chromatography. The eluent used in the chromatography process was: TGEE (40 mM Tris-Cl , pH7.8, 10% Glycerol, 0.5 mM EGTA, 1 mM EDTA) containing 400 mM NaCl and 30% ethylene glycol. like image 3 As shown in B, the E3 activity using GST-p12 as a substrate mainly exists in the Ft fraction (the supernatant after passing through the column Flow through), and is not detected in the 15th fraction of the elution peak of the Pol δ four-subunit protein complex. to E3 activity. The Pol delta four-subunit protein complex and E3 activity have been separated.

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

[0041]The Ft in Example 2 was dialyzed in dialysate TGEED (TGEE+1 mM DDT)/500 mM ammonium sulfate, and the dialyzed Ft was separated by Phenyl Sepharose hydrophobic chromatography, using TGEED containing 500 mM to 0 mM ammonium sulfate. Sulfate gradient elution was carried out, and E3 activity in each eluted fraction was detected. like Figure 4 shown, E3 activity with GST-p12 as a substrate was mainly present in fraction 50. Fractions 40-60 were pooled and dialyzed against TGEED dialysate to remove ammonium sulfate.