Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Bio-PROTAC artificial protein targeting UBE2C

A protein and artificial technology, applied in DNA/RNA fragments, animal/human peptides, recombinant DNA technology, etc., which can solve the problems of lack of small molecule inhibitors, difficulty in developing inhibitors, and missegregation of chromosomes.

Pending Publication Date: 2022-02-18
SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT +1
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current study shows that overexpression of UBE2C leads to chromosome missegregation, which in turn alters the cell cycle process and promotes cell proliferation
In many human tumor tissues, UBE2C has been detected to be overexpressed, and this phenomenon is related to the progression and poor prognosis of various tumors. These evidences indicate that it is involved in the development and invasion of tumors and is a potential cancer target. However, due to the relatively smooth surface of this protein, it is difficult to develop its inhibitors, so there is currently no small molecule inhibitors for it

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Bio-PROTAC artificial protein targeting UBE2C
  • Bio-PROTAC artificial protein targeting UBE2C
  • Bio-PROTAC artificial protein targeting UBE2C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Prokaryotic expression and isolation and purification of bio-PROTAC

[0048] The prokaryotic expression and separation and purification steps of the bio-PROTAC of the present invention are as follows:

[0049] (1) Prokaryotic expression vector construction:

[0050] The bio-PROTAC can be expressed using the pet28 series of prokaryotic vectors. First, pet28a was reverse-PCRed to obtain a linear vector. Use the 15bp-20bp sequence at the end of the linearized vector as a homologous sequence, and add it to the 5' end of the gene-specific forward / reverse amplification primer sequence, and then use the target gene as a template to amplify to obtain homologous Sequence inserts. Mix the linearized vector and the insert in proportion, and mix the linear template and the insert at a molar ratio of 1:2, then add 1 μl of Exnase II, 2 μl of 5×CE II Buffer, and use dd H 2 Make up O to 10 μl, and react at 37°C for 30 minutes to complete the recombination reaction. Then...

Embodiment 2

[0055] Example 2. In vitro ubiquitination activity assay

[0056] 0.25μM E1 (mouse UBA1, 120Kd), 2μM E2 (UBE2D2, 19.6Kd), 0.5μM E3 (bio-PROTAC WT , bio-PROTAC CA , 43.5Kd), 50μM Ub (human Ub, 8.6Kd), 5mM MgCl, 2.5mM ATP and 2MmUBE2C (20Kd) were mixed and incubated in PBS buffer (pH7.4) at room temperature. Samples were taken at different time points, and the reaction mixture was added to an equal volume of 2*SDS-PAGE loading, and heated in a boiling water bath for 10 min. Afterwards, 12% SDS-polyacrylamide gel was used for electrophoresis, and the gel after electrophoresis was stained with Coomassie Brilliant Blue staining solution, and then decolorized overnight in acetic acid destaining solution. Finally image the gel with a gel imager.

[0057] The result is as Figure 4 As shown, the bio-PROTAC (wild type) with the correct sequence can ubiquitinate UBE2C in vitro, while the mutant (the cysteine ​​at position 184 of the bio-PROTAC protein sequence provided by this paten...

Embodiment 3

[0059] Example 3. Verification of the activity of bio-PROTAC in degrading exogenous UBE2C in cells

[0060] (1) Construction of eukaryotic expression vector: Since the protein itself is difficult to pass through the cell membrane, it is necessary to construct a eukaryotic expression vector first. The construction method is similar to the construction method of the prokaryotic expression vector described above, and will not be repeated here. This bio-PROTAC can be constructed using the PCDNA3.1 series vectors.

[0061] (2) Transfection of eukaryotic vector into cells: inoculate a 6-well plate with culture medium without antibiotics one day before transfection, and perform transfection when the cell density grows to 70%-90% on the second day. It is recommended to use lipo 2000 (thermofisher) as transfection reagent. Cell culture medium should be replaced with opti-MEM before transfection. For transfection, first dilute the DNA and 8 μl lipo 2000 with 250 μl opti-MEM respectiv...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention belongs to the technical field of biology, and relates to a novel artificial protein targeting UBE2C and practical application of the novel artificial protein. The fusion protein disclosed by the invention is composed of two protein structural domains, namely a WHB structural domain and an NEL structural domain, wherein the WHB structural domain is derived from a structural domain of a natural APC2 protein which has direct interaction with UBE2C, and NEL is from a conserved E3 enzyme structural domain of an E3 enzyme IPAN9.8 of Shigella. The bio-PROTAC disclosed by the invention can be used for specifically recognizing UBE2C, successfully realizes ubiquitination modification of UBE2C protein in a cell-free environment, and can degrade UBE2C subjected to exogenous expression in cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a novel artificial protein targeting UBE2C, its preparation method and practical application. Background technique [0002] UBE2C (Gene ID: 11065) is an exclusive E2 enzyme (ubiquitin-conjugating enzyme) of the anaphase-promoting complex / loop body (APC / C), which is mainly responsible for the ubiquitin chain on Lys-11 (K11) on the APC / C substrate protein The initiation of the ubiquitin chain is further elongated by APC / C and another E2 enzyme UBE2S, thereby jointly regulating the progression of mitosis. Current studies have shown that overexpression of UBE2C leads to chromosome missegregation, which in turn alters the cell cycle process and promotes cell proliferation. In many human tumor tissues, UBE2C has been detected to be overexpressed, and this phenomenon is related to the progression and poor prognosis of various tumors. These evidences indicate that it is involved in the develo...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/47C12N9/00C12N15/62C12N15/70
CPCC07K14/47C12N9/93C12N15/70C12Y603/02019C07K2319/00Y02A50/30
Inventor 李子刚尹丰汪金鹏叶宇鑫
Owner SHENZHEN BAY LAB PINGSHAN TRANSLATIONAL MEDICINE CENT
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com