Difference between revisions of "Part:BBa K2653006"
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===Method=== | ===Method=== | ||
To validate the protein degradation efficiency of the ScFv-based ErbB3 Predator system, we | To validate the protein degradation efficiency of the ScFv-based ErbB3 Predator system, we | ||
| − | transfected MCF7 cells with plasmids expressing | + | transfected MCF7 cells with plasmids expressing ErbB3 ScFv-linker-IgG Fc fusion protein and HA-Trim21. |
| − | Trim21. | + | |
===Result=== | ===Result=== | ||
| − | Western Blotting analysis showed that with the high expression of | + | Western Blotting analysis showed that with the high expression of ErbB3 ScFv-linker- |
| − | Fc fusion protein and HA-Trim21, the ErbB3 protein level decreased dramatically in cells | + | IgG Fc fusion protein and HA-Trim21, the ErbB3 protein level decreased dramatically in cells transfected with ErbB3 Predator (approximately 30%, Fig 1A and B). This result demonstrated that the ScFv-based Predator system could successfully degrade the target membrane protein. |
| − | transfected with ErbB3 Predator (approximately 30%, Fig 1A and B). This result demonstrated that the ScFv-based Predator system could successfully degrade the | + | |
| − | https:// | + | <html> |
| + | <p><img src="https://2020.igem.org/wiki/images/c/c8/T--NUDT_CHINA--ContributionFig1.jpg" alt="" width="700"/></p> | ||
| + | </html> | ||
Figure 1. (A and B) Western blotting assay showing the components of ErbB3 Predator and the degradation of ErbB3. | Figure 1. (A and B) Western blotting assay showing the components of ErbB3 Predator and the degradation of ErbB3. | ||
Latest revision as of 15:38, 27 October 2020
erbB3-composite
Complex of recombinate scFv of erbB3 and hIgG1-Fc.
Usage and Biology
This part is the key unit of our demonstration part. Besides the necessary promoter CMV and terminator BHG, the composite part BBa_K2653006 is a crucial functional part in the demonstration part of the PR PREDATOR system. The PR PREDATOR is designed to recognize and bind with target proteins, and then recruit the proteasome to deplete the target protein through the ubiquitin-proteasome pathway.
In the demostration, we insert sequence of recombinate scFv of erbB3, which is made up of VL, VH and an improved linker 3XGS into this part, and express the recombinate antibody of erbB3 that includes the scFv and the hIgG1-Fc.
Trim21, the E3 ubiquitin ligase, plays the most important role in the degradation system. The C-terminal B30.2 domain on trim21 offers a site for the conservative Fc region of human IgG 1,2 and 4 to bind with[1]. When the recombinate scFv of erbB3 is linked to the hIgG1-Fc, and after the antibody-antigen interaction, a ternary complex is build up. The trim21 then functions as a E3 ubiquitin ligase and proceeds the complex to be depleted through the ubiquitin-proteasome pathway.
Contribution:NUDT_CHINA, 2020
Method
To validate the protein degradation efficiency of the ScFv-based ErbB3 Predator system, we transfected MCF7 cells with plasmids expressing ErbB3 ScFv-linker-IgG Fc fusion protein and HA-Trim21.
Result
Western Blotting analysis showed that with the high expression of ErbB3 ScFv-linker- IgG Fc fusion protein and HA-Trim21, the ErbB3 protein level decreased dramatically in cells transfected with ErbB3 Predator (approximately 30%, Fig 1A and B). This result demonstrated that the ScFv-based Predator system could successfully degrade the target membrane protein.
Figure 1. (A and B) Western blotting assay showing the components of ErbB3 Predator and the degradation of ErbB3.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2574
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 3243
Illegal BamHI site found at 3781 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2531
Illegal AgeI site found at 1376 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1886
Illegal BsaI site found at 3621
Illegal SapI.rc site found at 2234
References
[1]Rhodes D A, Trowsdale J. TRIM21 is a trimeric protein that binds IgG Fc via the B30.2 domain[J]. Molecular Immunology, 2007, 44(9):2406-2414.