Difference between revisions of "Part:BBa K2653006"

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===Contribution:NUDT_CHINA, 2020===
 
===Contribution:NUDT_CHINA, 2020===
  
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===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  
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transfected MCF7 cells with plasmids expressing ScFvErbB3-linker-Fc fusion protein and HA-
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transfected MCF7 cells with plasmids expressing ErbB3 ScFv-linker-IgG Fc fusion protein and HA-Trim21.
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Trim21. Western Blotting analysis showed that with the high expression of ScFvErbB3-linker-
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Fc fusion protein and HA-Trim21, the ErbB3 protein level decreased dramatically in cells
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transfected with ErbB3 Predator (approximately 30%, Fig 1A and B). This result demonstrated that the ScFv-based Predator system could successfully degrade the targeted membrane protein.
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https://static.igem.org/mediawiki/parts/c/c6/T--NUDT_CHINA--erbB3-composite.jpg
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===Result===
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Western Blotting analysis showed that with the high expression of ErbB3 ScFv-linker-
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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.
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<p><img src="https://2020.igem.org/wiki/images/c/c8/T--NUDT_CHINA--ContributionFig1.jpg" alt="" width="700"/></p>
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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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2574
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 3243
    Illegal BamHI site found at 3781
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2531
    Illegal AgeI site found at 1376
  • 1000
    INCOMPATIBLE 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.