Part:BBa_K2653016

GFP-nano-IgG1-FC-HA TAG-Trim21

Functional unit of the PR PREDATOR, expressing fusioned GFPnano-hIgG1-FC and HA tag-Trim21 by automatic cleavage of P2A.

Usage and Biology

Besides the necessary promoter CMV and terminator BHG, our composite part BBa_K2653016 is a crucial functional part in the whole 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.
GFP-nano is the nanobody of the green fluorescence protein, which has only VH site of normal GFP-antibody, and is linked together with hIgG1-Fc by the designed 3xGS linker to form a recombinate GFP antibody.
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 nanobody of GFP 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.

===Sequence and Features===

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2813
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 3482
  Illegal BamHI site found at 4020
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 2770
  Illegal AgeI site found at 1615
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 721
  Illegal BsaI site found at 2125
  Illegal BsaI site found at 3860
  Illegal SapI.rc site found at 2473
  

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.