Difference between revisions of "Part:BBa K3396005"

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<p style=" font-weight: bold; font-size:13px;"> Table 2: Binding affinity of tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS to protacs with different tau binding peptides models. </p>
 
<p style=" font-weight: bold; font-size:13px;"> Table 2: Binding affinity of tTrim21-(G<sub>4</sub>S)<sub>3</sub>-DocS to protacs with different tau binding peptides models. </p>
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Revision as of 14:55, 12 October 2022


Trim21-DocS

This composite part derives from TRIM21 and replace its PRYSPRY domain with DocS, and it perform the same function with the original TRIM21. What’s more, HA is added to the N-terminal of TRIM21, making it easier for TRIM21 to be detected by Western Blotting.


Usage and Biology

To demonstrate that the TRIM21 still works after replacing its PRYSPRY domain and keep its connection with all kinds of nanobodies, the PRYSPRY domain was replaced by DocS. Here is the mechanism of the recombined TRIM21-DocS:

1. The GFPnano tagged with Coh2 combines with targeted protein.

2. TRIM21-DocS connect Coh2-GFPnano-target through the DocS-Coh2 interaction.

3. The targeted protein is degraded by ubiquitin-proteasome system recruited by TRIM21.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 204
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 161
  • 1000
    COMPATIBLE WITH RFC[1000]

===Improvement by team CU_Egypt 2022)

Improvement by team CU_Egypt

There seemed to be some mistakes in the original sequence uploaded by NUDT 2020 (BBa_K3396005), there was an additional stop codon in the middle of the fusion protein and an extra Tyrosine residue in their Glycine-Serine Linker. These misconceptions hinder the usage of this part by any iGEM team, so we ought to provide them with the correct sequence that they could use easily if they want to assemble this part. 

            Figure 1.: Alignment of modified Trim-G4S-DocS sequence with the original sequence from NUDT_2020
(BBa_K3396007) showing the extra amino acid residues (Valine-Leucine-Glutamic acid- Lysine) and the stop codon in the middle.

The modified part was codon optimized and expressed in E. Coli to test its functionality in binding to our PROTAC (Coh2-G4S-Tau binding peptide), wet lab experiments showed significant results of expression and binding, these results were validated priorly by dry lab work of modeling and docking. 

      Figure 2.: Predicted 3D structure of our fusion protein Truncated trim (tTrim21)-(G4S)3-DocS.


Table 1: Quality assessment parameters of tTrim21-(G4S)3-DocS. model.

1.2. Docking

Docking is done to test the interaction of the whole fusion proteins together, and how it can change the binding affinity from the tagged part in our contribution to DocS (BBa_K3396000). The results prove that when DocS is fused to tTrim21, the resulted protein has higher affinity to GST-Coh2-linker-WWW than basic DocS and Coh2. 

                     Figure 3.: All docked structure of TLD by Galaxy and ClusPro displayed by Pymol.

Table 2: Binding affinity of tTrim21-(G4S)3-DocS to protacs with different tau binding peptides models.