Part:BBa_K3396005
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 204
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 161
- 1000COMPATIBLE 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.
Dry Lab Results
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.
WetLab Results
Trim21 is an E3 ligase that can add ubiquitin molecules to the target protein. We fused this part to the dockerin protein to form the Snitch first composite that can bind to their binding partner Cohesine protein fused to Tau binding peptide, these two partners can trigger the Tau ubiquitination upon the interaction between TBP and tau. We started with making suspension of this part and ligate it with pJET using T4 ligase, then we transform it into DH5 alpha to amplify it. Then we extract the plasmid using manual miniprep protocol and restrict it using XbaI and XhoI to ligate it with pGS-21a, and transformation into BL-21 to induce protein expression using IPTG. We extracted the protein using a chemical lysis buffer and purify the protein using Ni-NTA affinity chromatography. We tested the activity and specificity of this part by using a pull-down assay that detects the protein-protein interaction between Docs linked to Trim21 and Coh2 which is linked to TBP, then we use BCA assay to characterize the interaction.
Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a
We transformed His Trim21 (L) Doc in BL-21 using the pGS-21a vector, and we used the TSS buffer protocol as it shows the best results compared to Calcium Chloride buffer and the combination between Calcium Chloride and Magnesium Chloride buffer.
Figure 2. Transformed plate of His Trim21 (L) Doc + pGS-21a
Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET vector
Figure 3. Transformed plate of His Trim21 (L) Doc + pJET
Comparison between chemical lysis and sonication for His Trim21 (L) DOC
We extract the protein Trim21 (L) Doc using two methods, the chemical method which is based on the use of lysozyme to lysis the bacterial cell membrane, in the other side the physical method, which used sonication to degrade the bacterial cell membrane. We compared the lysis methods using the BCA assay. The results of the BCA assay show that the extraction using the physical method is more efficient.
Figure 4. This graph shows a significant difference between chemical lysis and sonication
for His Trim21 (L) DOC, after we had the results, we optimized our protocol to
use sonication for His Trim21 (L) DOC
SDS PAGE of induced and non-induced samples of His Trim 21 (L) DOC
Figure 5. This figure shows the comparison between the induced and non-induced samples of His Trim21
(L) DOC, where well no.1 is the non-induced sample while well no.3 is the induced sample showing that
our protein is induced effectively owing to our right choice of IPTG, time interval and concentration
Pull-down assay of His Trim21 (L) DOC against GST COH WWW and GST COH TD28 Rev
Pull-down assay is a one-step assay that is used to detect protein-protein interaction. We incubate Trim (L) Doc with GST Coh WWW and GST Coh TD28rev to see the best interaction, we used the BCA assay to characterize the interaction.
Figure 6. This graph shows the comparison of pull-down assay between His Trim (L) DOC against GST COH WWW and
GST COH TD28 Rev, showing that the interaction between His Trim21 (L) DOC and GST COH WWW is better than that
of His Trim (L) DOC and GST COH TD28 Rev as the concentration of the elution coming from the pull-down assay
of His Trim21 (L) DOC and GST COH WWW is more than that of His Trim21 (L) DOC and GST COH TD28rev
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