Part:BBa_K4165202
Trim-(GGGGS)3-Docs
This parts code for the Trim21 E3 ligase with the PRYSPRY domain truncated (BBa_K3396007), fused to type 1 Dockerin module derived from Clostridium thermocellum cellulosome scaffoldin by a Glycine Serine flexible linker repeated three times to maintain part flexibility needed during target ubiquitination.
Usage and Biology
This fusion protein is part of our Trim System (Snitch System), it is a modified version of the NUDT 2020 part (BBa_K3396005). It is supposed to bind to the PROTAC (Coh2-linker-tau_binding_peptide) to assemble a full system that will be able to target Tau protein through the binding peptide and recruit ubiquitin to tag the whole protein to initiate degradation by proteasomes through Trim21.
Trim21 (E3) is an integral part of the protein turnover processes which serves as a quality control step. In order to be degraded by proteasome 26S, the protein must be tagged with a ubiquitin tail. E3 ligase serves the function of transferring the ubiquitin to the protein of interest. So, in order for the process to be directed more specifically at certain proteins, we took advantage of the high affinity between the two modules (DocS and Coh2) which make up the cellulosome, to act as a protein pair that will guide trim to any chosen protein.
This process could be done by anchoring Trim21 to either one of the modules and anchoring its other counterpart with a targeting domain for the protein of interest, more feasible and directed ubiquitination of the target protein has been achieved, which eventually leads to specific degradation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 535
Improvement
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 of NUDT_2020 (BBa_K3396007) showing the extra amino acid residues (Valine-Leucine-Glutamic acid- Lysine) and the stop codon in the middle.
Dry lab Characterization
We simulated the fusion protein to detect its stability using 3D structure assessment, we then docked the protein with its binding partner Coh2 to check the binding affinity and whether the fusion of the trim to the Docs hindered the Interaction between the Coh2 and Docs Binding modules
Modeling
tTrim21-(G4S)3-DocS is modeled by AlphaFold2, ITASSER, MODELLER, Robetta and TrRosetta, best model obtained from TrRosetta. We assembled this part with a different linker and substituted the DocS with Coh2 (BBa_K4165200), (BBa_K4165201), and (BBa_K4165203) this was done to test the fusion N-terminally and C-terminally to Truncated trim with the two linkers combinations. The model assessment results showed that the combination of trim followed by 3xggggs linker and c-terminally fused dockerin gave the best model quality.
Figure 2.: Predicted 3D structure of our fusion protein tTrim21-(G4S)3-DocS.
Table 1: Quality assessment parameters of tTrim21-(G4S)3-DocS. model.
The modeling results show that fusing DocS to tTrim21 by (G4S)3 linker improves the quality of the model more than DocS basic part (BBa_K3396000).
1.2. Docking
Docking is done to test the whole fusion proteins' interaction 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 resulting protein has a higher affinity to GST-Coh2-linker-WWW than basic DocS and Coh2. For more information please check our Docking page.
Figure 3.: All docked structures 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.
1.2. Mathematical modeling
The rate of formation of the complex of Trim21-linker-DocS and GST-Coh2-linker-tau binding peptide is modeled using our Ubiquitin proteasome system simulating mathematical model.
Figure 4.: Graphical representation showing the change in concentration of Trim21-linker-DocS, GST-Coh2-
linker-tau binding peptides and their complex with time.
Figure 5.: Graphical representation showing the change in concentration of Tau with time when it is added to
the Ubiquitin proteasome system for degradation.
the high decline in the tau concentration at the beginning of the simulation is due to its binding with the complex formed above, then the rate of decrease slows down as all complex binds to tau, the slow decrease is dependent on the degradation of tau mainly.
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 transformed 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 Doc linked to Trim21 and Coh which is linked to TBP, then we use the BCA assay to characterize the interaction.
Ligation of His Trim21 (L) Doc with pJET cloning vector
We used T4 ligase to ligate the His Trim21 (L) Doc with pJET cloning vector so, we incubated His Trim21 (L) Doc with pJET cloning vector overnight at 15°C.
Figure 6. This figure shows the ligation reaction between His Trim21 (L) Doc and pJET cloning vector, The band
of pJET+His-TRIM21-L-DOCs appeared at size 4203.
Transformation of His Trim21 (L) Doc in DH-5 alpha using pJET cloning vector
We transform the His trim21 (L) Doc into DH5 alpha using the pJET cloning vector to be amplified. In the transformation, we compare 3 protocols, the TSS buffer, Calcium Chloride buffer, and a combination between Calcium Chloride and Magnesium Chloride. The results showed that the TSS buffer is the most efficient one.
Figure 7. Transformed plate of His Trim21 (L) Doc + pJET.
Transformation of His Trim21 (L) Doc in BL-21 using pGS-21a expression vector
We transformed His Trim21 (L) Doc in BL-21 using the pGS-21a vector. 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 8. The transformed plate of His Trim21 (L) Doc + pGS-21a.
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, and the physical method, which uses 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 9. 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
SDS-PAGE depends on molecular weight, so we performed SDS-PAGE to check our protein size and to compare induced and non-induced samples.
Figure 10. 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.2 is the induced sample showing that
our protein is induced effectively owing to our right choice of IPTG, time interval, and concentration.
Affinity chromatography of His Trim21 (L) Doc
Affinity chromatography is a technique to purify the proteins to get the protein alone without the cell lysate. Hence, we performed affinity chromatography to get His Trim21 (L) Doc alone to characterize it before performing a pull-down assay.
Figure 11. This figure shows the BCA assay results after performing affinity chromatography, The absorbance of
E1, E2 is almost the same. So, we expected that the elution buffer would have a problem. The graph shows that the
concentration of His Trim21 (L) Doc= 0.144754456 mg/mL
Pull-down assay of His Trim21 (L) Doc against GST Coh (L) WWW and GST Coh TD28Rev
Pull-down assay is a one-step assay that is used to detect protein-protein interaction. We incubated 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 12. This graph shows the comparison of pull-down assay between His Trim (L) Doc against GST Coh (L) WWW and
GST Coh (L) TD28 Rev, showing that the interaction between His Trim21 (L) Doc and GST Coh (L) 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 (L) WWW is more than that of His Trim21 (L) Doc and GST Coh (L) TD28rev.
References
1- Lytle BL, Volkman BF, Westler WM, Heckman MP, Wu JH. Solution structure of a type I dockerin domain, a novel prokaryotic, extracellular calcium-binding domain. J Mol Biol. 2001 Mar 30;307(3):745-53. doi: 10.1006/jmbi.2001.4522. PMID: 11273698.
2- Ronchi, V. P., & Haas, A. L. (2012). Measuring rates of ubiquitin chain formation as a functional readout of ligase activity. In Ubiquitin Family Modifiers and the Proteasome (pp. 197-218). Humana Press.
3- Collins, G. A., & Goldberg, A. L. (2017). The logic of the 26S proteasome. Cell, 169(5), 792-806.
| None |