Part:BBa_K4165003
CoH2 (Cohesin)
Cohesin type 2 is an enzyme that binds to its counterpart DocS (BBa_K3396000) to form a protein pair used for the assembly of our PROTAC system.
Usage and Biology
The Cohesin 2 module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Dockerin S. The Coh2–DocS pair represents the interaction between two complementary families of protein modules that exhibit divergent specificities and affinities, ranging from one of the highest known affinity constants between two proteins to relatively low-affinity interactions. This serves an essential role in the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome), this interaction happens in two different forms, called the dual binding mode, in a calcium-dependent manner due to the presence of a calcium-binding site in the dockerin protein.
We used the DocS-Coh2 binding in our Snitch system to form the PROTAC pair that will conjugate E3 ligase trim 21 (BBa_K4165001) with the binding peptide for our targeted protein tau.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Dry Lab Characterization
Optimization
This part is considered as an improved version of the NUDT 2020 team part (BBa_K3396001), it is optimized to be suitable for expression in E. coli.
Modeling
Coh2 was modeled tagged once with GST and once with His, to purify it and compare its stability and expression yield with the two tags, the models were done using (Alphafold - Modeller - trRosetta - Rosettafold) and the top models were obtained from Alphafold and trRosetta ranking 5 out of 6 according to our QA code.
GST-Coh2
Figure 1.: Predicted 3D structure of Coh2 protein tagged by GST designed by AlphaFold tool visualized on pymol.
His-Coh2
Figure 2.: Predicted 3D structure of Coh2 protein tagged by His designed by TRrosetta visualized by pymol.
Functional parameters
PI: 4.103
Charge at (pH 7): -7.149
Molecular weight: 14.741 kDa
WetLab Results
In the wet lab, we started with cloning in the pJET vector followed by the expression in the pGS-21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give a better yield, and quantified the protein expression before and after induction using BCA assay, in the end, we tested the His Coh affinity by the pulldown assay against the GST Doc and His Doc against GST Coh.
Transformation of His Coh in DH-5 alpha using pJET cloning vector
The transformation was done using the TSS buffer protocol, after trying three buffers which are Calcium chloride, Magnesium chloride, and a combination between Calcium chloride and Magnesium chloride, we optimized our protocol to use the TSS buffer protocol as it showed the best results with a transformation efficiency of His Coh in DH-5 alpha using pJET vector is 172000 No. of transformants/μg.
Figure 3. Transformed plate of His Coh + pJET
Transformation of His Coh in BL-21 using pGS-21a expression vector
After cloning the Coh protein in DH5 alpha we extract the plasmid using manual miniprep, as it extracts more yield. To initiate protein expression, we transform Coh protein in BL-21 using the pGS-21a vector. The transformation efficiency is 180000 No. of transformants/μg.
Figure 4. Transformed plate of His Coh + pGS-21a
Comparison between chemical lysis and sonication for His COH
Chemical lysis and physical lysis using sonication were done to check which of them gives better results in protein extraction. After comparing the results we optimized our protocol to use sonication for His Coh extraction.
Figure 5. This graph shows the difference between chemical lysis and sonication for His COH.
Pull-down assay of His Coh against GST Doc and GST Coh against His Doc.
Pull-down assay is a one-step technique performed to check the protein-protein interaction and to check if they bind properly. We performed a pull-down assay to check the binding affinity between His Coh against GST Doc, and between His doc and GST Coh (Illustrated in figure 6). We illustrate that the binding between His Doc with GST Coh is more stable than that His Coh with GST Doc.
Figure 6. This graph shows the pull-down assay of His Coh against GST Doc and His Doc against GST Coh.
BCA assay results for His COH and GST COH
BCA assay is a technique that is performed to quantify the protein concentration, and it depends on the color of the BCA working reagent which is directly proportional to the quantity of the protein. We performed BCA for His Coh to know its concentration and it is found to be 0.286725851.
Figure 7. This graph illustrates the results of BCA assay for His Coh showing that our protein
concentration is expected to be 0.286725851.
Figure 8. This graph illustrates the results of BCA assay for GST Coh showing that our protein
concentration is expected to be 0.1158.
pull down assay gel result =
We run the elusion from the pull-down interaction to check if the His Coh interacts well with GST Doc. If the binding between His Coh and GST Doc happened the gel of SDS-PAGE will contain 2 bands.
Figure 9. This figure shows the binding between His Coh and Gst Doc happened as there are two bands in the gel.
References
1. Brás, J. L., Carvalho, A. L., Viegas, A., Najmudin, S., Alves, V. D., Prates, J. A., Ferreira, L. M., Romão, M. J., Gilbert, H. J., & Fontes, C. M. (2012). Escherichia coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes. Methods in Enzymology, 510, 395-415. https://doi.org/10.1016/B978-0-12-415931-0.00021-5
2. Slutzki, M., Ruimy, V., Morag, E., Barak, Y., Haimovitz, R., Lamed, R., & Bayer, E. A. (2012). High-Throughput Screening of Cohesin Mutant Libraries on Cellulose Microarrays. Methods in Enzymology, 510, 453-463. https://doi.org/10.1016/B978-0-12-415931-0.00024-0
3. Stahl, S. W., Nash, M. A., Fried, D. B., Slutzki, M., Barak, Y., Bayer, E. A., & Gaub, H. E. (2012). Single-molecule dissection of the high-affinity cohesin–dockerin complex. Proceedings of the National Academy of Sciences, 109(50), 20431-20436.
4. Karpol A, Kantorovich L, Demishtein A, Barak Y, Morag E, Lamed R, Bayer EA. Engineering a reversible, high-affinity system for efficient protein purification based on the cohesin-dockerin interaction. J Mol Recognit. 2009 Mar-Apr;22(2):91-8. doi: 10.1002/jmr.926. PMID: 18979459.
5. Wojciechowski, M., Różycki, B., Huy, P.D.Q. et al. Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module. Sci Rep 8, 5051 (2018). https://doi.org/10.1038/s41598-018-23380-9
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