Difference between revisions of "Part:BBa K4165003"

Revision as of 15:40, 12 October 2022

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

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 pgs21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give 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 againt GST COH

Transformation of His COH in DH-5 alpha using pJET cloning vector

The transformation was done using 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 while the transformation efficiency of His coh in BL-21 using pGS-21a vector couldn't be calculated due to the crowded plate but we will use another plating method you can find the complete protocol in our wiki page

                                      Figure 3. Transformed plate of His COH + pJET 

Transformation of His COH in BL-21 using pGS-21a expression vector

                                   Figure 4. Transformed plate of His Coh + pGS-21a

Comparison between chemical lysis and sonication for His COH

Chemical lysis and sonication were done to check which of them gives better results in the protein extraction, and after comparing the results we optimized our protocol to use sonication for his coh

                Figure 5. This graph shows the difference between chemical lysis and sonication for His COH, 
                     after we had the results, we optimized our protocol to use sonication for His COH

Pull down assay of His COH against GST DOC and His DOC against GST COH

Pull down assay is a technique performed to check the interactions between the proteins and to check if they bind properly, we performed pull down assay to check the binding between His COH against GST DOC, and between his doc and GST Coh (Illustrated in figure 6)

                Figure 6. This graph shows the pull down assay of His COH against GST DOC and His DOC against GST COH 
                 illustrating that the binding between His DOC  with GST COH is more stable than that His COH with GST DOC 

BCA assay results for His COH and GST COH

BCA assay is a technique that is performed to quantify the proteins, and it depends on the color of the BCA dye which is directly proportional with the quantity of the protein, we performed BCA for His COH and GST COH to know its concentration and it is found to be 0.286725851 and 0.1158 respectively

               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 =

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