Difference between revisions of "Part:BBa K4165254"

Revision as of 19:18, 13 October 2022

GST-Coh2

This part encodes Cohesin 2 protein tagged with GST for its purification and characterization

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

Modeling

Coh2 was modeled tagged with GST to purify it and measure its expression yield, 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.

          Figure 1.: Predicted 3D structure of Coh2 protein tagged by GST designed by AlphaFold tool visualized on Pymol.

Table 1: Quality assessment parameters of GST-Coh2 model.

Docking

GST-Coh2 is docked to His-DocS

ΔG = -13.15 kcal/mol

          Figure 2.: 3D structure of GST-Coh2 docked with His-DocS on Galaxy and visualized on pymol.

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 GST Coh affinity by the pulldown assay against His Doc.

Ligation reaction between GST Coh and pJET cloning vector

We used T4 ligase to ligate GST Coh with the pJET cloning vector so, we incubated GST Coh with pJET overnight at 15°C.

                     Figure 3. This figure shows the ligation between GST Coh and pJET cloning vector.

Transformation of GST 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 GST Coh in DH-5 alpha using pJET vector is 400000 No. of transformants/μg, you can find the complete protocol on our wiki page.

                                        Figure 4. Transformed plate of GST COH + pJET. 

Miniprep for GST Coh

Miniprep is a technique that is done to extract the plasmid that contains our part. So, we performed miniprep for GST Coh in the pJET cloning vector to extract our protein.

                      Figure 5. This figure shows the miniprep of GST Coh in pJET cloning vector.

Transformation of GST Coh in BL-21 using pGS-21a expression vector

                                        Figure 6. Transformed plate of GST Coh + pGS-21a.

SDS PAGE for induced and non induced samples of GST Coh

SDS PAGE depends on molecular weight of the protein so, we performed SDS PAGE to check GST Coh size and to compare between induced and non induced samples

        Figure 7. This figure shows the SDS PAGE of induced and non induced samples of GST Coh where well no.1 is the 
         non induced sample while well no.2 is the induced sample, showing that the induced sample of GST Coh appeared 
         at the expected size which is 45Kda.

Comparison between chemical lysis and sonication for GST COH

Chemical lysis and physical using 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 GST Coh. after we had the result we optimized our protocol to use sonication for GST Coh extraction.

     Figure 8. This graph shows a highly significant difference between the chemical lysis and sonication for GST Coh. 

Pull-down assay of His Coh with GST Doc and His Doc with GST Coh

Pull-down assay is a one-step technique performed to check the protein-protein interaction. To check if they bind properly, we performed the pull-down assay to check the binding between His doc and GST Coh.

                    Figure 9. This graph illustrates that the binding between His Doc with GST Coh is more stable 
                                            than that of 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 working reagent which is directly proportional to the quantity of the protein, we performed BCA for GST Coh to know its concentration and it found to be 0.1158.

                Figure 10. This graph illustrates the results of the BCA assay for GST COH showing that our protein 
                                       concentration is expected to be 0.1158

Pull-down assay was performed to check the protein-protein interactions

         Figure 11. This figure shows that the binding between his doc and GST Coh happened as the concentration of 
                               the GST Coh and His Doc more than that of His Doc alone.

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