Difference between revisions of "Part:BBa K4165254"

Latest revision as of 03:04, 14 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

1.1.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.

1.2. Docking

The docking results showed that the fusion of GST to DocS increased its binding affinity score to Coh2 more than without the GST. Illustrating that GST increases the contact between both proteins. In addition, Galaxy and ClusPro docking results showed that the affinity when GST is fused to DocS is better than when fused to Coh2, this may be due to decreasing the overall non-interacting surface of the protein and consequently increasing the affinity. So, we ordered the four parts (His-DocS, GST-DocS, His-Coh2, GST-DocS) to prove our theory experimentally. All of the docking results were ranked using our code for calculating the binding affinity based on PRODIGY (please refer to our Programming club page a code under the name of son.

ΔG = -13.15 kcal/mol

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

His-Docs VS GST-Coh by ClusPro

                   Figure 3.: Docked structure of His-Docs VS GST-Coh2 designed by ClusPro visualized by Pymol.

Binding energies of Docs VS Coh2

             Table 3.: Binding energies of Docs VS Coh2 tagged with GST and His docked using Galaxy and ClusPro.

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 would 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

                                        Figure 4. The transformed plate of GST COH + pJET. 

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

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

        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.

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

                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

         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.

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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