Difference between revisions of "Part:BBa K4165254"

Revision as of 11:10, 12 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

Transfotmation of GST COH in BL-21 using pGS-21a vector

                                          Figure 3. Transformed plate of GST COH + pGS-21a 

Transformation of GST COH in DH-5 alpha using pJET vector

                                        Figure 4. Transformed plate of GST COH + pJET 

Comparison between chemical lysis and sonication for GST COH

                                  Figure 5. This graph shows a highly significant difference between the 
                                            chemical lysis and sonication for GST COH, after we had this 
                                            result we optimized our protocol to use sonication for GST COH

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

                      Figure 6. This graph illustrates that the binding between His DOC with GST COH is more 
                                            stable than that of His COH with GST DOC 

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