Difference between revisions of "Part:BBa K4165253"

Revision as of 15:42, 12 October 2022

GST-DocS

This composite part encodes for the Dockerin module (BBa_K3396000) tagged with a GST tag (BBa_K4165070).

Usage and Biology

The Dockerin S. module comes from the C. thermocellum scaffoldin and it could recognize and bind tightly to its complementary counterpart Cohesin 2. 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

Docs has been tagged with GST and His for purification and increasing the yield by the GST tag. then the model designed by several tools to get the best model.

GST-Docs

                Figure 1.: Predicted 3D structure of GST-Docs designed by RosettaFold tool displayed by Pymol.

WetaLab Results

Transformation of GST DOC in DH-5 alpha using pJET cloning vector

                                  Figure 2. Transformed plate of GST DOC + pJET 

Comparison between chemical lysis and sonication for GST DOC

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

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

                   Figure 3. This graph shows a significant difference between chemical lysis and sonication for GST 
                        DOC, after we had the results we optimized our protocol to use chemical lysis for GST DOC

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

                    Figure 4. This graph illustrates that the binding between His DOC with GST COH is more stable than that 
                                                          of His COH with GST DOC

pulldown 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