Coding

Part:BBa_K4165003

Designed by: Hossam Hatem   Group: iGEM22_CU_Egypt   (2022-09-29)
Revision as of 20:06, 11 October 2022 by Omnia Alaa11 (Talk | contribs)


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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

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

                                   Figure 3. Transformed plate of His Coh + pGS-21a

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

                                      Figure 4. Transformed plate of His COH + pJET 

Comparison between chemical lysis and 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

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



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