Coding

Part:BBa_K4165003

Designed by: Hossam Hatem   Group: iGEM22_CU_Egypt   (2022-09-29)
Revision as of 18:51, 5 October 2022 by Amr Gouda (Talk | contribs)


CoH2 (Cohesin)

Cohesin type 2 enzyme binds to its counterpart DocS (BBa_K3396000) to form the protein pair for the PROTAC system.


Usage and Biology

The DocS module comes from The C. thermocellum scaffolding and it could recognize and bind tightly to complementary Coh2 modules. 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.CoH2 (Cohesin) is a cellulolytic enzyme in Clostridium thermocellum [1]. The Cohesin family has great affinity for binding with its complementary counterpart family under the name of dockerins with 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 protac system to conjugate E3 ligase trim 21 [BBa_K4165001] with the binding peptide for our targeted protein.

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

Optimization

This part is considered as an improved version of that of NUDT 2020 team (BBa_K3396001), this part is optimized for expression in E.coli. After optimization, it gave the same protein as that of NUDT after translation.

Modeling

Coh2 has been modeled tagged by GST and His to purify it and increase its stability by GST tag.

Coh2-GST 

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


Coh2-His 

                  Figure 1.: Predicted 3D structure of Coh2 protein tagged by His designed by TRrosetta.


Dry Lab characterization

PI 4.103

Charge at (pH 7) -7.149

Molecular weight 14.741 kDa

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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