Part:BBa_K4165253
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 85
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
In the wet lab we started with cloning in the pJET vector followed by the expression in the pgs21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give better yield, and quantified the protein expression before and after induction using BCA assay, in the end, we tested the GST-docs affinity by the pulldown assay against the His Coh
Transformation of GST DOC in DH-5 alpha using pJET cloning vector and in BL-21 using pGS-21a expression vector.
Transformation effeciency was calculated for both GST Doc in pJET cloning vector and in pGS-21a expression vector and was found to be =155000 transformants/μg and =170000 No.of transformants/μg respectively </p>
Figure 2. Transformed plate of GST DOC + pJET
Transformation of GST DOC in BL-21 using pGS-21a expression vector
Figure 3. Transformed plate of GST Doc + pGS-21a
Comparison between chemical lysis and sonication for GST DOC
Chemical method and physical method which are: Chemical lysis and sonication respectively were used to extract our protein and to check which method is more effective.
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
BCA assay results of protein extraction
BCA assay is a technique used to check the concentration of the protein, it depends on the color of the BCA working solution which is directly proportional to the concentration of the protein.
Figure 4. This figure shows the concentration of our protein using BCA assay which is found to be 0.828894525
Pull-down assay of His COH with GST DOC and His DOC with GST COH
Pull-down assay is a one-step technique that is used to check protein-protein interaction. we performed pull-down assay for GST Doc to check its binding with His Coh.
Figure 5. This graph illustrates that the binding between His DOC with GST COH is more stable than that
of His COH with GST DOC
Pull-down assay gel result=
SDS PAGE was performed after pull-down assay to check the protein-protein interactions
Figure 6. This figure shows that the binding between His COH and GST doc happened as there are two bands in the gel
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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