Difference between revisions of "Part:BBa K4165003"

               Figure 3.: Docked structure of GST-Docs VS His-Coh2 designed by Cluspro displayed by Pymol.

GST-Docs VS His-Coh2 by Galaxy

                Figure 4.: Docked structure of GST-Docs VS His-Coh2 designed by Galaxy visualized by Pymol.

Binding energies of Docs VS Coh2

             Table 2.: Binding energies of Docs VS Coh2 tagged with GST and His docked using Galaxy and ClusPro.

WetLab Results

In the wet lab, we started with cloning in the pJET vector followed by the expression in the pGS-21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer would give a better yield and quantified the protein expression before and after induction using BCA assay, in the end, we tested the His Coh affinity by the pulldown assay against the GST Doc and His Doc against GST Coh.

Transformation of His Coh in DH-5 alpha using pJET cloning vector

                                      Figure 3. The transformed plate of His Coh + pJET 

Transformation of His Coh in BL-21 using pGS-21a expression vector

After cloning the Coh protein in DH5 alpha we extract the plasmid using manual miniprep, as it extracts more yield. To initiate protein expression, we transform Coh protein in BL-21 using the pGS-21a vector. The transformation efficiency is 180000 No. of transformants/μg.

                                   Figure 4. The transformed plate of His Coh + pGS-21a

Comparison between chemical lysis and sonication for His COH

Chemical lysis and physical lysis using sonication were done to check which gives better protein extraction results. After comparing the results, we optimized our protocol to use sonication for His Coh extraction.

                Figure 5. This graph shows the difference between chemical lysis and sonication for His COH.

Pull-down assay of His Coh against GST Doc and GST Coh against His Doc.

Pull-down assay is a one-step technique performed to check the protein-protein interaction and to check if they bind properly. We performed a pull-down assay to check the binding affinity between His Coh against GST Doc and between His doc and GST Coh (Illustrated in figure 6). We illustrate that the binding between His Doc with GST Coh is more stable than that of His Coh with GST Doc.

                Figure 6. This graph shows the pull-down assay of His Coh against GST Doc and His Doc against GST Coh. 

BCA assay results for His COH and GST COH

BCA assay is a technique that is performed to quantify the protein concentration. It depends on the color of the BCA working reagent, which is directly proportional to the quantity of the protein. We performed BCA for His Coh to know its concentration, which was found to be 0.286725851 mg/ml.

               Figure 7. This graph illustrates the results of the BCA assay for His Coh, showing that our protein 
                                       concentration is expected to be 0.286725851 mg/ml.

                Figure 8. This graph illustrates the results of the BCA assay for GST Coh, showing that our protein 
                                       concentration is expected to be 0.1158 mg/ml.

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