Difference between revisions of "Part:BBa K3396000"

               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.

His-Docs VS GST-Coh by ClusPro

                   Figure 5.: Docked structure of His-Docs VS GST-Coh2 designed by ClusPro visualized by Pymol.

His-Docs VS GST-Coh2 by Galaxy

                  Figure 6.: Docked structure of His-Docs VS GST-Coh2 designed by Galaxy visualized by Pymol.

Binding energies of Docs VS Coh2

             Table 3.: Binding energies of Docs VS Coh2 tagged with GST and His docked using Galaxy and ClusPro.

1. Functional Parameters

Wet Lab Results

As we investigated the literature, we found that the Docs part yields little amount due to its suitability to proteolytic attack. Hence we tried to configure its expression by adding two different tags and followed this pipeline to prove our concept experimentally: 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 will give better yield, and quantified the protein expression before and after induction using BCA assay. Ultimately, we tested the His-Doc affinity by the pulldown assay against the GST-Coh and GST Doc against His Coh.

Results of His-DocS

Ligation of His Doc with pJET cloning vector

We used T4 ligase to ligate His Doc with pJET cloning vector, so we incubated His Doc with pJET overnight at 15°C.

              Figure 4. This figure shows the ligation between His Doc and pJET, Results show the size of the 
                               the ligated plasmid is 3372 bp, as expected.

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

                      Figure 5. The transformed plate of His Doc + pJET using TSS buffer protocol

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

                                       Figure 6. The transformed plate of His Doc + pGS-21a 

Affinity chromatography results for His Doc

Affinity chromatography is a technique to purify the protein to get the protein alone without the cell lysate. So, we performed affinity chromatography for total protein extraction to get pure His Doc to characterize it before the pull-down assay.

        Figure 7. This figure shows the BCA assay results of affinity chromatography for His Doc, As shown from the graph, 
         E1 of His Doc purified is higher than E2, meaning we purified our protein successfully.

Comparison between chemical lysis and sonication for His DOC

We used two protein extraction methods: chemical lysis (chemical method) and sonication (physical method) to check which method is more effective for extracting His Doc. After we had the results, we optimized our protocol to use chemical lysis for His Doc

                  Figure 8. This graph shows the difference between chemical lysis and sonication for His Doc.
                      

SDS PAGE for induced and non-induced samples of His Doc

SDS-PAGE depends on the molecular weight of the protein. We performed SDS-PAGE to ensure that our protein is in the exact size and to show the difference between induced and non-induced samples of His Doc.

           Figure 9. This figure shows the comparison between induced and non-induced samples of His Doc where well no.2 is 
           the non-induced sample, while well no.4 is the induced sample showing that our protein is induced effectively 
                            owing to our right choice of IPTG, time interval, and concentration

Pull-down assay of His Coh with GST Doc and His Doc with GST Coh

Pull-down assay is a one-step technique used to test protein-protein interaction. Pull-down was performed to check and compare the interaction between His Doc against GST Coh and His Coh against GST Doc.

              Figure 10. This graph illustrates that the binding between His Doc with GST Coh is more stable than that 
                                                of His Coh with GST Doc.

Reference

[1] BARAK Y, HANDELSMAN T, NAKAR D, et al. Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction [J]. J Mol Recognit, 2005, 18(6): 491-501.

[2] Kazutaka Sakka, Yuka Sugihara, Sadanari Jindou, Makiko Sakka, Minoru Inagaki, Kazuo Sakka, Tetsuya Kimura, Analysis of cohesin–dockerin interactions using mutant dockerin proteins, FEMS Microbiology Letters, Volume 314, Issue 1, January 2011, Pages 75–80, https://doi.org/10.1111/j.1574-6968.2010.02146.x

[3] Lawrie, J., Song, X., Niu, W., & Guo, J. (2018). A high throughput approach for the generation of orthogonally interacting protein pairs. Scientific Reports, 8. https://doi.org/10.1038/s41598-018-19281-6

[4] Wojciechowski, M., Różycki, B., Huy, P. D. Q., Li, M. S., Bayer, E. A., & Cieplak, M. (2018). Dual binding in cohesin-dockerin complexes: the energy landscape and the role of short, terminal segments of the dockerin module. Scientific reports, 8(1), 1-14.