Difference between revisions of "Team:TU-Eindhoven/Project"
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| + | <figcaption>Figure 2: Assembly of the CT33 construct with Strep-tactin to get a valency of 4</figcaption></div></div> | ||
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Revision as of 10:21, 1 November 2017
Overview
In the last few years, quite some papers have been published about gelation and liquid-liquid phase separation. [1-4] These papers based the proteins used on naturally occurring proteins that were discovered to form a cluster inside living cells. Recently, work has also been done on the design of protein-based smart gels, which show that multivalent constructs with valency of at least 3 lead to gelation. [5-7]
Based on this information, we designed a whole new system based on proteins with a totally different natural function than clustering inside living cells, but with known interactions and broad applications. In our project, we created two constructs that will interact with each other to form a network of proteins, the basics of a gel.
The System
Our designed system is named GUPPI, after Gelation Using Protein Protein Interactions. Just as the iGEM team before us, we will use the 14-3-3 protein as basis of our designed system. Last year, the 14-3-3 protein was used as scaffold and they mutated many different sites to tune the interaction strength. This year we will go back to one type of 14-3-3, and instead of mutating sites of the monomer, we will use it as building block for a very large protein, consisting out of multiple monomers. The 14-3-3 protein naturally forms a dimer with itself, and thus has a valency of 2. We increased the amount of monomers via DNA design, which led to a valency of 4. However, with a simple DNA mutation in the last monomer of the construct, we blocked the functionality of the last monomer, leading to a functional valency of 3.
Figure 1: Mutation of the last 14-3-3 pocket to make it non-functional
The second construct needed for our project is the protein CT33. CT33 is a H(+)-ATPase derivative and can bind into the pocket of a 14-3-3 monomer. This interaction is relatively weak, but can be increased excessively by adding a small molecule, fusicoccin, that acts as a stabilizer. This means that CT33 is a very nice basis for a construct to combine with 14-3-3 for forming a network after inducing the system with fusicoccin. As the C-terminus of CT33 is necessary for the correct folding of the protein, it was not possible to design one protein out of multiple CT33 parts. The solution we found is to use a Strep-tag together with Strep-tactin. A Strep-tag is mostly known for its application in protein purification, just like a His-tag. For the purification with a Strep-tag, a resin is needed on which the tag can bind strongly. The resin is called streptavidin, but an engineered form of streptavadin is also available, which is called Strep-tactin. Strep-tactin is a protein with four binding sites for Strep-tag. We used Strep-tactin to create a construct consisting out of four CT33 proteins, leading to our second construct, which has thus a valency of 4.
Figure 2: Assembly of the CT33 construct with Strep-tactin to get a valency of 4
[1] S. F. Banani, H. O. Lee, A. A. Hyman, and M. K. Rosen, “Biomolecular condensates: organizers of cellular biochemistry,” Nat. Rev. Mol. Cell Biol., vol 18, pp 285-298 2017.
[2] S. Banjade and M. K. Rosen, “Phase transitions of multivalent proteins can promote clustering of membrane receptors”, eLife, 2014
[3] S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, M. K. Rosen, S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, and M. K. Rosen, “Compositional Control of Phase-Separated Cellular Bodies,” Cell, vol. 166, pp. 651–663, 2016.
[4] P. Li, S. Banjade, H.C. Cheng, B. Chen, L. Guo, M. Llaguno, J.V. Hollingsworth, D.S. King, S.F. Banani, P.S. Russo, Q.X. Jiang, B.T. Nixon and M.K. Rosen, “Phase transitions in the assembly of multivalent signalling proteins”, Nature, vol. 483, pp 336-340, 2012.
[5] T. Z. Grove, C. O. Osuji, J. D. Forster, E. R. Dufresne, and L. Regan, “Stimuli-Responsive Smart Gels Realized via Modular Protein Design,” J. Am. Chem. Soc., vol. 132, pp. 14024–14026, 2010.
[6] T. Z. Grove, J. Forster, G. Pimienta, E. Dufresne, and L. Regan, “A Modular Approach to the Design of Protein-Based Smart Gels A Modular Approach to the Design of Protein-Based Smart Gels,” Biopolymers, vol. 97, no. 7, pp. 508–517, 2012.
[7] C. T. S. Wong, P. Foo, J. Seok, W. Mulyasasmita, A. Parisi-amon, and S. C. Heilshorn, “Two-component protein-engineered physical hydrogels for cell encapsulation,” Proc. national Acad. Sci. United States Am., vol. 106, no. 52, pp. 22067–22072, 2009.
[2] S. Banjade and M. K. Rosen, “Phase transitions of multivalent proteins can promote clustering of membrane receptors”, eLife, 2014
[3] S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, M. K. Rosen, S. F. Banani, A. M. Rice, W. B. Peeples, Y. Lin, S. Jain, R. Parker, and M. K. Rosen, “Compositional Control of Phase-Separated Cellular Bodies,” Cell, vol. 166, pp. 651–663, 2016.
[4] P. Li, S. Banjade, H.C. Cheng, B. Chen, L. Guo, M. Llaguno, J.V. Hollingsworth, D.S. King, S.F. Banani, P.S. Russo, Q.X. Jiang, B.T. Nixon and M.K. Rosen, “Phase transitions in the assembly of multivalent signalling proteins”, Nature, vol. 483, pp 336-340, 2012.
[5] T. Z. Grove, C. O. Osuji, J. D. Forster, E. R. Dufresne, and L. Regan, “Stimuli-Responsive Smart Gels Realized via Modular Protein Design,” J. Am. Chem. Soc., vol. 132, pp. 14024–14026, 2010.
[6] T. Z. Grove, J. Forster, G. Pimienta, E. Dufresne, and L. Regan, “A Modular Approach to the Design of Protein-Based Smart Gels A Modular Approach to the Design of Protein-Based Smart Gels,” Biopolymers, vol. 97, no. 7, pp. 508–517, 2012.
[7] C. T. S. Wong, P. Foo, J. Seok, W. Mulyasasmita, A. Parisi-amon, and S. C. Heilshorn, “Two-component protein-engineered physical hydrogels for cell encapsulation,” Proc. national Acad. Sci. United States Am., vol. 106, no. 52, pp. 22067–22072, 2009.
