"
Team:Heidelberg/pages/Reconstitution
From 2014.igem.org
Contents |
Introduction
A common strategy for investigating molecular and cellular biological questions is the use of fusion proteins and to control the functions of these proteins in a spatial and temporal manner. Instead of manipulating proteins on the genomic level, we aimed at editing proteins post-translationally. By using our intein toolbox, one is able to fuse proteins and/or protein tags(Link), as well as reconstitute the function by complementing two formerly split halves of a single protein, and thereby recreate the function of the protein. Mechanistically, the reconstitution of split proteins is identical with the fusion of different proteins to their tags.
To demonstrate the restoration of function of a formerly split protein, we choose a set of fluorescent proteins, whose function can, when properly reassembled, easily be read out using their florescence. Split fluorescent proteins are rarely used in the context of intein splicing. However they are widely applied in bimolecular fluorescence complementation (BiFC) assays [1]. This approach is based on the complementation between fragments of fluorescent proteins that reconstitute its fluorescence when brought into proximity by associated interacting proteins.
Methods
Selection of Split Sites
For each of the fluorescent proteins, we selected split sites according to previous published research. Additionally to fluorescent proteins we choose to create two versions of split luciferases. A list of all designed constructs are depicted in the following table:
| Protein | Split site | Comment |
|---|---|---|
| mRFP | 154/155 | Split between β - barrel 7 and 8 [2] |
| mCherry | 168/169 | Split between β - barrel 8 and 9 [3] |
| GFP | 157/158 | Split between barrel β - 7 and 8 [4] |
| sfGFP | 64/65 | In front of the chromphore region [5] |
| Firefly Luciferase | 437/438 | In flexible tether between the two subunits [6] |
| Renilla Luciferase | 229/230 | split between barrel β - 7 and 8 [7] |
Results
Discussion
Outlook
References
[1] Hu, C-D, & T.,K., Kerppola. Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol. 21(5), 539-545 (2003).
[2] Jach, G., Pesch, M., Richter, K., Frings, S., & Uhrig, J., F. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nature Methods, 3, 597-600 (2006).
[3] Furman, J., L., Badran, A., H., Shen, S., Stains, C., I., Hannallah, J., Segal, D., J., Ghosh, I. Systematic evaluation of split-fluorescent proteins for the direct detection of native and methylated DNA. Bioorg Med Chem Lett, 19(14), 3748-3751 (2009).
[4] Oyawa, T., Takeuchi, M., Kaihara, A., Sato, M., Umezawa, Y. Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time. Anal. Chem, 73, 5866-5874 (2001).
[5] Aranko, A., S., Oeemig, J., S., Kajander, T., & Iwai, H. Intermolecular domain swapping induces intein-mediated protein alternative splicing. Nat. Chem. Bio., 9, 616-622 (2013).
[6] Ozawa, T., Kaihara, A., Sato, M., Tachihara, K., Umezawa, Y. Split luciferase as an optical probe for detecting protein- protein interactions in mammalian cells based on protein splicing. Anal. Chem, 73, 2516-2521 (2001).
[7] Kim, S. B., Ozawa, T., Watanabe, S., Umezawa, Y. High- throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split renilla luciferase. PNAS, 101, 11542–11547 (2004).
