Team:FAU Erlangen/Tour21
Gluing it together
One of the prerequisites for our goal was the co-localization of the DNA binding protein and the histone modifying protein. This could be achieved by a fusion protein combining those two, as in the work of Mendenhall et al. (2013). However, this method would force its users to synthesize new fusion proteins for every new locus and every new modification they want to analyze. A more flexible approach lies in the non-covalent bond between biotin and streptavidin. Although this would have allowed us to design interchangeable fusion proteins for easier large-scale applications, we were unwilling to commit to the trade-off between flexibility and stability, as the bond in question, albeit strong, doesn't react well to low pH or mechanical forces (Chivers et al., 2010). To get the best of both worlds, we chose to utilize the recently established SpyTag/SpyCatcher system, which provides an isopeptide bond to connect two proteins and hold them together.
The SpyTag/SpyCatcher system as it is used in our project was developed in 2013 by the group of Associate Professor Mark Howarth from the University of Oxford. It (the system, not the university) is derived from the adhesin domain CnaB2 of a fibronectin-binding protein of Streptococcus pyogenes, a Gram-positive species of bacteria pathogenic to humans (Zakeri et al.). A spontaneous reaction within the mostly hydrophobic protein core irreversibly binds the nitrogen atom of a lysin's side chain to an oxygen atom of an aspartate's side chain. This intramolecular bond can be turned into an intermolecular bond by splitting the protein in two, which is exactly what Howarth's group did.
During the course of several steps, the Catcher was optimized and shortened from 138 to 84 amino acids (Zakeri et al. 2012, and Li et al., 2014). The Catcher we decided to use shares its 113 amino acids with the protein from the Registry part BBa_K1159200, which was submitted in 2013 by the team from the TU Munich. Since there are more different DNA binding proteins than histone modificating proteins, we paired the Tag with the former and the Catcher with the latter. This way, one could perform the same epigenetic modification at several gene loci without needing to produce the larger binding partner, the Catcher, several times. Once different Tag/Catcher systems are discovered or designed and become widely available, as will surely be the case in the near future, our method could be expanded to faciliate two or more kinds of modifications simultaneously.
References
- Eric M. Mendenhall, Kaylyn E. Williamson, Deepak Reyon, James Y. Zou, Oren Ram, J. Keith Joung, and Bradley E. Bernstein (2013). Locus-specific editing of histone modifications at endogenous enhancers using programmable TALE-LSD1 fusions. Nat Biotechnol. 2013 Dec; 31(12): 1133–1136.
- Claire E. Chivers, Estelle Crozat, Calvin Chu, Vincent T. Moy, David J. Sherratt, and Mark Howarth (2010). A streptavidin variant with slower biotin dissociation and increased mechanostability. Nat Methods. 2010 May; 7(5): 391–393.
- Bijan Zakeri, Jacob O. Fierer, Emrah Celik, Emily C. Chittock, Ulrich Schwarz-Linek, Vincent T. Moy, and Mark Howarth (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 2012 Mar 20; 109(12): E690-E697.
- Gianluca Veggiani, Bijan Zakeri, and Mark Howarth (2014). Superglue from bacteria: unbreakable bridges for protein nanotechnology. Trends in Biotechnology, Volume 32, Issue 10, Pages 506-512.
- Long Li, Jacob O. Fierer, Tom A. Rapoport, and Mark Howarth (2014). Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag. J Mol Biol. 426(2): 309–317.