Team:Slovenia/Protease signaling/Overview
Achievements
We successfully designed and tested four new active split site-specific proteases in addition to the previously published TEVp and therefore extended the set of functional split proteases from one to five, including TEVpE, PPVp, SuMMVp and SbMVp. Upon overexpression none of the tested proteases is toxic to mammalian cells, demonstrating that they do not interfere with essential cellular processes.
We proved that PPVp, SuMMVp, SbMVp and TEVp are fully orthogonal to each other. This provides the building blocks for the design of complex logic operations based on post-translational protein modifications instead of the widely used transcriptional regulation. We demonstrated higher cleavage activity of the novel split proteases against their respective substrates in comparison to already existing split TEVp. We demonstrated that activity of new split proteases can be regulated by different external stimuli, such as chemical ligands and light.