Split orthogonal proteases
The split protein system based on the inducible dimerization is an attractive method to regulate the protease activity. Wehr et al.
Our team hypothesized that the same inducible dimerization approach could also be used with TEVp homologues. We converted all of the tested orthogonal potyviral proteases
to split proteases by splitting them at positions corresponding to the position of the previously described split TEV protease. We selected three different types of
dimerization domains to induce the activity of the split proteases. The first pair of dimerization domains was the rapamycin responsive FKBP/FRB system
Interactions among different proteins play a key role among all living organisms. Chemically induced dimerization (CID) is one of such interactions,
which allows two different protein domains to dimerize after the addition of a small molecule. The most widely used CID to date is the FKBP/FRB system
which heterodimerizes upon rapamycin addition
Rapamycin is a 31-membered macrolide antifungal antibiotic that was first isolated from the Streptomyces hygroscopicus and binds with high affinity to the
12-kDa FK506 binding protein (FKBP) as well as to a 100-aminoacid domain (E2015 to Q2114) of the mammalian target of rapamycin (mTOR) protein known as the
FKBP-rapamycin binding domain (FRB) (4.10.1)
We tested the full set of four orthogonal proteases with the rapamycin inducible system by measuring their activity with the cycLuc reporter. Increasing luciferase activity was detected correlating with the amount of the transfected protease fragments in stimulated cells (4.10.2.). Luciferase in unstimulated cells remained inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin.
Additionally, we tested the kinetics of rapamycin induction with PPVp and its corresponding cycLuc reporter. We showed that luciferase activity starts increasing just a few minutes after the addition of rapamycin, resulting in activity comparable to activity of the reporter in the presence of constitutively active whole protease within one hour after the induction (4.10.3.).
After demonstrating that the new proteases are active as split enzymes with rapamycin-induced complementation, we adapted the same split system to other inputs. To connect the split protease-based signaling to mechanosensing, we prepared Ca2+ inducible proteases based on calmodulin and M13 interaction. The first results look promising; however we have to confirm them in the repeated experiments. As an additional type of input we prepared light inducible split proteases.