Demonstration: Testing the TALebots
In addition, we tried to linearize the circular protein again. Using TEV Protease and ProTEV Plus Buffer by Promega, we tried to show that the circularization is reversible. Due to the special TEV sequence that is included in our vector, the circular TALE can be cut by the protease.
Figure 6 and 7 show SDS gels after incubating purified TALE protein for different time periods with TEV protease. The SDS gels were blotted onto a membrane using Western blot and afterward treated with Strep antibodies (anti-mouse) or Flag antibodies for immunostaining.
The results for TALEs purified with and without DTT are almost the same. There is also no difference between the two TALE proteins (Ax7L-DS and Ax7R-RR). The amount of protein that could be detected decreases over time.
Previous experiments have proven that the vector contains the desired TALE and that the purification using a Strep-Column by IBA could work. We also got promising results that led us to believe circular TALebots are formed in vitro.
However, we did not detect circular TALE proteins on Figure 7. If there would be circular proteins, we would have detected bands with Strep and Flag tag since both tags are part of the protein. Since we could only detect bands on the membranes using Flag antibodies, we can be sure that the proteins are linear. The Strep-tag is located right next to the TEV site in the protein and therefore a TEV digest cuts the Strep-tag off.
After getting those results, we tried E. coli Origami 2 (DE3) to express circular TALEs, but we did not get any results out of those experiments either.
To test our constructs for heat stability and resistance against storage, we performed several stability tests after purifying the protein. We exposed the circular TALEs to 37°C, 4°C, room temperature and three freeze-thaw cycles. Of course, we also used linear TALE constructs as a negative control.
We had several problems during the stability test. Therefore, we cannot show our results. If you want to find out more about why this experiment did not show results, take a look on our “Results” page.