Team:UCLA/Proof

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Proof of Concept

Overview

Our intended final product was a protein cage capable of disassembling due to specific proteolytic cleavage. This would disassemble the cage, exposing the inside of the cage and its contents to the environment where the targeted enzyme was in.

Using Protein Cage Mutants

We demonstrated proof of concept using thrombin as our targeted enzyme, and successfully mutated thrombin cleavage sites into a variety of positions along two protein cages, O3-33 and PCQuad.

Not all of these mutations yielded viable cages however. Some caused the subunits of the cage to no longer be compatible, preventing formation of the cage, and some caused the subunits of the cage not to form in the first place.

One mutant for each of the cages yielded properly formed cages, with nearly identical characteristics to their respective wild types.

We mixed a solution of each of these wild types with thrombin, allowing them to incubate for 16 hours at room temperature. Additionally, we created an identical mixture with the same conditions using the wild types of each cage as a control.

Assay Results

Detailed results can be found here

After the incubation period, we ran DLS and SDS-PAGE assays to determine the effects of the added thrombin. The wild types had no change, while the mutants showed clear signs of cleavage.

We further ran time variation experiments of the same thrombin assay. This involved allowing the thrombin-protein mixture to incubate for 4 hours, 8 hours, 12 hours, and 16 hours. The longer the period of time, the more cleavage that occurred, as could be seen from the intensity of bands representing the unaffected subunits (fading over time) and the cleaved subunits (intensifying over time).

DLS Results for PCQuad Mutant M1 before addition of thrombin and after addition of thrombin:

DLS Results for PCQuad Mutant M1 before addition of thrombin and after addition of thrombin:

Explanation of Results

All of these results pointed to the explanation that thrombin was indeed causing subunit level cleavage in the proteins at the added cleavage sites, as expected. DLS results further confirmed that the subunit level cleavage was disrupting the formation of the full cage.

These results thus demonstrate that specifically through the addition of a targeted enzyme, we were able to disassemble our protein cages, exposing the inside of the cage to the environment. The enzyme did not affect the wild type cages, thus it was only through our designed introduction of the cleavage site that this was made possible.

We thus show the concept that any similar proteolytic cleavage site could be added in place of the thrombin cleavage site we used, allowing for targeting of other enzymes. Additionally, the cage may be loaded with drug molecules which would be exposed only when in presence of the targeted enzyme to disassemble the cage.