Team:UCLA/Protein Cages
Introduction
Higher order self-assembling protein assemblies are commonplace in nature, such as ferritin, which carries iron in many single-celled organisms. Due to their encapsulating function and box-like structure, such assemblies are often called protein “cages.” Inspired by these natural sources, an abundance of research has been done into creating synthetic cages with new customized properties such as stability, size, and subunit types. Furthermore, while applications for cages have been suggested, many have not been tested experimentally.
An application that we have centered our project around is targeted drug delivery. By specifying the site of delivery, the effects of the drug can be limited to desired areas with maximized efficiency and we can avoid damaging undesired targets in the body.
Thrombosis, the process by which a clot forms in the blood stream, is associated with widespread diseases including stroke and many heart conditions. Anticoagulants prevent clot formation by interfering with clotting factors and allowing for smoother blood flow and restoring normal circulation. However, these drugs can have detrimental side effects, such as an inability to inhibit fatal blood loss through cuts or other wounds. By localizing the site of delivery and reducing the necessary drug dosage, we can mitigate these kinds of problems. A protein cage encapsulating an anticoagulant drug that would selectively release its payload at the site of a clot would fulfill this purpose. To specify the target, we should select an associated enzyme or byproduct of the clotting cascade. Thrombin, a protease present at blood clots, cleaves a specific amino acid sequence. This sequence, if properly inserted into a protein cage containing an anticoagulant, would result in disassembly of the cage, release of the drug molecule to the targeted area, and destruction of the clot.
The goal of our project is to modify protein cages that disassemble in the presence of thrombin protease. We are using two previously created synthetic cages, one composed of 12 subunits forming a tetrahedral cage while the other is composed of 24 subunits forming an octahedral cage. For each of these protein cages, we have designed mutants with inserted thrombin cleavage sites with the intention to induce disassembly of the cages by treatment with thrombin protease. Future directions of this project consist of loading anticoagulant drug molecules into our mutant cages and assaying for the release of the drug molecules upon disassembly in the presence of thrombin.
Design
The main goal of the project is to develop a mechanism for targeted drug delivery. By incorporating protease cleavage sites into our protein cages, we control disassembly of the cage simply by providing it with a specific protease. The protease we decided to work with is Thrombin, a crucial member in the blood clotting cascade. Its role in Thrombosis makes it a key target in many of our leading causes of death including, heart attacks and strokes. By incorporating Thrombin cleavage sites into our cage and using Thrombin as our targeting molecule, we hope to directly combat heart attacks and strokes using our drug delivery mechanism.
The design of the project involves three distinct steps.
1. The first step revolves around the design of our mutants derived from the wild type cage. Because of the sensitive nature of these protein cages, there were specific criteria that we had set to maximize mutant formation and guarantee disassembly of our cage once it came into contact with the protease
- Design mutations away from any possibly secondary or tertiary structures.-The main reasoning behind this criteria is to avoid disruption of the protein cage. It is difficult to gauge which sequences are crucial in cage formation so by avoiding mutations in secondary or tertiary structures, we hoped to mutate a region that would have a minimal impact in cage formation and thus cage disruption.
- Design mutations on the exterior of the cage.-The main reasoning behind this criteria is to allow for easy access by the Thrombin protease. By placing the mutation on the exterior of the cage, we hoped to reduce steric hindrance to the protease and maximize chances of cleavage.
- Design mutations near the linker region (ONLY FOR PC Quad)-The main reasoning behind this criteria is to help disassemble the cage. In the original design of the wild type PCquad cage, it is understood that the there is a linker region that serves as the backbone of a subunit thus proving crucial to the cage structure. By mutating a region close to the linker, we hoped to maximize chances of cage disassembly.
Using these criteria, we designed 10 mutant cages for PCquad and 3 mutant cages for O3-33.
2. The second step of the project is protein expression and verification of cage formation. We attempted expression of five of our best PCquad designs and all three of our O3-33 designs. Expression was done using standard expression protocols and purification was done using his -tag nickel column purification while cage formation was verified using Dynamic Light Scattering.
3. The final step of the project involved testing our successfully formed mutant cages for disassembly in the presence of Thrombin protease. The mutant cages that had successfully formed cages were treated to Thrombin protease at various concentrations and durations. Disassembly of cage was verified using a combination of SDS page to see if the individual subunits cut at the locations of the cleavage site insert and DLS to see if the entire cage structure fell apart.
