The aim of this project is to develop a targeted drug delivery platform by engineering protein cages known as ‘encapsulins’. In the past year, we have made significant progress towards achieving the stepwise goals of this project.

Outcome 1: Developed cell-free protocols for producing encapsulin protein cages.
The crucial enabling step for this project is the ability to produce encapsulins outside of a living cell, so that non-biological molecules, such as cytotoxic drugs, can be loaded into the cages. We successfully established a cell-free protein synthesis workflow in our lab, based off the protocols and reagents provided by Prof. Otting at ANU. Once optimisation of the yields from cell-free expression are completed, we will prepare a publication on this preliminary work.

Outcome 2: Cargo-peptide conjugates synthesised for encapsulation within cages.
We have obtained dye-labelled versions of the targeting peptide required for packaging into encapsulin cages. In addition, we have made fluorescent protein conjugates as a secondary measure of the encapsulation process. We find that while cell-free approaches can produce encapsulin, using a disassembly-reassembly strategy on encapsulins produced from bacteria may be a more efficient approach due to higher yields. We are finalising the right conditions for this packaging process, after which this work will be published.

Outcome 3: Successful fusion of various protein domains to the exterior of encapsulins, demonstrating feasibility of antibody fusion for cell-specific targeting.
We have managed to fuse a range of proteins (fluorescent proteins, dioxygenase enzymes etc.) to the exterior of encapsulins, and shown that they still form cages that have similar assembly properties to the unmodified encapsulin. This shows that it is possible to fuse antibodies to the exterior of the cages, and work is currently underway to continue developing this aspect of the project.

Budget: As planned, funds were used to employ my 2018 Honours student to continue as a Research Assistant to work on outcomes 1 and 2. Even though the funds provided were less than what was originally requested, we managed to save costs that were budgeted for visiting Prof. Otting, by doing the experiments in our lab and getting collaborative advice remotely.