Overview

Our team has dedicated the past year to investigating protein nanocompartments as a mechanism for drug delivery. Specifically, we have focused on the use of encapsulins, a specific class of bacterial nanocompartments that self-assemble and envelop cargo, and that are relatively straightforward to work with [1]. We worked to test both the encapsulins’ ability to envelope cargo and how organisms take up the cargo-carrying proteins. In our experimentation, we utilized baker’s yeast (S. cerevisiae) and mNeonGreen (mNG) as organisms and cargo, respectively. These were chosen due to yeast’s well documented endocytosis pathway and the ability to measure mNeonGreen absorption using fluorescence microscopy.

Figure 1: T. Maritima Encapsulin

Our main motivation for choosing to investigate encapsulins was the widespread benefits that we believe can come from investing in the subject. Once protein compartments assemble around the cargo, they are incredibly stable [2]. The nanocompartments also allow for more efficiency in delivering medication as they can better penetrate tissue and circulate in the body for longer [3]. The primary reason we looked to encapsulins over other classes of protein nanocompartments was the ease of working with encapsulins. As a purely student-run design team, our resources are relatively limited, but we felt that we could use encapsulins as a “proof of concept” for the use of nanocompartments in pharmaceutical transport more generally.

Figure 2: Encapsulin Loading of Cargo

Over the course of our project, we primarily focused on two areas. The first was adding an alpha factor tag to the encapsulin to facilitate endocytosis in yeast. Alpha factor is a yeast mating hormone known to facilitate receptor-mediated endocytosis in yeast [4]. This was attempted through inverse fusion PCR and then Gibson assembly without definitive success. The second aspect of the project involved purifying the encapsulin protein (without alpha factor) and then attempting to endocytose into yeast cells as a negative control (meaning endocytosis shouldn’t happen without the alpha factor). We also performed some computational modeling of the encapsulin.

Future Directions

1. Our team plans to complete cloning experiments to incorporate alpha-factor on the outside of the encapsulin and perform endocytosis assays to verify that the encapsulin is properly taken up by the cell. We plan to track the encapsulin in the cell using more specific assays such as endosome fluorescent staining.
2. We plan to investigate the possibility of delivering nucleic acid cargo to eukaryotic cells in addition to fluorescent protein. We plan to construct an encapsulin with alpha-factor on the outside and attach an RNA-binding protein to the inside of the encapsulin to load the RNA. We plan to add a specific nucleic acid sequence that codes for a protein that causes measurable response such as apoptosis or fluorescence.
3. Our project focuses on using yeast cells to demonstrate proof-of-concept that encapsulins can be endocytosed. We also plan to test endocytosis in mammalian cells using other targeting peptides such as EGFR or attaching mannose to the outside of the encapsulin to trigger endocytosis in its corresponding cell line.

References

1. Giessen, T., Silver, P. Widespread distribution of encapsulin nanocompartments reveals functional diversity. Nat Microbiol 2, 17029 (2017). https://doi.org/10.1038/nmicrobiol.2017.29
2. Jones, JA, Giessen, TW. Advances in encapsulin nanocompartment biology and engineering. Biotechnology and Bioengineering. 2021; 118: 491– 505. https://doi.org/10.1002/bit.27564
3. Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform Hyojin Moon, Jisu Lee, Junseon Min, and Sebyung Kang Biomacromolecules 2014 15 (10), 3794-3801 DOI: 10.1021/bm501066m
4. Toshima JY, Toshima J, Kaksonen M, Martin AC, King DS, Drubin DG. Spatial dynamics of receptor-mediated endocytic trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives. Proc Natl Acad Sci U S A. 2006 Apr 11;103(15):5793-8. doi: 10.1073/pnas.0601042103. Epub 2006 Mar 30. PMID: 16574772; PMCID: PMC1458652.