Sortase A-based post-translational modifications on encapsulin nanocompartments

Protein-based encapsulin nanocompartments, known for their well-defined structures and versatile functionalities, present promising opportunities in the biotechnology and nanomedicine area. In this investigation, we effectively developed a sortase A-mediated protein ligation system in E. coli to site-specifically attach target proteins to encapsulin, both internally and on its surfaces without any further in vitro steps. We explored the potential applications of fusing sortase enzyme and a protease for post-translational ligation of encapsulin to a green fluorescent protein (GFP) and anti-CD3 scFv. Our results demonstrated that this system could attach other proteins to the nanoparticles’ exterior surfaces without adversely affecting their folding and assembly processes. Additionally, this system enabled the attachment of proteins inside encapsulins which varied shapes and sizes of the nanoparticles due to cargo overload. This research opens up avenues for further exploration of the applications and engineering possibilities of encapsulins in the rapidly advancing field of nanotechnology.