Surface Engineeringof the Encapsulin Nanocompartmentof Myxococcus xanthus for Cell-TargetedProtein Delivery

Encapsulin nanocompartments (ENCs), or simply encapsulins, are a novel type of protein nanocage found in bacteria and archaea. The complete encapsulin systems include protein cargoes involved in specific metabolic tasks. Cargoes are selectively encapsulated due to the presence of a specific cargo-loading peptide (CLP). However, heterologous proteins fused to the CLP have also been successfully encapsulated, making encapsulins a very promising system for protein-carrying and delivery. Nevertheless, for precise cell or tissue delivery, encapsulins require the addition of tagging peptides or proteins. In this study, the external surface of the Myxococcus xanthus ENC (MxENC) was analyzed and modified to carry the bioorthogonal conjugation peptide (SpyTag) to further decorate the MxENCs with any targeting protein previously fused to the SpyTag orthogonal pair, the SpyCatcher protein. The structural analysis of MxENC led to the selection of the surface loop 155–159 and the C-terminus of the encapsulin shell protein (EncA) for the genetic fusion of the SpyTag peptide. The engineered EncA forms retained the competence for self-assembly into ENCs. To provide cellular specificity, the PreS1_21–47 hepatocyte-targeting peptide, genetically fused to the SpyCatcher protein, was successfully conjugated to both engineered versions of the MxENC. The modified nanocompartments underwent comprehensive characterization for stability, cargo loading, cellular uptake, and cargo release in HepG2 cells, demonstrating their potential as protein-delivery vehicles. These results provide valuable insights into the design and customization of nanocompartments, opening up possibilities for improved drug delivery applications in biotechnology and nanomedicine.