Targeted expression of a designed fusion protein containing BMP2 into the lumen of exosomes

Exosomes are 30-150 nm membrane vesicles, originating from the endocytic pathway. By acting as natural carriers of biomolecules, they can transfer various materials to recipient cells. Therefore, discovering novel strategies for cargo packaging into exosomes is crucial.The fusion constructs, consisting of protein of interest (BMP2) along with the targeting motif, linkers, tracking proteins, and enzyme cleavage sites, were computationally designed. Following the homology modeling, the best structure was selected and subjected to molecular dynamics (MD) simulation and docking analyses. The fusion protein gene was expressed in the HEK-293LTV cell line. The high-efficiency transfected and transduced cells were screened and their exosomes were isolated. Finally, cell and exosome lysates were evaluated for expression of the fusion protein.A total of 12 constructs with lengths ranging from 483 to 496 were designed. The top three templates, 1REW, 2H5Q, and 2MOF were screened. MD simulation and docking analyses of the structures revealed their stability and functionality. In the protein expression analyses, three bands at sizes of approximately 60, 25, and 12.5 kDa were observed, consistent with the sizes of the complete fusion protein, dimeric, and monomeric BMP2 protein. The presence of a 12.5 kDa band at exosome lysate analysis might suggest that it was loaded and cleaved inside exosomes.In summary, these findings revealed that the proposed idea for cargo sorting within the exosome lumen through incorporating an appropriate cleavage site was effective, thus providing further insight into the potential of exosomes as nano-shuttles bearing therapeutic biomolecules.