A protein cage system for producing bioactive peptides inEscherichia coli

Abstract New therapeutics are urgently needed to curb the spread of drug-resistant diseases. Bioactive peptides (BAPs), including antimicrobial peptides, are emerging as an exciting new class of compounds with advantages over current drug modalities, especially small molecule drugs that are prone to resistance development. However, production of BAPs currently relies heavily on chemical synthesis, which is environmentally and economically costly. To address this limitation, we developed a platform that enables the biosynthetic production of BAPs via encapsulation within self-assembling protein cages in the cytoplasm of Escherichia coli . Using bacteriophage P22 virus-like particles (VLPs), we assessed the encapsulation of three structurally distinct classes of BAPs with activities against bacteria, cancer, and malaria. We demonstrated that encapsulation within P22 VLPs enables successful production of BAPs that are otherwise recalcitrant to recombinant expression. This approach is especially attractive for positively charged BAPs and we observed an increase in packaging efficiency within P22 VLPs that corresponded to an increase in overall positive charge. Both cyclic and linear BAPs were recovered following a simple downstream processing pipeline that involved VLP disassembly, then extraction of BAPs via enzymatic processing. This work demonstrates that encapsulation of BAPs within P22 VLPs is an effective method for biosynthetic production of BAPs in E. coli . By providing an alternative to chemical synthesis, this approach has the potential to increase accessibility and reduce the cost and burden of chemical waste for producing therapeutically valuable BAPs.