Design, preparation and characterization of octopus-like self-releasing intracellular protein transporter LEB5 based on Escherichia coli heat-labile enterotoxin

Abstract

Despite the great potential of protein drugs as intracellular therapeutic agents, the unmet challenge in breaking through the cell membrane barrier and delivering them to intracellular targets remains. Therefore, developing safe and effective delivery vehicles is critical for fundamental biomedical research and clinical applications. In this study, we designed an octopus-like self-releasing intracellular protein transporter, the LEB5, based on the heat-labile enterotoxin. This carrier comprises five identical units, each of which has three main components: a linker, a self-releasing enzyme sensitivity loop, and the LTB transport domain. The LEB5 comprises five purified monomers that self-assemble to create a pentamer with ganglioside GM1 binding capacity. The fluorescent protein EGFP was used as a reporter system to identify the LEB5 features. The high-purity fusion protein ELEB monomer was produced from modified bacteria carrying pET24a(+)-eleb recombinant plasmids. EGFP protein could effectively detach from LEB5 by low dosage trypsin, according to electrophoresis analysis. The transmission electron microscopy results indicate that both LEB5 and ELEB5 pentamers exhibit a relatively regularly spherical shape, and the differential scanning calorimetry measurements further suggest that these proteins possess excellent thermal stability. Fluorescence microscopy revealed that LEB5 translocated EGFP into different cell types. Flow cytometry showed cellular differences in the transport capacity of LEB5. According to the confocal microscopy, fluorescence analysis and western blotting data, EGFP was transferred to the endoplasmic reticulum by the LEB5 carrier, detached from LEB5 by cleavage of the enzyme-sensitive loop, and released into the cytoplasm. Within the dosage range of LEB5 10–80 μg/mL, cell counting kit-8 assay revealed no significant changes in cell viability. These results demonstrated that LEB5 is a safe and effective intracellular self-releasing delivery vehicle capable of transporting and releasing protein medicines into cells.