Production and Purification of A Cell-Penetrating Peptide-Erythropoietin Fusion Protein and Optimization of an in Vitro Blood-Brain Barrier Model for Central Nervous System Drug Delivery

Abstract Drug delivery into the central nervous system (CNS) is a brilliant research field, and the development of protein production and purification procedures for novel therapeutic proteins is crucial. Erythropoietin (EPO) is a glycoprotein with tremendous neuroprotective potential, but its bulky size prevents easy penetration across the blood-brain barrier (BBB). EPO-HBHAc is a promising cell-penetrating peptide modified protein for CNS diseases, necessitating an appropriate in vitro BBB model for further evaluation. The plasmid of EPO-HBHAc was constructed by DNA recombinant technology, and the Chinese Hamster Ovary (CHO-K1) cell expression system was selected to generate target proteins. His-tag and size exclusion purification were used to purify the target protein from the cell-conditioned medium; target proteins were further evaluated by western blotting and Coomassie blue staining. Moreover, the endothelial cells (bEnd.3) and astrocytes (CTX TNA2) were used to generate the in vitro BBB model, and transepithelial electrical resistance (TEER) and paracellular diffusion were measured to evaluate barrier integrity. The EPO-HBHAc plasmid was successfully constructed, and a stable cell line expressing EPO-HBHAc was generated. A higher protein expression level was observed in serum-containing medium than in serum-free medium. His-tag purification is not sufficient to remove impurities from target proteins, and thus size exclusion purification was performed to increase the purity of the protein of interest. In contrast, a higher TEER value and lower paracellular diffusion were observed in the co-culture model than in the mono-culture model. Furthermore, the higher TEER value was observed in inserts with a larger growth area (4.67 cm2) than in those with a smaller area (0.33 cm2). In conclusion, we demonstrated that some critical points might impact protein production and the in vitro BBB model construction in this study. Importantly, our research will provide valuable information in the field of CNS drug delivery.