Optimization of Activity Retention for Recombinant APE1/Ref-1 through PEGylation

PEGylation of recombinant proteins enhances their bioavailability by improving stability, solubility, and biological activity. This study aimed to optimize conditions for PEGylating apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) to preserve its reducing activity and ensure it remains non-immunogenic in vivo. The PEGylation reaction of recombinant APE1/Ref-1 showed a progressive pattern dependent on the molar ratio with methoxy polyethylene glycol (PEG)-succinimidyl-glutarate. Optimal PEGylation was achieved with a 30 molar ratio of PEG, modifying 29 lysine residues of recombinant APE1/Ref-1. Activity analysis demonstrated that PEGylated APE1/Ref-1 retained reducing activity for over 4 weeks under refrigerated conditions, comparable to its pre-preservation state. To evaluate the immunogenicity of PEGylated APE1/Ref-1 for in vivo applications, an enzyme linked immunosorbent assay confirmed that PEGylation did not alter its antigenicity. PEGylation is a critical strategy for developing biopharmaceuticals and is expected to be highly beneficial for in vivo applications.