High-Level Secretory Production of Recombinant E2-Spy Antigen Protein via Combined Strategy in Pichia pastoris

E2-Spy (abbreviated as ES) plays a vital role as a component in the Bacterial-Like Particles (BLPs) vaccine against classical swine fever virus (CSFV). This vaccine demonstrates remarkable immunoprotection, highlighting the importance of augmenting ES production in the development of CSFV subunit vaccines. In this study, a Pichia pastoris strain capable of high-yield secretory production of ES was developed through signal peptide engineering, gene dosage optimization and co-expression of molecular chaperones. Initially, a hybrid signal peptide cSP3 was engineered, leading to a 3.38-fold increase in ES production when compared to the control strain 1-alpha-ES. Subsequently, cSP3 was evaluated for its expression efficiency alongside different commonly used signal peptides under multicopy conditions. SDS-PAGE analysis revealed that 2-alpha d14-ES exhibited the highest ES production, displaying a 4.38-fold increase in comparison to 1-alpha-ES. Afterwards, SSA1, YDJ1, BIP, LHS1, and their combinations were integrated into 2-alpha d14-ES, resulting in a 1.92-fold rise in ES production compared to 2-alpha d14-ES (equivalent to a 6.18-fold increase compared to 1-alpha-ES). The final yield of ES was evaluated as 168.3 mg/L through comparison with serially diluted BSA protein bands.