Key result
FMDV VLPs were successfully produced in P. pastoris independently of 3C protease by co-expressing VP0, VP3, and VP1, avoiding the toxicity of 3C protease in host cells.
Why the study?
Inactivated foot-and-mouth disease vaccines carry safety concerns, while virus-like particle production in Pichia pastoris avoids uncontrollable cleavage efficiency and host toxicity of 3C protease.
A 3C protease-independent approach enables the successful production of foot-and-mouth disease virus-like particles in Pichia pastoris, providing a safer alternative for vaccine development.
May enable safer, low-cost FMD VLPs via Pichia pastoris; leaves open in vivo efficacy and regulatory translation.
The inactivated vaccines have played a pivotal role in the control and eradication of foot-and-mouth disease (FMD). However, certain safety concerns remain. Recently, virus-like particles (VLPs) have gradually become a research hotspot. As the eukaryotic expression system with the lowest production costs, the production of VLPs using Pichia pastoris has significant potential. During the natural infection process of FMD virus (FMDV), the polyprotein P1 is cleaved by 3C protease to form VP0, VP3, and VP1, which are subsequently assembled into VLPs. In this study, we adopted an alternative approach, co-expressing VP0, VP3, and VP1 without 3C protease for the production of FMDV VLPs in P. pastoris. The western blot (WB) assays showed variable protein expression on the same plasmid. VP0 was the highest, while VP3 and VP1 were similar. Furthermore, the order of proteins on the plasmid also mattered. The results indicated that His6 tags at VP0, VP3, and VP1 N-termini significantly affected VLPs assembly. The three-dimensional structure of FMDV revealed that the N-terminus of VP3 and VP1, which are situated in the external space of VLPs, can be fused with His6 tag. Inserting His6 tags into the G-H loop region of VP1 did not hinder assembly, thus providing a reference for the affinity purification of capsid and VLPs assembly. Here, FMDV VLPs were successfully produced independently of 3C protease, avoiding the uncontrollable cleavage efficiency and toxicity of 3C protease in host cells and demonstrating the potential of P. pastoris for FMDV VLPs production. • FMDV VLPs could be produced in P. pastoris by a 3 C protease–independent approach • Optimal expression of FMDV VLPs in P. pastoris is achieved at pH 7 with 72-h induction • His6 can be fused to the G-H region and C-terminus of VP1 and C-terminus of VP3 without affecting the VLPs assembly
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Li et al. (2025) studied Foot-and-mouth disease. 3C protease-independent co-expression of VP0, VP3, and VP1 was evaluated on Production and assembly of FMDV VLPs. FMDV VLPs were successfully produced in P. pastoris independently of 3C protease by co-expressing VP0, VP3, and VP1, avoiding the toxicity of 3C protease in host cells.
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