Key result
The 3C mutant protease (L127P) cleaved the FMDV P1 precursor protein with a peak efficiency of 89% at 30°C for 2 hours, enabling the in vitro assembly of virus-like particles.
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Nonstructural protein 3C, a master protease of Picornaviridae, plays a critical role in viral replication by directly cleaving the viral precursor polyprotein to form the viral capsid protein and antagonizing the host antiviral response. Additionally, 3C protease, as a tool enzyme, is involved in regulating polyprotein expression. Here, the 3C mutant gene (3Cm), fused with a small ubiquitin-like modifier (SUMO) tag at the N-terminal and featuring a mutation at position 127, was inserted into the cold-shock plasmid pCold of Escherichia coli for expression. Meanwhile, the P1-∆2A plasmid was constructed for expression in Pichia pastoris. The expressions of 3C protein and P1 precursor protein were confirmed by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (SDS-PAGE), and western blot (WB) analysis. The results showed that the wild-type 3C protease is toxic to the host, not only inhibiting protein expression but also inducing the degradation of the host. Moreover, mutation of the 127th amino acid from leucine (L) to proline (P) on the β-ribbon of 3C enhanced the overexpression capacity of 3C in E. coli while maintaining enzymatic activity. Subsequently, 100 µg P1 protein was utilized as a substrate to investigate the cleavage efficiency of 3C protease at various concentrations, temperatures, durations, and pH levels. The results showed that the target protein was cleaved when the protease reached 8 μg. We also found that the presence of the N-terminal SUMO tag did not affect the cleavage activity of 3Cm. The optimal cleavage activity was observed between 25 and 37 °C, with the peak cleavage efficiency of 89% at 30 °C for 2 h. More than 50% of the substrate was degraded within 1 h at 30 °C. Its optimal pH range is between 7 and 8. Remarkably, the P1 protein, cleaved by 3Cm protease, can further form virus-like particles (VLPs) in vitro. KEY POINTS: • Expression and purification of toxic protein 3C protease in E. coli • Cleavage efficiency assessment of 3C protease at various temperatures, durations, and pH • Assembly of virus-like particles of FMDV by cleaving the precursor polyprotein in vitro.
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The L127P mutation in 3C protease allows for efficient in vitro cleavage of FMDV P1 precursor protein and subsequent assembly into virus-like particles, overcoming the toxicity of wild-type 3C protease in host cells.
Li et al. (2024) studied Foot-and-mouth disease virus (FMDV). 3C protease mutant (L127P) vs. Wild-type 3C protease was evaluated on Peak cleavage efficiency of P1 protein. The 3C mutant protease (L127P) cleaved the FMDV P1 precursor protein with a peak efficiency of 89% at 30°C for 2 hours, enabling the in vitro assembly of virus-like particles.