Key result
Extracellular production of the Taiwan-native norovirus P domain in P. pastoris achieved a yield of 345 mg/L with 94.8% purity and 66.9% recovery, forming particles with biological binding activity.
Why the study?
Intracellularly expressed norovirus P proteins in Escherichia coli and Pichia pastoris suffered from low yield and purification difficulties, limiting their application in vaccine development.
The successful extracellular secretion of the norovirus P domain in P. pastoris provides a scalable strategy for antiviral vaccine development.
Extracellular P domain secretion in P. pastoris reaches 345 mg/L with simplified purification; leaves open scalable NoV vaccine manufacturing pending further immunogenicity and clinical data.
Many efforts in norovirus vaccine development have focused on subunit or recombinant protein vaccines, such as subviral P particles formed by the protruding (P) domain of VP1. P particles are immunogenic and have a region with a human histo-blood group antigen binding site, an interaction critical for infecting the host. In the past, only intracellular NoV P proteins expressed in Escherichia coli and Pichia pastoris were reported, and the low yield and difficulty in purification limited their applications. In this study, the Taiwan-native NoV P domain was successfully expressed and secreted by P. pastoris. The secretion efficiency was greatly enhanced by integrating oligosaccharyl transferase (Ost1) into the α-factor signal peptide and coexpressing Hac1. The production of NoV P in fermentation cultures reached 345 mg/L, and the purity and recovery were 94.8% and 66.9%, respectively, after only ion-exchange chromatography. Transmission electron microscopy analysis showed that the small P particles were mostly ring-, square-, and triangle-shaped, with diameters of 10-15 nm. The biological activity of NoV P was confirmed by saliva-binding assay using human histo-blood group antigen. This study describes the secretion and characterization of the Taiwan-native norovirus P domain in P. pastoris. Particles formed from the P domain were similar in size, morphology, and binding ability to those expressed intracellularly. The strategy described in this study provides great potential in scale-up production and antiviral vaccine development.
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Chien et al. (2023) studied Norovirus vaccine development. Extracellular production of Taiwan-native norovirus P domain in Pichia pastoris was evaluated on Production yield, purity, and recovery. Extracellular production of the Taiwan-native norovirus P domain in P. pastoris achieved a yield of 345 mg/L with 94.8% purity and 66.9% recovery, forming particles with biological binding activity.
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