https://scholars.lib.ntu.edu.tw/handle/123456789/634035
Title: | Extracellular Production of the Taiwan-Native Norovirus P Domain Overexpressed in Pichia pastoris | Authors: | Chien, Man Ling Yu, Chun Fu CHING-TSAN HUANG |
Keywords: | Hac1 | norovirus | oligosaccharyl transferase 1 (Ost1) | P particle | tag-free P protein | Issue Date: | 1-Jun-2023 | Publisher: | MDPI | Journal Volume: | 9 | Journal Issue: | 6 | Start page/Pages: | Article number 498 | Source: | Fermentation | Abstract: | 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. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/634035 | ISSN: | 2311-5637 | DOI: | 10.3390/fermentation9060498 |
Appears in Collections: | 生化科技學系 |
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