Small P particles formed by Taiwan native norovirus P domain overexpressed in Pichia pastoris and purified by tag-free purification schemes

Joint Event on International Conference on Cancer Research & Diagnostics & 16^th Asia Pacific Biotechnology Congress

August 15-16, 2018 Singapore

Yu-Ling Chen and Ching-Tsan Huang

National Taiwan University, Taiwan

Scientific Tracks Abstracts: J Tissue Sci Eng

Abstract :

Statement of the Problem: The Protrusion (P) domain of the major structural protein VP1 of Norovirus (NoV) is critical for host immune response and receptor binding. Most heterologous P domain expressed in Escherichia coli or Pichia pastoris forms P particles consisted of 24 P monomers through intermolecular contact in the P regions and end-linked cysteine tag. Particularly, the small P particle consisted of 12 P monomers is only found in the P domain with terminal modification. P. pastoris expression system exist advantages over bacteria including no endotoxin risk, low costs and easy scale-up. Currently, the purification of the recombinant P protein from the crude extract mainly relied on purification tags. However, the purification tags increase the cost and labor in downstream processes and the risk on authentic structure alternation. Method: The NoV P domain of the most predominant NoV strain GII.4 isolated from Taiwan was expressed in the P. pastoris. A high-yield fermentation process of the recombinant NoV P complexes and the tag-free purification schemes were developed. The recombinant NoV P complexes were also verified by LC-MS/MS, ELISA and saliva binding assay and its polymer formation was analyzed by gel filtration, dynamic light scattering and transmitted electronic microscopy. Findings: The majority of NoV P proteins expressed in P. pastoris formed small P particles, composed by 12 copies of the P domain with a diameter of 14 nm and in square and ring shapes. Conclusion: The high cell density fermentation processes and tag-free purification schemes of NoV P protein were successfully developed. The small P particles formed by Taiwan native norovirus P domain might provide further morphogenesis study and vaccine development.

Biography :

Yu-Ling Chen is currently a doctoral student at Department of Biochemical Science and Technology, National Taiwan University in Taiwan. Her research interests include heterologous gene expression systems of the methylotrophic yeast Pichia pastoris, the edible mushroom Flammulina velutipes and antigen presentation system of norovirus P particle.

E-mail: d01b22006@ntu.edu.tw