Establishment of a two-step purification scheme for tag-free recombinant Taiwan native norovirus P and VP1 proteins

摘要

The protruding (P) domain of the major capsid protein VP1 of norovirus (NoV) is the crucial element for immune recognition and host receptor binding. The heterologous P protein expressed by Pichia pastoris self-assembles into P particles. However, tag-free NoV protein purification schemes have rarely been reported due to the low iso-electric point of NoV proteins, which leads to highly competitive binding between the target protein and yeast host cell proteins at alkaline pH. In this study, a two-step purification scheme based on surface histidines and the charge on the NoV GII.4 strain P protein was developed. Using HisTrap and ion exchange chromatography, the P protein was directly purified, with a recovery of 28.1% and purity of 82.1%. Similarly, the NoV capsid protein VP1 was also purified using HisTrap and gel filtration chromatography based on native surface histidines and self-assembly ability, with 20% recovery and over 90% purity. Dynamic light scattering and transmission electron microscopy analyses of the purified NoV P revealed that most of these small P particles were triangle-, squareand ring-shaped, with a diameter of approximately 14 nm, and that the purified NoV VP1 self-assembles into particles with a diameter of approximately 47 nm. Both the purified NoV P and VP1 particles retained human histo-blood group antigen-binding ability, as evidenced by a saliva-binding assay.