Expression of a Truncated H1N1 Neuraminidase and High-throughput Screening of its Inhibitor

Abstract

Aim To produce the H1N1 neuraminidase (NA) with catalytic activity by Pichia pastoris and screen novel inhibitors of NA from the extracts of microbial fermentation broths. Methods The truncated H1N1 NA coding gene was cloned into plasmid pPICZαA to construct the recombinant plasmid pPICZαA-NA, which was linearized and then electroporated into P. pastoris SMD1168. The correct transformants were induced by methanol for the expression of recombinant NA, which was then used to construct a high-throughput screening model for the screening of novel NA inhibitors from the extracts of fermentation broths. Results The recombinant P. pastoris could secrete the truncated NA of H1N1 with catalytic activity. Moreover, its activity was significantly inhibited by oseltamivir and zanamivir, two licensed inhibitors of H1N1 NA. Based on these, more than 20 000 microibal fermentation extracts were screened, and 6 of them showed < 50% inhibition rate in the secondary screening experiment. Conclusion The truncated NA with catalytic activity could be used to construct the high-throughput model for screening new inhibitors of NA. Compared with purification from virion, the preparation of NA is more convenient and safer from P. pastoris, which would facilitate the development of high-throughput assay for screening novel NA inhibitors.