Secretory expression of norovirus GII.4 VP1 protein in Pichia pastoris

Abstract

Objective To construct recombinant plasmid pPIC9K-VP1 with viral RNA for the secretory expression of norovirus (NoV) GⅡ.4 VP1 protein in P. pastoris. Methods GⅡ.4 NoV-VP1 was inserted into pPIC9K to obtain recombinant plasmid pPIC9K-VP1. Then, the recombinant plasmid pPIC9K-VP1 was linearized by Bam H I and Sal I and transformed into the yeast host strain P. pastoris GS115 by electroporation. At the initial stage of fermentation, glycerol was added into the culture, and after depletion of glycerol, induction of NoV-VLPs production was initiated by the addition of methanol. The supernatant from the shake flask culture was collected by centrifugalization, concentrated by ultrafiltration and purified by ion-exchange chromatography, and the expressed plasmids were assayed by SDS-PAGE, TEM and Western blot. Results NoV-VP1 protein gene was successfully cloned into the expression vector pPIC9K, and VLPs protein was secreted into the fermentation supernatant. The SDS-PAGE gel showed a band at 62 000 corresponding to the molecular weight of the VLPs protein. Western blot by using the anti-NoVs serum also revealed a positive band at 62 000, thus confirming the expression of the full-length VP1 protein. Protein concentration was over 1 g/L in the fermentation supernatant, and approximately 600 mg after protein purification. The morphology and size of the NoV-VLPs were studied using transmission electron microscopy (TEM), fully confirming that the formed particles had an average size of 40 nm. Conclusions In this study we reported a method for the production of fully assembled NoV-VLPs with a high yield from P. pastoris. These production and purification systems described above could be used for large-scale production of norovirus virus-like particles and lay a good foundation for the development of NoV vaccine. Key words: Norovirus; Pichia pastoris; Secretory expression