Synthesis of the human respiratory syncytial virus nucleoprotein in yeast Saccharomyces cerevisiae

Anotacija

The development of a simple, efficient and cost-effective system for generation of respiratory syncytial virus (RSV) nucleoprotein (NP) might help to upgrade reagents for virus serology, and facilitate an investigation of viral replication and RNA encapsidation mechanisms. To this aim, the gene encoding human RSV NP of antigenic subgroup A was cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. For the recombinant nucleoprotein the yeast expression system proved to be efficient. After purification by CsCl gradient centrifugation, the recombinant RSV–A N protein from yeast yielded 0.9–1.2 mg/1g of wet yeast biomass. A similar electrophoretic mobility of rN and native N proteins was exhibited by SDSPAGE analysis. Recombinant nucleoproteins reacted with immune sera in a Western blot assay. Electron microscopy of purified recombinant nucleoproteins demonstrated an assembly of herring-bone structures typical of viruses of the family Paramyxoviridea. The yeast-expressed RSV-A N protein appeared to be stable during purification and storage. A change of amino acid Y337 to C precluded formation of nucleocapsid-like particles. Keywords: human respiratory syncytial virus, nucleoprotein, yeast, nucleocapsid-like particles