title: Enhanced expression of secretable influenza virus neuraminidase in suspension mammalian cells by influenza virus nonstructural protein 1.
abstract: Influenza neuraminidase (NA) is a major target for anti-influenza drugs. With an increasing number of viruses resistant to the anti-NA drug oseltamivir, functionally active recombinant NA is needed for screening novel anti-NA compounds. In this study, the secretable NA (sNA) head domain of influenza A/Vietnam/DT-036/05 (H5N1) virus was expressed successfully in human embryonic kidney (HEK-293T) cells and shown to be enzymatically active. The inclusion of a plasmid encoding nonstructural protein 1 (NS1) of influenza A/Puerto Rico/8/34 virus with the sNA plasmid in the cotransfection demonstrated an increase in H5N1 sNA expression by 7.4 fold. Subsequently, the sNA/NS1 cotransfection protocol in serum-free 293-F suspension cell culture was optimized to develop a rapid transient gene expression (TGE) system for expression of large amounts of H5N1 sNA. Under optimized conditions, NS1 enhanced H5N1 sNA expression by 4.2 fold. The resulting H5N1 sNA displayed comparable molecular weight, glycosylation, K(m) for MUNANA, and K(i) for oseltamivir carboxylate to those of H5N1 NA on the virus surface. Taken together, the NS1-enhancing sNA expression strategy presented in this study could be used for rapid high-level expression of enzymatically active H5N1 sNA in suspension mammalian cells. This strategy may be applied for expression of sNA of other strains of influenza virus as well as the other recombinant proteins.
authors: Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Pathumthani 12120, Thailand. tarangsri.niv@biotec.or.th
authors: Tarangsri
authors: T
authors: Nivitchanyong
authors: Suganya
authors: S
authors: Yongkiettrakul
authors: Jarin
authors: J
authors: Kramyu
authors: Supitcha
authors: S
authors: Pannengpetch
authors: Nanchaya
authors: N
authors: Wanasen
mesh_terms: D001709:Biotechnology / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D003001:Cloning, Molecular; D005347:Fibroblasts / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
chemical_list: C085820:INS1 protein, influenza virus; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C487630:NA protein, influenza A virus; D009439:Neuraminidase