title: A generic system for the expression and purification of soluble and stable influenza neuraminidase.
abstract: The influenza surface glycoprotein neuraminidase (NA) is essential for the efficient spread of the virus. Antiviral drugs such as Tamiflu (oseltamivir) and Relenza (zanamivir) that inhibit NA enzyme activity have been shown to be effective in the treatment of influenza infections. The recent 'swine flu' pandemic and world-wide emergence of Tamiflu-resistant seasonal human influenza A(H1N1) H(274)Y have highlighted the need for the ongoing development of new anti-virals, efficient production of vaccine proteins and novel diagnostic tools. Each of these goals could benefit from the production of large quantities of highly pure and stable NA. This publication describes a generic expression system for NAs in a baculovirus Expression Vector System (BEVS) that is capable of expressing milligram amounts of recombinant NA. To construct NAs with increased stability, the natural influenza NA stalk was replaced by two different artificial tetramerization domains that drive the formation of catalytically active NA homotetramers: GCN4-pLI from yeast or the Tetrabrachion tetramerization domain from Staphylothermus marinus. Both recombinant NAs are secreted as FLAG-tagged proteins to allow for rapid and simple purification. The Tetrabrachion-based NA showed good solubility, increased stability and biochemical properties closer to the original viral NA than the GCN4-pLI based construct. The expressed quantities and high quality of the purified recombinant NA suggest that this expression system is capable of producing recombinant NA for a broad range of applications including high-throughput drug screening, protein crystallisation, or vaccine development.
authors.affiliation: Division of Materials Science & Engineering, CSIRO, Parkville, Victoria, Australia. Peter.Schmidt@CSIRO.au
authors.forename: Peter M
authors.initials: PM
authors.lastname: Schmidt
authors.forename: Rebecca M
authors.initials: RM
authors.lastname: Attwood
authors.forename: Peter G
authors.initials: PG
authors.lastname: Mohr
authors.forename: Susan A
authors.initials: SA
authors.lastname: Barrett
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: McKimm-Breschkin
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D024882:Drug Resistance, Viral / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology* / Q000235:genetics*; D009439:Neuraminidase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D010802:Phylogeny; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D009439:Neuraminidase