title: Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase.
abstract: The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.
authors.affiliation: Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos, GR 118 55 Athens, Greece.
authors.forename: Thanasis
authors.initials: T
authors.lastname: Dalakouras
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Smith
authors.forename: Dimitris
authors.initials: D
authors.lastname: Platis
authors.forename: Manon M J
authors.initials: MM
authors.lastname: Cox
authors.forename: Nikolaos E
authors.initials: NE
authors.lastname: Labrou
mesh_terms: D000327:Adsorption; D000345:Affinity Labels; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology / Q000276:immunology*; D007252:Influenza Vaccines / Q000138:chemical synthesis* / Q000276:immunology; D007313:Insecta; D008958:Models, Molecular; D018716:Molecular Mimicry; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D000345:Affinity Labels; D017931:DNA Primers; D007252:Influenza Vaccines; D014614:Vaccines, Synthetic; D009439:Neuraminidase