title: Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.
abstract: PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.
authors.forename: M
authors.initials: M
authors.lastname: Kamencayová
authors.forename: I
authors.initials: I
authors.lastname: Košík
authors.forename: J
authors.initials: J
authors.lastname: Hunková
authors.forename: Z W
authors.initials: ZW
authors.lastname: Subr
mesh_terms: D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D017801:Plum Pox Virus / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C442816:PB1-F2 protein, Influenza A virus; D014764:Viral Proteins