title: A potyvirus-based gene vector allows producing active human S-COMT and animal GFP, but not human sorcin, in vector-infected plants.
abstract: Potato virus A (PVA), a potyvirus with a (+)ssRNA genome translated to a large polyprotein, was engineered and used as a gene vector for expression of heterologous proteins in plants. Foreign genes including jellyfish GFP (Aequorea victoria) encoding the green fluorescent protein (GFP, 27 kDa) and the genes of human origin (Homo sapiens) encoding a soluble resistance-related calcium-binding protein (sorcin, 22 kDa) and the catechol-O-methyltransferase (S-COMT; 25 kDa) were cloned between the cistrons for the viral replicase and coat protein (CP). The inserts caused no adverse effects on viral infectivity and virulence, and the inserted sequences remained intact in progeny viruses in the systemically infected leaves. The heterologous proteins were released from the viral polyprotein following cleavage by the main viral proteinase, NIa, at engineered proteolytic processing sites flanking the insert. Active GFP, as indicated by green fluorescence, and S-COMT with high levels of enzymatic activity were produced. In contrast, no sorcin was detected despite the expected equimolar amounts of the foreign and viral proteins being expressed as a polyprotein. These data reveal inherent differences between heterologous proteins in their suitability for production in plants.
authors.affiliation: Department of Applied Biology, PO Box 27, FIN-00014 University of Helsinki, Finland.
authors.forename: Jani
authors.initials: J
authors.lastname: Kelloniemi
authors.forename: Kristiina
authors.initials: K
authors.lastname: Mäkinen
authors.forename: Jari P T
authors.initials: JP
authors.lastname: Valkonen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D011993:Recombinant Fusion Proteins; C503113:SRI protein, human; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D002394:Catechol O-Methyltransferase