title: Influence of elastin-like polypeptide and hydrophobin on recombinant hemagglutinin accumulations in transgenic tobacco plants.
abstract: Fusion protein strategies are useful tools to enhance expression and to support the development of purification technologies. The capacity of fusion protein strategies to enhance expression was explored in tobacco leaves and seeds. C-terminal fusion of elastin-like polypeptides (ELP) to influenza hemagglutinin under the control of either the constitutive CaMV 35S or the seed-specific USP promoter resulted in increased accumulation in both leaves and seeds compared to the unfused hemagglutinin. The addition of a hydrophobin to the C-terminal end of hemagglutinin did not significantly increase the expression level. We show here that, depending on the target protein, both hydrophobin fusion and ELPylation combined with endoplasmic reticulum (ER) targeting induced protein bodies in leaves as well as in seeds. The N-glycosylation pattern indicated that KDEL sequence-mediated retention of leaf-derived hemagglutinins and hemagglutinin-hydrophobin fusions were not completely retained in the ER. In contrast, hemagglutinin-ELP from leaves contained only the oligomannose form, suggesting complete ER retention. In seeds, ER retention seems to be nearly complete for all three constructs. An easy and scalable purification method for ELPylated proteins using membrane-based inverse transition cycling could be applied to both leaf- and seed-expressed hemagglutinins.
authors.affiliation: Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany; Department of Plant Cell Biotechnology, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST), Hanoi, Vietnam.
authors.forename: Hoang Trong
authors.initials: HT
authors.lastname: Phan
authors.affiliation: Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry (IPB), Halle, Germany.
authors.forename: Bettina
authors.initials: B
authors.lastname: Hause
authors.affiliation: Microscopy Unit, Biocenter, University of Halle-Wittenberg, Halle, Germany.
authors.forename: Gerd
authors.initials: G
authors.lastname: Hause
authors.affiliation: Molecular Plant Physiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.affiliation: Molecular Plant Physiology, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.affiliation: Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Department of Chemistry, University of Natural Resources and Applied Life Sciences, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: VTT Technical Research Centre of Finland, Espoo, Finland.
authors.forename: Jussi
authors.initials: J
authors.lastname: Joensuu
authors.affiliation: Department of Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Conrad
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D004549:Elastin / Q000302:isolation & purification / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000378:metabolism; D006031:Glycosylation; D006388:Hemagglutinins / Q000378:metabolism*; D053124:Influenza A Virus, H5N1 Subtype / Q000378:metabolism; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D006388:Hemagglutinins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004549:Elastin