title: Transgenic Production of an Anti HIV Antibody in the Barley Endosperm.
abstract: Barley is an attractive vehicle for producing recombinant protein, since it is a readily transformable diploid crop species in which doubled haploids can be routinely generated. High amounts of protein are naturally accumulated in the grain, but optimal endosperm-specific promoters have yet to be perfected. Here, the oat GLOBULIN1 promoter was combined with the legumin B4 (LeB4) signal peptide and the endoplasmic reticulum (ER) retention signal (SE)KDEL. Transgenic barley grain accumulated up to 1.2 g/kg dry weight of recombinant protein (GFP), deposited in small roundish compartments assumed to be ER-derived protein bodies. The molecular farming potential of the system was tested by generating doubled haploid transgenic lines engineered to synthesize the anti-HIV-1 monoclonal antibody 2G12 with up to 160 μg recombinant protein per g grain. The recombinant protein was deposited at the periphery of protein bodies in the form of a mixture of various N-glycans (notably those lacking terminal N-acetylglucosamine residues), consistent with their vacuolar localization. Inspection of protein-A purified antibodies using surface plasmon resonance spectroscopy showed that their equilibrium and kinetic rate constants were comparable to those associated with recombinant 2G12 synthesized in Chinese hamster ovary cells.
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Corrensstr. 3, 06466, Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Goetz
authors.initials: G
authors.lastname: Hensel
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Phytoantibodies, Corrensstr. 3, 06466, Stadt Seeland/OT Gatersleben, Germany; Medical Faculty, Institute of Biochemistry and Molecular Biology II, Heinrich-Heine-University, Universitätsstrasse 1, 40225, Düsseldorf, Germany.
authors.forename: Doreen M
authors.initials: DM
authors.lastname: Floss
authors.affiliation: Department for Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.affiliation: Institute for Molecular Biotechnology, Biology VII, RWTH Aachen, Worringerweg 1, 52074, Aachen, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Sack
authors.affiliation: Department for Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Melnik
authors.affiliation: Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Altmann
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Structural Cell Biology, Corrensstr. 3, 06466, Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Twan
authors.initials: T
authors.lastname: Rutten
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Plant Reproductive Biology, Corrensstr. 3, 06466, Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
authors.affiliation: Department for Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.affiliation: Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Phytoantibodies, Corrensstr. 3, 06466, Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Conrad
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D018554:Avena / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D056625:Endosperm / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015483:HIV Antibodies / Q000096:biosynthesis*; D015488:HIV Antigens / Q000378:metabolism; D006238:Haploidy; D001467:Hordeum / Q000235:genetics*; D007700:Kinetics; D009928:Organ Specificity / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D011134:Polysaccharides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis; D019076:Transgenes
chemical_list: D000911:Antibodies, Monoclonal; D015483:HIV Antibodies; D015488:HIV Antigens; D010940:Plant Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins