title: Non-food/feed seeds as biofactories for the high-yield production of recombinant pharmaceuticals.
abstract: We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Morandini
authors.forename: Linda
authors.initials: L
authors.lastname: Avesani
authors.forename: Luisa
authors.initials: L
authors.lastname: Bortesi
authors.forename: Bart
authors.initials: B
authors.lastname: Van Droogenbroeck
authors.forename: Kirsten
authors.initials: K
authors.lastname: De Wilde
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.forename: Flavia
authors.initials: F
authors.lastname: Bazzoni
authors.forename: Luca
authors.initials: L
authors.lastname: Santi
authors.forename: Annalisa
authors.initials: A
authors.lastname: Brozzetti
authors.forename: Alberto
authors.initials: A
authors.lastname: Falorni
authors.forename: Eva
authors.initials: E
authors.lastname: Stoger
authors.forename: Ann
authors.initials: A
authors.lastname: Depicker
authors.forename: Mario
authors.initials: M
authors.lastname: Pezzotti
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D008070:Lipopolysaccharides / Q000276:immunology; D008264:Macrophages / Q000187:drug effects / Q000276:immunology; D051379:Mice; D008854:Microscopy, Electron; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011384:Proinsulin / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000096:biosynthesis; D012639:Seeds / Q000378:metabolism* / Q000648:ultrastructure; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D019076:Transgenes; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
chemical_list: D008070:Lipopolysaccharides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D011384:Proinsulin; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2