title: Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant.
abstract: The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.
authors.affiliation: Dipartimento di Biotecnologie, Università degli Studi di Verona, Verona, Italy.
authors.forename: Linda
authors.initials: L
authors.lastname: Avesani
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pedrazzini
authors.forename: Maddalena
authors.initials: M
authors.lastname: Devirgilio
authors.forename: Andrea
authors.initials: A
authors.lastname: Pompa
authors.forename: Alessandra
authors.initials: A
authors.lastname: Barbante
authors.forename: Elisa
authors.initials: E
authors.lastname: Gecchele
authors.forename: Paola
authors.initials: P
authors.lastname: Dominici
authors.forename: Francesca
authors.initials: F
authors.lastname: Morandini
authors.forename: Annalisa
authors.initials: A
authors.lastname: Brozzetti
authors.forename: Alberto
authors.initials: A
authors.lastname: Falorni
authors.forename: Mario
authors.initials: M
authors.lastname: Pezzotti
mesh_terms: D019295:Computational Biology; D005968:Glutamate Decarboxylase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D005968:Glutamate Decarboxylase