title: Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing.
abstract: Plants have emerged as competitive production platforms for pharmaceutical proteins that are required in large quantities. One example is the 65-kDa isoform of human glutamic acid decarboxylase (GAD65), a major autoimmune diabetes autoantigen that has been developed as a vaccine candidate for the primary prevention of diabetes. The expression of GAD65 in plants has been optimized but large-scale purification is hampered by its tendency to associate with membranes. We investigated the potential for large-scale downstream processing by evaluating different combinations of plant-based expression systems and engineered forms of GAD65 in terms of yield, subcellular localization and solubility in detergent-free buffer. We found that a modified version of GAD65 lacking the first 87 amino acids accumulates to high levels in the cytosol and can be extracted in detergent-free buffer. The highest yields of this variant protein were achieved using the MagnICON transient expression system. This combination of truncated GAD65 and the MagnICON system dramatically boosts the production of the recombinant protein and helps to optimize downstream processing for the establishment of a sustainable plant-based production platform for an autoimmune diabetes vaccine candidate.
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Matilde
authors.initials: M
authors.lastname: Merlin
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Elisa
authors.initials: E
authors.lastname: Gecchele
authors.affiliation: Department of Applied Genetic and Cell Biology, University of Natural Resources and Life Science, Vienna, Austria.
authors.forename: Elsa
authors.initials: E
authors.lastname: Arcalis
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Remelli
authors.affiliation: Department of Internal Medicine, University of Perugia, Perugia, Italy.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Brozzetti
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Mario
authors.initials: M
authors.lastname: Pezzotti
authors.affiliation: Department of Biotechnology, University of Verona, Verona, Italy. linda.avesani@univr.it.
authors.forename: Linda
authors.initials: L
authors.lastname: Avesani
mesh_terms: D003600:Cytosol / Q000378:metabolism; D005968:Glutamate Decarboxylase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D009154:Mutation; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development
chemical_list: D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase
keywords: Molecular farming; Protein engineering; Protein purification; Protein solubility; Type 1 diabetes