Enhanced GAD65 production in plants using the MagnICON transient expression system: Optimization of upstream production and downstream processing
| Autor: | Linda Avesani, Mario Pezzotti, Matilde Merlin, Elsa Arcalis, Sabrina Remelli, Annalisa Brozzetti, Elisa Gecchele |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gene isoform Molecular farming Protein engineering Protein purification Protein solubility Type 1 diabetes Applied Microbiology and Biotechnology Molecular Medicine Biology law.invention 03 medical and health sciences Cytosol law Tobacco Humans chemistry.chemical_classification Downstream processing Glutamate Decarboxylase business.industry General Medicine Plants Genetically Modified Subcellular localization Recombinant Proteins Biotechnology Amino acid Cell biology 030104 developmental biology chemistry Mutation Recombinant DNA business |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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