GFP-based expression of truncated GSD65mut forms in high-throughput platforms

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by the destruction of insulin-secreting pancreatic beta cells, causing the need of life-long insulin therapy. The 65 kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen involved in the disease development. Recent phase II clinical trials have shown that administration of alum-formulated GAD65 lead to a significant preservation of residual insulin secretion without serious adverse effects (Ludvigsson et al., 2008); large-scale confirmatory studies are underway in Europe and in the USA. Current systems for the production of large quantity of purified recombinant protein are very expensive and inadequate to provide enough GAD65 to meet the global demand. We have previously shown that GAD65 and a mutated catalytically-inactive form of the protein (GAD65mut) can be expressed in transgenic tobacco plants (Avesani et al., 2003; Avesani et al., 2010). GAD65mut accumulates 10-fold higher than GAD65 and retains the immunogenic properties required to treat T1DM (Avesani et al., 2010). In order to develop a system for the highefficient production and purification of GAD65, we engineered GAD65mut to various extents to obtain soluble forms of the molecule. It is well documented that GAD65 undergoes some posttranslational modifications in the N-terminal domain that result in a firmly membrane-anchored protein, which is only released by the use of detergents, thus complicating downstream processing of the protein. In the present work we described and discussed the solubility and accumulation levels of three N-truncated forms of GAD65mut in comparison with full-length GAD65mut and GAD65 in a plant-based and a yeast-based platform. The two platforms have been chosen for the high-throughput and fast expression of the molecules. Furthermore, in both the systems, GAD65- based forms have been C-terminal fused with GFP in order to easily determine the recombinant proteins sub-cellular localization.