Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant Linda Avesani 1 , Alessandro Vitale 2 , Emanuela Pedrazzini 2 , Maddalena deVirgilio 2 , Andrea Pompa 2 , Alessandra Barbante 2 , Elisa Gecchele 1 , Paola Dominici 1 , Francesca Morandini 1 , Annalisa Brozzetti 3 , Alberto Falorni 3 and Mario Pezzotti 1 * 1 Dipartimento di Biotecnologie, Universita` degli Studi di Verona, Verona, Italy 2 Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Milano, Italy 3 Dipartimento di Medicina Interna, Universita ` di Perugia, Perugia, Italy Received 6 July 2009; revised 19 January 2010; accepted 28 January 2010. *Correspondence (Tel +39 045 8027951; fax +39 045 8027929; email mario.pezzotti@univr.it) Keywords: T1DM, GAD65, transgenic plant. Summary The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoanti- gen 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 recombi- nant 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. Introduction Plant-based systems may offer economic advantages for the large-scale production of therapeutic proteins in high demand (Ma et al., 2005a,b; Basaran and Rodriguez-Cer- ezo, 2008). However, the overall yields of some proteins can be low, reflecting a combination of factors including inefficient synthesis, incorrect folding, sub-optimal codon usage and poor stability (Doran, 2006; Daniell et al., 2009). The 65 kDa isoform of glutamic acid decarboxylase (GAD65) plays an important role in the development of type 1 diabetes mellitus (T1DM), a chronic disease charac- terized by the autoimmune destruction of pancreatic islet beta cells (Gepts, 1965). Plant-based systems have been developed to produce GAD65 (Porceddu et al., 1999; Ma et al., 2004; Wang et al., 2008) and its chimeric derivative GAD67 ⁄ 65 (Avesani et al., 2003), and transgenic plants expressing GAD65 have been used in combination with those expressing murine interleukin-4 to prevent autoim- mune diabetes in NOD mice (Ma et al., 2004). However, the yields were low, with a maximum of 0.04% total solu- ble protein (TSP) for GAD65 (Porceddu et al., 1999; Ma et al., 2004; Wang et al., 2008) and 0.2% TSP for GAD67 ⁄ 65 (Avesani et al., 2003). GAD is an essential enzyme present in all eukaryotes and many prokaryotes, catalysing the decarboxylation of glutamate to c-aminobutyrate (GABA) and CO 2 (Sogho- monian and Martin, 1998; Capitani et al., 2003; Gut et al., 2006). Plant GAD has a distinctive C-terminal auto- regulatory calcium ⁄ calmodulin (CaM)-binding domain (Baum et al., 1993) whose in vivo role was determined by expressing a mutant GAD lacking this domain in trans- genic tobacco plants (Baum et al., 1996). The constitutive calcium-independent activity of the enzyme produced ª 2010 The Authors 862 Plant Biotechnology Journal ª 2010 Society for Experimental Biology and Blackwell Publishing Ltd Plant Biotechnology Journal (2010) 8, pp. 862–872 doi: 10.1111/j.1467-7652.2010.00514.x