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 therefore a potential issue in the fight against T1DM but current production platforms are expensive. GAD65 has previously been expressed in transgenic tobacco plants. Here, we show that a catalytically-inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein, which is more than10-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 due to 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.
Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant
Vitale A;Pedrazzini E;Pompa A;Vitale A;Pedrazzini E;de Virgilio M;Pompa A;
2010
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 therefore a potential issue in the fight against T1DM but current production platforms are expensive. GAD65 has previously been expressed in transgenic tobacco plants. Here, we show that a catalytically-inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein, which is more than10-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 due to 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.