Open AccessRecombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant
Author(s) -
Avesani Linda,
Vitale Alessandro,
Pedrazzini Emanuela,
DeVirgilio Maddalena,
Pompa Andrea,
Barbante Alessandra,
Gecchele Elisa,
Dominici Paola,
Morandini Francesca,
Brozzetti Annalisa,
Falorni Alberto,
Pezzotti Mario
Publication year - 2010
Publication title -
plant biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.525
H-Index - 115
eISSN - 1467-7652
pISSN - 1467-7644
DOI - 10.1111/j.1467-7652.2010.00514.x
Subject(s) - biology , transgene , endoplasmic reticulum , mutant , recombinant dna , gene isoform , glutamate decarboxylase , biochemistry , genetically modified crops , genetically modified tomato , enzyme , microbiology and biotechnology , gene
Summary 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 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 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.