title: Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human alpha-L-iduronidase in transgenic tobacco leaves.
abstract: We are examining various plant-based systems to produce enzymes for the treatment of human lysosomal storage disorders. Constitutive expression of the gene encoding the human lysosomal enzyme, alpha-L-iduronidase (IDUA; EC 3.2.1.76) in leaves of transgenic tobacco plants resulted in low-enzyme activity, and the protein appeared to be subject to proteolysis. Toward enhancing production of this recombinant enzyme in vegetative tissues, transgenic tobacco plants were generated to co-express a CaMV35S:Chamaecyparis nootkatensis Abscisic Acid Insensitive3 (CnABI3) gene construct, along with the human gene construct. The latter contained regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene (5'-flanking, signal-peptide-encoding, and 3'-flanking regions). Ectopic synthesis of the CnABI3 protein led to the transactivation of the arcelin promoter and accordingly high activity (e.g., 25,000 pmol/min/mg total soluble protein) and levels of recombinant IDUA mRNA and protein were induced in leaves of transgenic tobacco, particularly in the presence of 150-200 microM S-(+)-ABA. Synthesis of human IDUA containing a carboxy-terminal ER retention (SEKDEL) sequence was also inducible by ABA in leaves co-transformed with the CnABI3 gene. As compared to the natural S-(+)-ABA, two persistent ABA analogues, (+)-8' acetylene ABA and (+)-8'methylene ABA, led to greater levels of beta-glucuronidase (GUS) reporter activities in leaves co-expressing the CnABI3 gene and a vicilin:GUS chimeric gene. In contrast, (+)-8' acetylene ABA and natural ABA appeared to be equally effective in stimulating the CnABI3-induced expression of an arcelin:GUS gene, and of the human IDUA gene, the latter also driven by arcelin-gene-regulatory sequences. Various stress-related treatments, particularly high concentrations of NaCl, had an even greater effect than ABA in promoting accumulation of human IDUA in co-transformed tobacco leaves. This strategy provides the means of enhancing the yields of recombinant proteins in transgenic plant vegetative tissues and potentially in cultured plant cells. The human recombinant protein can be readily induced in the presence of chemicals such as NaCl that can be added to cell cultures or even whole plants without a significant increase in production costs.
authors.affiliation: Department of Biological Sciences, Simon Fraser University, Burnaby, BC, Canada. kermode@sfu.ca
authors.forename: Allison R
authors.initials: AR
authors.lastname: Kermode
authors.forename: Ying
authors.initials: Y
authors.lastname: Zeng
authors.forename: Xiaoke
authors.initials: X
authors.lastname: Hu
authors.forename: Samantha
authors.initials: S
authors.lastname: Lauson
authors.forename: Suzanne R
authors.initials: SR
authors.lastname: Abrams
authors.forename: Xu
authors.initials: X
authors.lastname: He
mesh_terms: D000040:Abscisic Acid / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D029780:Chamaecyparis / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D006801:Humans; D007068:Iduronidase / Q000235:genetics / Q000378:metabolism*; D010937:Plant Growth Regulators / Q000494:pharmacology; D018515:Plant Leaves / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism
chemical_list: D010937:Plant Growth Regulators; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D000040:Abscisic Acid; D007068:Iduronidase