Ectopic expression of a conifer Abscisic Acid Insensitive3 transcription factor induces high-level synthesis of recombinant human α-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.

An ABI3-interactor of conifers responds to multiple hormones

“…Characterization of the gymnosperm ABI3 in both yellow-cedar, and in heterologous (angiosperm) hosts, shows that the CnABI3 protein has similar roles as its angiosperm counterparts. [1][2][3] As reported in a recent publication, using a yeast 2-hybrid approach, 4 we further identified a new protein of yellow-cedar, CnAIP2, that interacts with CnABI3. CnAIP2 plays important roles during several key transitions of the plant lifecycle, and this protein appears to act as a global regulator with functions opposite to those of ABI3 proteins.…”

A Conifer ABI3-Interacting Protein Plays Important Roles during Key Transitions of the Plant Life Cycle

“…Indeed, particularly relevant to this study, our previous research showed that yellow cedar CnABI3 functions nearly perfectly in the Arabidopsis ABI3 null mutant abi3-6, restoring almost all of the mutant's severe phenotypes (Zeng and Kermode, 2004). Furthermore, ectopic expression of the conifer CnABI3 gene in transgenic tobacco (Nicotiana tabacum) activates the promoters of seed storage protein genes, similar to that of the Arabidopsis ABI3 gene (Zeng et al, 2003;Kermode et al, 2007). Thus, due to the severe limitations of conventional approaches for the functional characterization of conifer genes/proteins (i.e.…”

Biocatalysis, Enzyme Engineering and Biotechnology

“…Plants also have high-fidelity expression, folding and post-translational modification mechanisms, which could produce human proteins of substantial structural and functional equivalency compared to proteins from mammalian expression systems (Gomord andFaye 2004, Joshi andLopez 2005). Additionally, plant-made human proteins of clinical interest (Schillberg et al 2005, Twyman et al 2005, Vitale and Pedrazzini 2005, Tiwari et al 2009, such as antibodies (Hassan et al 2008, Ko et al 2009, De Muynck et al 2010, Lai et al 2010, vaccines (Hooper 2009, Alvarez andCardineau 2010), cytokines (Elias-Lopez et al 2008) and enzymes (Kermode et al 2007, Stein et al 2009, are free of potentially hazardous human diseases, viruses or bacterial toxins. However, there is considerable concern regarding the potential hazards of contamination of the natural gene pool by the transgenes and possible additional safety precautions will raise the production cost.…”