Post-transcriptional factors are important for high-level expression of the human α-l-iduronidase gene in Arabidopsis cgl (complex-glycan-deficient) seeds

We are developing various plant-based systems to produce enzymes for the treatment of lysosomal storage disorders. One such system is seeds of the Arabidopsis thaliana cgl mutant, which are deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), and thus produce recombinant proteins containing glycans primarily in the high-mannose form. We sought to identify gene regulatory sequences that would enhance the expression of a human recombinant gene encoding α-l-iduronidase (IDUA; EC 3.2.1.76) in mature seeds of the cgl mutant. Three promoters derived from temporally-regulated seed genes, those of the napin, vicilin, and arcelin genes, led to similar human IDUA activities at seed maturity for the highest-expressing lines (0.6–1.1 units/mg total soluble protein; TSP). In constructs containing the arcelin (arc 5-I) gene promoter, exchange of the 5′-UTR and signal peptide sequences of the IDUA gene with those of the arcelin gene resulted in a considerable increase in IDUA enzyme activity level which was also reflected in an increase in the accumulation of protein and in steady-state mRNA levels. Replacement of the nopaline synthase gene 3′ end with the arcelin gene 3′ end also resulted in a significant increase in IDUA activity. However, the increase was highest in the presence of other gene regulatory sequences from the arcelin gene (5′-UTR and signal peptide), which appeared to act synergistically to enhance expression at the protein and activity level. Three lines expressing this ‘optimized’ construct exhibited extremely high IDUA protein levels and activities (e.g., 820 ± 63 units/mg TSP), which may have been due to transcriptional and post-transcriptional factors. The human enzyme possessed moderate stability in seeds stored in the dry state following harvest.