Abstract
Sugarcane and energy cane (Saccharum spp. hybrids) are ideal for plant-based production of recombinant proteins because their high resource-use efficiency, rapid growth and efficient photosynthesis enable extensive biomass production and protein accumulation at a cost-effective scale. Here, we aimed to develop these species as efficient platforms to produce recombinant Galanthus nivalis L. (snowdrop) agglutinin (GNA), a monocot-bulb mannose-specific lectin with potent antiviral, antifungal and antitumor activities. Initially, GNA levels of 0.04% and 0.3% total soluble protein (TSP) (0.3 and 3.8 mg kg–1 tissue) were recovered from the culms and leaves, respectively, of sugarcane lines expressing recombinant GNA under the control of the constitutive maize ubiquitin 1 (Ubi) promoter. Co-expression of recombinant GNA from stacked multiple promoters (pUbi and culm-regulated promoters from sugarcane dirigent5-1 and Sugarcane bacilliform virus) on separate expression vectors increased GNA yields up to 42.3-fold (1.8% TSP or 12.7 mg kg–1 tissue) and 7.7-fold (2.3% TSP or 29.3 mg kg–1 tissue) in sugarcane and energy cane lines, respectively. Moreover, inducing promoter activity in the leaves of GNA transgenic lines with stress-regulated hormones increased GNA accumulation to 2.7% TSP (37.2 mg kg–1 tissue). Purification by mannose-agarose affinity chromatography yielded a functional sugarcane recombinant GNA with binding substrate specificity similar to that of native snowdrop-bulb GNA, as shown by enzyme-linked lectin and mannose-binding inhibition assays. The size and molecular weight of recombinant GNA were identical to those of native GNA, as determined by size-exclusion chromatography and MALDI-TOF mass spectrometry. This work demonstrates the feasibility of producing recombinant GNA at high levels in Saccharum species, with the long-term goal of using it as a broad-spectrum antiviral carrier molecule for hemopurifiers and in related therapeutic applications.
Highlights
The production of recombinant therapeutic proteins on a large scale is a fast-growing sector of biopharmaceutical research and industry
Recombinant Snowdrop-Bulb Galanthus nivalis L. (snowdrop) agglutinin (GNA) Is Synthesized in Transgenic Sugarcane
Southern blot analysis revealed that the majority of the promoter and 35STNOST double terminator (pUbi):GNA lines exhibited a simple integration pattern (Figure 1B; representative pUbi:GNA line 1D; Supplementary Figure S1)
Summary
The production of recombinant therapeutic proteins on a large scale is a fast-growing sector of biopharmaceutical research and industry. The need for a platform that offers low production costs, safety and high scalability has led to the use of plants as biofactories (Williams et al, 2014; Chen and Davis, 2016). Sugarcane and energy cane (Saccharum spp. hybrids) are ideal platforms for the production of native and recombinant protein-based therapeutics at commercial levels, due to their high resource-use efficiencies, rapid growth, efficient photosynthesis and high biomass production capacity, with potential yields of up to 49 tons of dry biomass per hectare per annum (Ando et al, 2011; Matsuoka et al, 2014). Sugarcane offers a high level of transgene containment (Altpeter and Oraby, 2010). Energy cane has not been studied for its use as a biofactory for recombinant therapeutic proteins
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