Commercial Expression Levels of a Potent Recombinant Antiviral Therapeutic Protein in Sugarcane

Expression of recombinant proteins in plants represent a promising system to produce therapeutic proteins since plants can be grown in large quantities at low cost. In addition to those benefits, plant-produced proteins are free of human pathogen contaminants, reducing the risk of potential adverse effects. However, to be a reliable system, plant-synthesized recombinant proteins should be expressed in large-enough quantities to be commercially extracted. Sugarcane ( Saccharum spp. hybrids) has been proposed as an ideal crop for recombinant protein production because of its rapid growth rate and high light and water to biomass conversion efficiency. Moreover, sugarcane is an excellent biological gene containment crop due to its vegetative propagation system. This project aims to develop an efficient expression system in sugarcane for the production of a proprietary recombinant protein with antiviral and antitumor activities. Initial work generated several transgenic sugarcane varieties that overexpress the therapeutic protein under the control of the constitutive maize ubiquitin 1 (Ubi1) promoter, and we have recovered protein expression levels up to 0.1% of total soluble protein (TSP). In order to increase recombinant protein yield, sugarcane varieties were transformed with five new constructs carrying the proprietary therapeutic protein under the control of sugarcane stem-regulated promoters from DIRIGENT5-1 (SHDIR5-1), Sugarcane bacilliform virus (SCBV21), DIRIGENT16 (SHDIR16), PROLINE RICH PROTEIN (SHPRP), and ELONGATION FACTOR1a (SHEF1a). A 35S-NOS double terminator was used to stabilize transcription. Two sugarcane varieties (cv. CP72-1210 and CP89-2143) were transformed using a biolistic method with single, or up-to a five-construct combination to stack stem specific promoters driving the expression of the recombinant protein. Newly generated T₀ staked lines show yields of up to 0.4% TSP equivalent to 4mg/Kg fresh weight of stalk tissue. This represents a four-fold yield increase from previously generated transgenic lines.