Publication:

High-density seedling expression system for the production of bioactive human cardiotrophin-1, a potential therapeutic cytokine, in transgenic tobacco chloroplasts

Histidine‐tagged human cardiotrophin‐1 (hCT‐1), a recently discovered cytokine with excellent therapeutic potential, was expressed in tobacco chloroplasts under the transcriptional and translational control of two different promoters (rrn and psbA) and 5′‐untranslated regions (5′‐UTRs) (psbA and phage T7 gene 10). The psbA 5′‐UTR promotes recombinant hCT‐1 (rhCT‐1) accumulation in chloroplasts at higher levels (eight‐fold) than those obtained for the phage T7 gene 10 5′‐UTR, regardless of the promoter used, indicating that the correct choice of translational control element is most important for protein production in chloroplasts. The maximum level of rhCT‐1 achieved was 1.14 mg/g fresh weight (equivalent to 5% of total soluble protein) with the psbA promoter and 5′‐UTR in young leaves harvested after 32 h of continuous light, although the bioactivity was significantly lower (~35%) than that of commercial hCT‐1. However, harvesting in the dark or after 12 h of light did not result in a significant decrease in the bioactivity of rhCT‐1, suggesting that 32 h of over‐lighting affects the biological activity of rhCT‐1. Because high levels of rhCT‐1 accumulation took place mainly in young leaves, it is proposed that seedlings should be used in a ‘closed system’ unit, yielding up to 3.2 kg per year of rhCT‐1. This amount would be sufficient to meet the estimated annual worldwide needs of hCT‐1 for liver transplantation surgery in a cost‐effective manner. Furthermore, our strategy is an environmentally friendly method for the production of plant‐based biopharmaceuticals.