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

摘要

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?￠???2?￠????untranslated regions (5?￠???2?￠????UTRs) ( psbA and phage T7 gene 10 ). The psbA 5?￠???2?￠????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?￠???2?￠????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?￠???2?￠????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.