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 (approximately 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.
Farran
Imma
I
Instituto de Agrobiotecnología, UPNA-CSIC-Gobierno de Navarra, Campus Arrosadía, 31006 Pamplona, Spain.
Río-Manterola
Francisco
F
Iñiguez
María
M
Gárate
Sonia
S
Prieto
Jesús
J
Mingo-Castel
Angel M
AM
D002736:Chloroplasts / Q000235:genetics* / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors; D018745:Genome, Plant; D006801:Humans; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D036226:Seedlings / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*
D016207:Cytokines; D012333:RNA, Messenger; D011994:Recombinant Proteins; C092073:cardiotrophin 1