Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco PlantsReport as inadecuate




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Asialo-erythropoietin, a desialylated form of human erythropoietin EPO lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO rhuEPOM by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase GalT genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene GapC promoters, respectively. Plant-produced asialo-rhuEPO asialo-rhuEPOP was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS-MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP 20 U-ml provides 2-fold better cytoprotection 44% to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM 21%. The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 JAK2 and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.



Author: Farooqahmed S. Kittur, Mamudou Bah, Stephanie Archer-Hartmann, Chiu-Yueh Hung, Parastoo Azadi, Mayumi Ishihara, David C. Sane, Ji

Source: http://plos.srce.hr/



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