Abstract
Recombinant human erythropoietin (rhEPO), a glycohormone, is one of the leading biopharmaceutical products. The production of rhEPO is currently restricted to mammalian cell expression systems because of rhEPO's highly complex glycosylation pattern, which is a major determinant for drug-efficacy. Here we evaluate the ability of plants to produce different glycoforms of rhEPO. cDNA constructs were delivered to Nicotiana benthamiana (N. benthamiana) and transiently expressed by a viral based expression system. Expression levels up to 85 mg rhEPO/kg fresh leaf material were achieved. Moreover, co-expression of rhEPO with six mammalian genes required for in planta protein sialylation resulted in the synthesis of rhEPO decorated mainly with bisialylated N-glycans (NaNa), the most abundant glycoform of circulating hEPO in patients with anemia. A newly established peptide tag (ELDKWA) fused to hEPO was particularly well-suited for purification of the recombinant hormone based on immunoaffinity. Subsequent lectin chromatography allowed enrichment of exclusively sialylated rhEPO. All plant-derived glycoforms exhibited high biological activity as determined by a cell-based receptor-binding assay. The generation of rhEPO carrying largely homogeneous glycosylation profiles (GnGnXF, GnGn, and NaNa) will facilitate further investigation of functionalities with potential implications for medical applications.
Highlights
Erythropoietin (EPO) is a glycoprotein mainly produced in the adult kidney, and was initially highlighted for its action on the hematopoietic system [1, 2]
We demonstrate the efficient expression of sialylated rhEPOELD in N. benthamiana, already at 4 dpi
Terminal sialylation of rhEPOELD seemed to have a positive impact on the stability compared with WTrhEPOELD
Summary
Erythropoietin (EPO) is a glycoprotein mainly produced in the adult kidney, and was initially highlighted for its action on the hematopoietic system [1, 2]. Native and rhEPOs, isolated from urine and Chinese hamster ovary (CHO) cells, respectively, carry large amounts of elongated and branched structures (i.e. sialylated bi-, tri-, and tetra-antennary oligosaccharides). Native hEPO isolated from serum, which presents the physiologically active form of the hormone, exhibits in general the same type of N-linked oligosaccharides in terms of branching patterns as CHO-derived rhEPO but the relative amounts of the individual structures are different. A major difference is the presence of relatively large amounts of non-sialylated GnGnF structures and the absence of tetra-antennary oligosaccharides in circulatory hEPO [13]. Large glycan variations can occur between different individuals’ urinary EPO [14]. These observations indicate that N-glycans actively contribute to the modulation of hEPO activities in vivo
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
