Journal
BIOTECHNOLOGY JOURNAL
Volume 8, Issue 3, Pages 371-382Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201200363
Keywords
Erythropoietin; Glycoengineering; Plants; Purification; Sialylation
Funding
- Laura Bassi Centres of Expertise [822757]
- Fonds zur Forderung der wissenschaftlichen Forschung FWF [L575-B13]
- Austrian Science Fund (FWF) [L575] Funding Source: Austrian Science Fund (FWF)
- Austrian Science Fund (FWF) [L 575] Funding Source: researchfish
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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.
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