Expression of functionally active sialylated human erythropoietin in plants.
Expression of functionally active sialylated human erythropoietin in plants.
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DOI：
10.1002/biot.201200363
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发表时间：
2013-03
影响因子：
4.7
通讯作者：
Steinkellner, Herta
中科院分区：
文献类型：
--
作者：
Jez, Jakub;Castilho, Alexandra;Grass, Josephine;Vorauer-Uhl, Karola;Sterovsky, Thomas;Altmann, Friedrich;Steinkellner, Herta
重组人促红细胞生成素(RhEPO)是一种糖类激素，是目前主要的生物制药产品之一。由于重组人促红细胞生成素高度复杂的糖基化模式是药物疗效的主要决定因素，目前重组人促红细胞生成素的生产仅限于哺乳动物细胞表达系统。在这里，我们评估了植物产生不同形式的重组人促红细胞生成素的能力。将构建的表达载体导入本氏烟草中，并通过病毒表达系统进行瞬时表达。重组人促红细胞生成素的表达水平达到85 mg/kg鲜叶。此外，重组人促红细胞生成素与植物蛋白唾液酸化所需的6个哺乳动物基因的共表达导致了以双唾液酸化N-糖链(NANA)修饰的重组人促红细胞生成素的合成，双唾液酸化的N-糖链是贫血患者循环中hEPO最丰富的糖形式。一种新建立的与hEPO融合的多肽标签(ELDKWA)特别适合于基于免疫亲和力的重组激素的纯化。随后的凝集素层析使专门的唾液酸化的重组人促红细胞生成素得以浓缩。通过基于细胞的受体结合分析，所有植物来源的糖类都显示出很高的生物活性。携带基本均一的糖基化图谱(GnGnXF、GnGn和NaNA)的重组人EPO的产生将有助于进一步研究具有潜在医学应用意义的功能。
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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影响因子：
4.3
作者：
Castilho, Alexandra;Gattinger, Pia;Steinkellner, Herta
通讯作者：
Steinkellner, Herta
影响因子：
50.5
作者：
Bendandi, M.;Marillonnet, S.;Gleba, Y.
通讯作者：
Gleba, Y.
影响因子：
2.9
作者：
Darling, RJ;Kuchibhotla, U;Beals, JM
通讯作者：
Beals, JM
DOI：
10.1073/pnas.0606631103
发表时间：
2006-10-03
影响因子：
11.1
作者：
Giritch, Anatoli;Marillonnet, Sylvestre;Gleba, Yuri
通讯作者：
Gleba, Yuri
影响因子：
13.8
作者：
Conley, Andrew J.;Zhu, Hong;Menassa, Rima
通讯作者：
Menassa, Rima