Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles.
Glycoengineering tobacco plants to stably express recombinant human erythropoietin with different N-glycan profiles.
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DOI：
10.1016/j.ijbiomac.2020.04.199
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发表时间：
2020-08-15
影响因子：
8.2
通讯作者：
Xie J
中科院分区：
文献类型：
--
作者：
Kittur FS;Hung CY;Zhu C;Shajahan A;Azadi P;Thomas MD;Pearce JL;Gruber C;Kallolimath S;Xie J
基于植物的表达系统可用于生产具有许多潜在优势的生物制药，但植物不能直接用于表达功能性人类糖蛋白，因为它们的糖基化能力与哺乳动物不同。为了利用基于植物的表达来生产重组人促红细胞生成素 (rhuEPO)，我们通过将七到八个哺乳动物基因（包括目标人 EPO）稳定引入烟草基因组中，对烟草植物进行了糖基化改造，以产生 β1,4-半乳糖基化、平分 N-乙酰氨基葡萄糖 (GlcNAc) 和唾液酸化的能力。野生型人 β1,4-半乳糖基转移酶基因 (GalT) 或嵌合 GalT 基因 (ST/GalT)（其中 GalT 细胞质跨膜干区 (CTS) 被来自大鼠 2,6-唾液酸转移酶 (ST) 的 CTS 取代）共表达，以产生带有 β1,4-半乳糖延伸的 N-聚糖链的 rhuEPO，并比较它们的β1,4-半乳糖基化效率。哺乳动物UDP-N-乙酰氨基葡萄糖2-差向异构酶/N-乙酰甘露糖胺激酶基因（GNE）、N-乙酰神经氨酸磷酸合酶基因（NANS）、CMP-N-乙酰神经氨酸合成酶基因（CMAS）、CMP-唾液酸转运蛋白基因（CST）和α-2,6-唾液酸转移酶基因（ST）共表达，以在植物中构建唾液酸化能力。此外，编码N-乙酰葡糖胺基转移酶III（GnTIII）的人MGAT3也被共表达以产生具有平分GlcNAc的N-聚糖链。我们的PCR和RT-PCR结果表明，上述转基因不仅整合到烟草基因组中，而且正确转录。研究发现 GalT 和 ST/GalT 均可将 β1,4-半乳糖残基添加到 N-聚糖链上，但后者效率更高。此外，共表达 MGAT3 可以产生二等分的 GlcNAc。然而，我们目前的努力确实产生了唾液酸化能力。表达 EPO 和 ST/GalT 的转基因植物可用于生产具有高比例 β1,4-半乳糖延伸 N-聚糖链的 rhuEPO，用于组织保护目的。
Plant-based expression system can be used to produce biopharmaceuticals with many potential advantages, but plants cannot be directly used to express functional human glycoproteins because of their differences in glycosylation abilities from mammals. To exploit a plant-based expression for producing recombinant human erythropoietin (rhuEPO), we glycoengineered tobacco plants by stably introducing seven to eight mammalian genes including a target human EPO into tobacco genome in order to generate capacities for β1,4-galactosylation, bisecting N-acetylglucosamine (GlcNAc) and sialylation. Wild type human β1,4-galactosyltransferase gene (GalT) or a chimeric GalT gene (ST/GalT) with GalT cytoplasmic-transmembrane-stem region (CTS) replaced by the CTS from the rat 2,6-sialyltransferase (ST) was co-expressed to produce rhuEPO bearing β1,4-galactose-extended N-glycan chains as well as to compare their β1,4-galactosylation efficiencies. Mammalian UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE), N-acetylneuraminic acid phosphate synthase gene (NANS), CMP-N-acetylneuraminic acid synthetase gene (CMAS), CMP-sialic acid transporter gene (CST) and α−2,6-sialyltransferase gene (ST) were co-expressed to build sialylation capacity in plants. In addition, the human MGAT3 encoding N-acetylglucosaminyltransferase III (GnTIII) was also co-expressed to produce N-glycan chains with bisecting GlcNAc. Our PCR and RT-PCR results demonstrated that the above transgenes were not only incorporated into tobacco genome but also properly transcribed. Both GalT and ST/GalT were found to add β1,4-galactose residues to the N-glycan chains, but the latter was more efficient. Furthermore, co-expressing MGAT3 could generate bisected GlcNAc. However, our current efforts did result in generating sialylation capacity. Created transgenic plants expressing EPO and ST/GalT could be used to produce rhuEPO with high proportion of β1,4-galactose-extended N-glycan chains for tissue protective purposes.
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影响因子：
4.6
作者：
Greif, Franklin;Ben-Ari, Ziv;Hochhauser, Edith
通讯作者：
Hochhauser, Edith
DOI：
10.1073/pnas.1031753100
发表时间：
2003-05-27
影响因子：
11.1
作者：
Erbayraktar, S;Grasso, G;Brines, M
通讯作者：
Brines, M
影响因子：
4.7
作者：
Jez, Jakub;Castilho, Alexandra;Grass, Josephine;Vorauer-Uhl, Karola;Sterovsky, Thomas;Altmann, Friedrich;Steinkellner, Herta
通讯作者：
Steinkellner, Herta
影响因子：
4.3
作者：
Castilho, Alexandra;Gattinger, Pia;Steinkellner, Herta
通讯作者：
Steinkellner, Herta
影响因子：
13.8
作者：
Frey, Alexander D.;Karg, Saskia R.;Kallio, Pauli T.
通讯作者：
Kallio, Pauli T.