Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants.
Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants.
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DOI：
10.1016/j.ijbiomac.2014.10.033
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发表时间：
2015-01
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;Arthur E;Nguyen M;Hung CY;Sane DC;Xie J
脱唾液酸促红细胞生成素（asialo-EPO）是人糖蛋白激素促红细胞生成素（EPO）的去唾液酸化形式，已报道其在器官损伤的动物模型中具有神经保护、心脏保护和肾保护作用。由于目前通过酶促去唾液酸化从哺乳动物细胞制备的重组人EPO（rhuEPOM）生产无唾液酸-EPO的方法在商业上是不可行的，我们和其他人使用基于植物的表达系统来生产重组人无唾液酸-EPO（无唾液酸-rhuEPOP）。尽管在植物中实现了高表达水平，但从植物提取物中纯化它仍然是一个更大的挑战，这阻碍了研究其组织保护作用并将其转化为临床实践。本研究建立了一种从转基因烟草叶片组织中分离纯化去唾液酸rhuEPOP的方法，该方法分为两步：一步是利用去唾液酸rhuEPOP的高pI（8.75），通过离子交换层析将其从酸性植物蛋白中分离出来;使用这个过程中，高达31%的asialo-rhuEPOP可以从植物提取物中回收到接近均匀。这项工作表明，在烟草植物中表达的无唾液酸-rhuEPOP可以用最少的步骤以高产率和纯度纯化，这可能适合于放大。此外，离子交换层析步骤与蛋白特异性抗体柱的使用一起可用于从转基因叶组织中纯化各种各样的碱性重组蛋白。
Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPOM) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPOP). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPOP from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPOP. Using this process, up to 31% of the asialo-rhuEPOP could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPOP expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues.
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影响因子：
2.8
作者：
Hu, YL;Chen, S;Zhang, SQ
通讯作者：
Zhang, SQ
影响因子：
4.3
作者：
Castilho, Alexandra;Gattinger, Pia;Steinkellner, Herta
通讯作者：
Steinkellner, Herta
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：
2.9
作者：
Agnello, D;Bigini, P;Ghezzi, P
通讯作者：
Ghezzi, P