Kittur Farooqahmed S, Arthur Elena, Nguyen Maikhanh, Hung Chiu-Yueh, Sane David C, Xie Jiahua
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC 27707, USA.
Carilion Clinic and Virginia Tech Carilion School of Medicine, Roanoke, VA 24014, USA.
Int J Biol Macromol. 2015 Jan;72:1111-6. doi: 10.1016/j.ijbiomac.2014.10.033. Epub 2014 Oct 25.
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 (rhuEPO(M)) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). 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-rhuEPO(P) 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-rhuEPO(P). Using this process, up to 31% of the asialo-rhuEPO(P) could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPO(P) 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.
去唾液酸促红细胞生成素（去唾液酸 - EPO）是人类糖蛋白激素促红细胞生成素（EPO）的去唾液酸化形式，据报道在器官损伤的动物模型中具有神经保护、心脏保护和肾脏保护作用。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（rhuEPO(M)）生产去唾液酸 - EPO的方法在商业上不可行，我们和其他人使用基于植物的表达系统来生产重组人去唾液酸促红细胞生成素（去唾液酸 - rhuEPO(P)）。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，这阻碍了对其组织保护作用的研究以及将其转化为临床应用。在本研究中，开发了一种从转基因烟草叶组织中两步纯化去唾液酸 - rhuEPO(P)的方法：基于其高pI（8.75）的离子交换色谱法将其与酸性植物蛋白分离，以及免疫亲和色谱法以获得纯的去唾液酸 - rhuEPO(P)。使用该方法，高达31%的去唾液酸 - rhuEPO(P)可以从植物提取物中回收至接近均一性。这项工作表明，在烟草植物中表达的去唾液酸 - rhuEPO(P)可以使用最少的步骤以高产率和高纯度进行纯化，这可能适用于扩大规模。此外，离子交换色谱步骤与使用蛋白质特异性抗体柱一起可用于从转基因叶组织中纯化多种碱性重组蛋白。
