Kittur Farooqahmed S, Lalgondar Mallikarjun, Hung Chiu-Yueh, Sane David C, Xie Jiahua
Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, 1801 Fayetteville Street, Durham, NC, 27707, USA.
Plant Cell Rep. 2015 Mar;34(3):507-16. doi: 10.1007/s00299-014-1730-4. Epub 2014 Dec 14.
C -terminally fused Strep -tag II is removed from rhuEPO expressed in tobacco plants. The finding suggests that direct fusion of purification tags at the C -terminus of rhuEPO should be avoided. Asialo-erythropoietin (asialo-EPO), a desialylated form of EPO, is a potent tissue-protective agent. Recently, we and others have exploited a low-cost plant-based expression system to produce recombinant human asialo-EPO (asialo-rhuEPO(P)). To facilitate purification from plant extracts, Strep-tag II was engineered at the C-terminus of EPO. Although asialo-rhuEPO(P) was efficiently expressed in transgenic tobacco plants, affinity purification based on Strep -tag II did not result in the recovery of the protein. In this study, we investigated the stability of Strep-tag II tagged asialo-rhuEPO(P) expressed in tobacco plants to understand whether this fused tag is cleaved or inaccessible. Sequencing RT-PCR products confirmed that fused DNA sequences encoding Strep-tag II were properly transcribed, and three-dimensional protein structure model revealed that the tag must be fully accessible. However, Western blot analysis of leaf extracts and purified asialo-rhuEPO(P) revealed that the Strep-tag II was absent on the protein. Additionally, no peptide fragment containing Strep-tag II was identified in the LC-MS/MS analysis of purified protein further supporting that the affinity tag was absent on asialo-rhuEPO(P). However, Strep-tag II was detected on asialo-rhuEPO(P) that was retained in the endoplasmic reticulum, suggesting that the Strep-tag II is removed during protein secretion or extraction. These findings together with recent reports that C-terminally fused Strep-tag II or IgG Fc domain are also removed from EPO in tobacco plants, suggest that its C-terminus may be highly susceptible to proteolysis in tobacco plants. Therefore, direct fusion of purification tags at the C-terminus of EPO should be avoided while expressing it in tobacco plants.
在烟草植物中表达的重组人促红细胞生成素（rhuEPO）上，C末端融合的链霉亲和标签II（Strep-tag II）被去除。这一发现表明，应避免在rhuEPO的C末端直接融合纯化标签。脱唾液酸促红细胞生成素（asialo-EPO），即促红细胞生成素的去唾液酸化形式，是一种有效的组织保护剂。最近，我们和其他人利用低成本的植物表达系统生产重组人脱唾液酸促红细胞生成素（asialo-rhuEPO(P)）。为便于从植物提取物中纯化，在促红细胞生成素的C末端设计了链霉亲和标签II。尽管asialo-rhuEPO(P)在转基因烟草植物中高效表达，但基于链霉亲和标签II的亲和纯化未得到该蛋白。在本研究中，我们研究了在烟草植物中表达的带有链霉亲和标签II的asialo-rhuEPO(P)的稳定性，以了解该融合标签是否被切割或无法接近。对RT-PCR产物测序证实，编码链霉亲和标签II的融合DNA序列被正确转录，三维蛋白质结构模型显示该标签必须完全可及。然而，对叶片提取物和纯化的asialo-rhuEPO(P)进行的蛋白质印迹分析表明，该蛋白上不存在链霉亲和标签II。此外，在纯化蛋白的LC-MS/MS分析中未鉴定到含有链霉亲和标签II的肽段，进一步支持asialo-rhuEPO(P)上不存在亲和标签。然而，在内质网中滞留的asialo-rhuEPO(P)上检测到了链霉亲和标签II，这表明链霉亲和标签II在蛋白质分泌或提取过程中被去除。这些发现与最近的报道一致，即在烟草植物中，C末端融合的链霉亲和标签II或IgG Fc结构域也会从促红细胞生成素上去除，这表明其C末端在烟草植物中可能对蛋白水解高度敏感。因此，在烟草植物中表达促红细胞生成素时，应避免在其C末端直接融合纯化标签。
