Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase.
Soluble prokaryotic overexpression and purification of bioactive human granulocyte colony-stimulating factor by maltose binding protein and protein disulfide isomerase.
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麦芽糖结合蛋白和蛋白质二硫化物异构酶对生物活性人粒细胞刺激因子的生物活性人粒细胞群刺激因子的纯化。
DOI：
10.1371/journal.pone.0089906
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Choe H
中科院分区：
文献类型：
--
作者：
Do BH;Ryu HB;Hoang P;Koo BK;Choe H
人粒细胞集落刺激因子（hGCSF）是一种促进嗜中性粒细胞生成的细胞因子，是一种治疗中性粒细胞减少症的有效药物。利用E.大肠杆菌是具有挑战性的，因为激素倾向于聚集并形成包涵体。本研究检测了七种不同的N-末端融合标签在E.杆菌四种标签蛋白，即麦芽糖结合蛋白（MBP）、N-利用物质蛋白A、蛋白质二硫键异构酶（PDI）和PDI的b 'a'结构域（PDIb 'a'），在正常条件下增加hGCSF的溶解度。将表达温度从30°C降低至18°C也增加了硫氧还蛋白标记和谷胱甘肽S-转移酶标记的hGCSF的溶解度。相比之下，六组氨酸标记的hGCSF在两个温度下都不溶。使用简单的常规层析方法从过表达的PDIb“a”-hGCSF和MBP-hGCSF蛋白质中纯化hGCSF。从500 mL大肠杆菌培养物中总共获得11.3 mg或10.2 mg纯hGCSF。分别表达PDIb“a”-hGCSF和MBP-hGCSF的大肠杆菌。SDS-PAGE分析和银染色证实分离的hGCSF蛋白质具有高纯度，并且内毒素水平小于0.05 EU/µg蛋白质。随后，使用小鼠M-NFS-60骨髓性白血病细胞系证实了纯化的hGCSF蛋白的生物活性与市售hGCSF相似。从MBP-hGCSF和PDIb 'a'-hGCSF纯化的hGCSF蛋白的细胞增殖剂量-反应曲线的EC 50分别为2.83±0.31 pM和3.38±0.41 pM。总之，本研究描述了一种有效的方法，可溶性表达和纯化的生物活性hGCSF在E。杆菌
Human granulocyte colony-stimulating factor (hGCSF), a neutrophil-promoting cytokine, is an effective therapeutic agent for neutropenia patients who have undergone several cancer treatments. Efficient production of hGCSF using E. coli is challenging because the hormone tends to aggregate and forms inclusion bodies. This study examined the ability of seven different N-terminal fusion tags to increase expression of soluble hGCSF in E. coli. Four tag proteins, namely maltose-binding protein (MBP), N-utilization substance protein A, protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), increased the solubility of hGCSF under normal conditions. Lowering the expression temperature from 30°C to 18°C also increased the solubility of thioredoxin-tagged and glutathione S-transferase-tagged hGCSF. By contrast, hexahistidine-tagged hGCSF was insoluble at both temperatures. Simple conventional chromatographic methods were used to purify hGCSF from the overexpressed PDIb'a'-hGCSF and MBP-hGCSF proteins. In total, 11.3 mg or 10.2 mg of pure hGCSF were obtained from 500 mL cultures of E. coli expressing PDIb'a'-hGCSF or MBP-hGCSF, respectively. SDS-PAGE analysis and silver staining confirmed high purity of the isolated hGCSF proteins, and the endotoxin levels were less than 0.05 EU/µg of protein. Subsequently, the bioactivity of the purified hGCSF proteins similar to that of the commercially available hGCSF was confirmed using the mouse M-NFS-60 myelogenous leukemia cell line. The EC50s of the cell proliferation dose-response curves for hGCSF proteins purified from MBP-hGCSF and PDIb'a'-hGCSF were 2.83±0.31 pM, and 3.38±0.41 pM, respectively. In summary, this study describes an efficient method for the soluble overexpression and purification of bioactive hGCSF in E. coli.
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2.9
作者：
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通讯作者：
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