Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli
Expression and Purification of Recombinant Human Granulocyte Colony-Stimulating Factor in Fed-Batch Culture of Escherichia coli
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DOI：
10.1007/s12010-013-0708-y
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发表时间：
2014-03-01
影响因子：
3
通讯作者：
Oh, Jae-Wook
中科院分区：
文献类型：
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作者：
Kim, Chang-Kyu;Choi, Jun-Ha;Oh, Jae-Wook
粒细胞集落刺激因子（G-CSF）是一种细胞因子，在造血细胞中具有多种作用，如调节增殖和分化。在这里，我们描述了补料分批培养，复性和纯化rhG-CSF。测试尿素或肌氨酸用于增溶包涵体（IB）的适用性。据观察，尿素是更有效的溶解和折叠的IB比肌氨酸。pH5.5沉淀法可提高rhG-CSF的纯度和纯化过程中rhG-CSF异构体的去除率。纯化后的rhG-CSF纯度为99%，每升培养液中蛋白含量为0.5g。我们的重组蛋白纯化的协议，使用离子交换色谱和半制备高效液相色谱的pH沉淀复性溶液可能是有益的生物制药的工业规模生产。
Granulocyte colony-stimulating factor (G-CSF) is a cytokine that has multiple roles in hematopoietic cells such as the regulation of proliferation and differentiation. Here, we describe fed-batch culture, refolding, and purification of rhG-CSF. The suitability of urea or sarcosine for solubilizing inclusion bodies (IBs) was tested. It was observed that urea is more efficient for solubilizing and refolding IBs than sarcosine is. The purity of rhG-CSF and the removal percentage of the rhG-CSF isoforms during purification were increased by pH 5.5 precipitation. The purity and the yield of purified rhG-CSF were 99 % and 0.5 g of protein per liter culture broth, respectively. Our protocols of recombinant protein purification using ion exchange chromatography and semipreparative high performance liquid chromatography of pH-precipitated refolded solution may be informative to the industrial scale production of biopharmaceuticals.