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首页> 外文期刊>Microbial Cell Factories >Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization
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Human granulocyte colony stimulating factor (hG-CSF): cloning, overexpression, purification and characterization

机译：人粒细胞集落刺激因子（hG-CSF）：克隆，过表达，纯化和鉴定

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Background Biopharmaceutical drugs are mainly recombinant proteins produced by biotechnological tools. The patents of many biopharmaceuticals have expired, and biosimilars are thus currently being developed. Human granulocyte colony stimulating factor (hG-CSF) is a hematopoietic cytokine that acts on cells of the neutrophil lineage causing proliferation and differentiation of committed precursor cells and activation of mature neutrophils. Recombinant hG-CSF has been produced in genetically engineered Escherichia coli (Filgrastim) and successfully used to treat cancer patients suffering from chemotherapy-induced neutropenia. Filgrastim is a 175 amino acid protein, containing an extra N-terminal methionine, which is needed for expression in E. coli . Here we describe a simple and low-cost process that is amenable to scaling-up for the production and purification of homogeneous and active recombinant hG-CSF expressed in E. coli cells. Results Here we describe cloning of the human granulocyte colony-stimulating factor coding DNA sequence, protein expression in E. coli BL21(DE3) host cells in the absence of isopropyl-β-D-thiogalactopyranoside (IPTG) induction, efficient isolation and solubilization of inclusion bodies by a multi-step washing procedure, and a purification protocol using a single cationic exchange column. Characterization of homogeneous rhG-CSF by size exclusion and reverse phase chromatography showed similar yields to the standard. The immunoassay and N-terminal sequencing confirmed the identity of rhG-CSF. The biological activity assay, in vivo , showed an equivalent biological effect (109.4%) to the standard reference rhG-CSF. The homogeneous rhG-CSF protein yield was 3.2 mg of bioactive protein per liter of cell culture. Conclusion The recombinant protein expression in the absence of IPTG induction is advantageous since cost is reduced, and the protein purification protocol using a single chromatographic step should reduce cost even further for large scale production. The physicochemical, immunological and biological analyses showed that this protocol can be useful to develop therapeutic bioproducts. In summary, the combination of different experimental strategies presented here allowed an efficient and cost-effective protocol for rhG-CSF production. These data may be of interest to biopharmaceutical companies interested in developing biosimilars and healthcare community.
机译：背景技术生物药物主要是通过生物技术工具产生的重组蛋白。许多生物制药的专利已经过期，因此目前正在开发生物仿制药。人粒细胞集落刺激因子（hG-CSF）是一种造血细胞因子，作用于嗜中性粒细胞谱系的细胞，引起定型前体细胞的增殖和分化以及成熟嗜中性粒细胞的活化。重组hG-CSF已在基因工程大肠杆菌（Filgrastim）中生产，并成功用于治疗患有化学疗法诱发的中性粒细胞减少症的癌症患者。 Filgrastim是一种175个氨基酸的蛋白质，含有一个额外的N末端甲硫氨酸，这是在大肠杆菌中表达所必需的。在这里，我们描述了一个简单且低成本的过程，该过程适合扩大规模生产和纯化在大肠杆菌细胞中表达的均质和活性重组hG-CSF。结果在这里我们描述了在没有异丙基-β-D-硫代半乳糖吡喃糖苷（IPTG）诱导，有效分离和增溶的情况下克隆人粒细胞集落刺激因子编码DNA序列，大肠杆菌BL21（DE3）宿主细胞中蛋白质表达的克隆。通过多步洗涤程序和使用单个阳离子交换柱的纯化方案来制备包涵体。通过尺寸排阻和反相色谱对均相的rhG-CSF进行表征显示出与标准品相似的收率。免疫测定和N末端测序证实了rhG-CSF的身份。体内生物活性测定显示出与标准参考rhG-CSF相当的生物效应（109.4％）。均一的rhG-CSF蛋白产量为每升细胞培养物3.2 mg生物活性蛋白。结论在不使用IPTG诱导的情况下重组蛋白表达是有利的，因为它可以降低成本，并且使用单一色谱步骤的蛋白纯化方案应进一步降低成本，以进行大规模生产。物理化学，免疫学和生物学分析表明，该方案可用于开发治疗性生物产品。总而言之，这里提出的不同实验策略的结合为rhG-CSF生产提供了一种有效且具有成本效益的方案。这些数据可能对有兴趣发展生物仿制药和医疗保健社区的生物制药公司感兴趣。

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