Expression, purification and characterization of a functional extracellular domain of porcine Fc gamma RII

文献类型： 外文期刊

第一作者： Tian, Xiaohui

作者： Tian, Xiaohui;Qiao, Songlin;Zhang, Gaiping;Xi, Jun;Liu, Yunchao;Tian, Xiaohui;Wang, Aiping;Li, Xuewu;Xu, Yuqing

作者机构：

关键词： poFc gamma RII;Extracellular domain;Expression;Rapid dilution refolding;Polyclonal antibody

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

ISSN： 1046-5928

年卷期： 2009 年 68 卷 1 期

页码：

收录情况： SCI

摘要： Fc gamma Rs are involved in regulating a multitude of innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches. In this report, we describe a simple method for the production of a large quantity of recombinant porcine Fc gamma RII. The extracellular domain of the porcine Fc gamma RII (poFc gamma RII) gene was constructed and cloned into the Escherichia coli expression vector pET-28a. The recombinant protein was expressed at high level in E. coli BL21 (DE3) and existed mainly as inclusion bodies. The inclusion bodies were solubilized in 6 M guanidine hydrochloride and purified by Ni-chelation, and refolded by rapid dilution. After purification and renaturation, the recombinant soluble protein (rsFc gamma RII) coated on high-binding ELISA plates, showed concentration dependent binding of porcine IgG and the binding of porcine IgG to the surface bound rsFc gamma RII was inhibited in a dose-dependent manner by soluble rsFc gamma RII itself. Then by the inhibition assay we evaluated the effectiveness of the rsFc gamma RII in inhibiting the IgG binding to the whole molecule of poFc gamma RII expressed on the Marc-145 cell surface, the rsFc gamma RII inhibited the binding of porcine IgG to the transfected Marc-145 cell's surface, with an IC50 value of 0.87 mu M, demonstrating that rsFc gamma RII manifests the similar specificity as native poFc gamma RII. The method for highly efficient production of biologically active poFc gamma RII may be employed for both basic research and potential clinical applications. (C) 2009 Elsevier Inc. All rights reserved.

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