Expression, refolding, and characterization of recombinant thrombopoietin/stem cell factor fusion protein in Escherichia coli

文献类型： 外文期刊

第一作者： Zang, Yuhui

作者： Zang, Yuhui;Zhang, Xu;Jiang, Xiaoling;Li, Haoran;Zhu, Jie;Zhang, Chi;Peng, Wei;Qin, Junchuan

作者机构：

关键词： TPO/SCF;megakaryopoiesis;bacterial expression;inclusion body;refolding;COLONY-STIMULATING FACTOR;GROWTH-RESPONSE;IN-VIVO;INTERLEUKIN-3;HEMATOPOIESIS;LEUKEMIA;IL-3;LINE

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

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收录情况： SCI

摘要： Thrombopoietin/stem cell factor (TPO/SCF) is a novel fusion protein that combines the complementary biological effects of TPO and SCF into a single molecule. In this study, TPO/SCF gene was cloned into pET32a and expressed as a thioredoxin (Trx) fusion protein with a C-terminal 6His-tag in Escherichia coli BL21(DE3) under the control of T7 promoter. Trx-TPO/SCF protein approximately accounted for 20% of the total bacterial proteins and was found to accumulate in inclusion bodies. Inclusion bodies were separated from cellular debris, washed with buffer containing 2 M urea, and solubilized with 8 M urea. The refolding of Trx-TPO/SCF was then carried out by an on-column method. Soluble Trx-TPO/SCF was characterized for its dose-dependent effects on promoting cells proliferation in both TF1 and Mo7e cell lines. rhTPO/SCF was released by thrombin digestion and further purified by Ni2+ affinity chromatography. Western blot analysis confirmed the identities of Trx-TPO/SCF and rhTPO/SCF.

分类号： Q93

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