Secretion expression and activity assay of a novel fusion protein of thrombopoietin and interleukin-6 in Pichia pastoris.
Secretion expression and activity assay of a novel fusion protein of thrombopoietin and interleukin-6 in Pichia pastoris.
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DOI：
10.1093/jb/mvm111
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发表时间：
2007-07
影响因子：
2.7
通讯作者：
H. Zhiyi;Lai Wenshan;Zhang Wenze;Deng Ning;Z. Chi;Y. Kenan;W. Ping;Wang Qianqian;Z. Qing
中科院分区：
文献类型：
--
作者：
H. Zhiyi;Lai Wenshan;Zhang Wenze;Deng Ning;Z. Chi;Y. Kenan;W. Ping;Wang Qianqian;Z. Qing
血小板生成素（TPO）是巨核细胞活动和血小板生成的重要造血因子。白细胞介素6（IL-6）可协同刺激TPO依赖的巨核细胞集落形成单位（CFU-Meg）的生长，这可能是TPO缺陷或c-mpl缺陷动物中残余血小板形成的原因。在本报告中，我们展示了一个高水平表达系统的发展，以产生一个78 kDa的人IL-6/TPO融合蛋白（命名为ZH 646）。通过构建表达载体pPICZ α-A-IL-6-linker-TPO，获得重组酵母GS115，然后从烧瓶中的酵母培养物上清液中以30 mg/l的产量有效分泌到培养基中。ZH 646经DEAE-Sephacel柱层析和Mono Q柱两步纯化。活性测定结果表明，ZH 646能显著促进Dami细胞CFU-Meg的形成和增殖，并呈剂量依赖性。此外，ZH 646在正常小鼠中也显示出血小板生成作用，并且能够促进骨髓抑制小鼠后正常血小板计数的恢复。这些结果表明，ZH 646是一种新的蛋白质，其活性比单独的TPO或IL-6强得多。因此，ZH 646具有与血小板产生相关的广谱巨核细胞生成活性。
Thrombopoietin (TPO) is an important haematopoietic factor in megakaryocytic activities as well as in platelet production. Interleukin 6 (IL-6) can co-stimulate TPO-dependent formation of colony forming unit of megakaryocyte (CFU-Meg) growth which could be responsible for residual platelet formation in TPO-deficient or c-mpl-deficient animals. In this report, we demonstrated the development of a high-level expression system to produce a 78-kDa human fusion protein IL-6/TPO (named ZH646). This was achieved by constructing the expression vector pPICZalpha-A-IL-6-linker-TPO, and obtained the recombinant yeast GS115, which then efficiently secreted into a medium with a yield of 30 mg/l from the supernatant of the yeast culture in flask. ZH646 was then purified using two steps via DEAE-Sephacel chromatography and Mono Q columns. Activity assay showed that ZH646 could significantly stimulate the formation of CFU-Meg and the proliferation of Dami cells in vitro in a dose-dependent manner. In addition, ZH646 also showed thrombopoietic effect in normal mice, and the ability to enhance recovery of normal platelet counts after myelosuppression mice. These results suggested that ZH646 is a novel protein, and its activities are much stronger than that of TPO or IL-6 alone. ZH646 therefore has a broad spectrum of megakaryopoiesis activity associated with platelet production.