Su Lin, Chen Song-Sen, Yang Ke-Gong, Liu Chang-Zheng, Zhang Yan-Li, Liang Zhi-Quan
National Laboratory of Medical Molecular Biology, Department of Biochemistry and Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, China.
Protein Expr Purif. 2006 Jun;47(2):477-82. doi: 10.1016/j.pep.2005.11.002. Epub 2005 Nov 28.
Stem cell factor (SCF) and erythropoietin are essential for normal erythropoiesis and induce proliferation and differentiation synergistically for erythroid progenitor cells. Here, we report our work on construction of SCF/erythropoietin mimetic peptide (EMP) fusion protein gene, in which human SCF cDNA (1-165aa) and EMP sequence (20aa) were connected using a short (GGGGS) or long (GGGGSGGGGGS) linker sequence. The SCF/EMP gene was cloned into the pBV220 vector and expressed in the Escherichia coli DH5alpha strain. The expression level of the fusion protein was about 30% of total cell protein. The resulting inclusion bodies were solubilized with 8 M urea, followed by dilution refolding. The renatured protein was subsequently purified by Q-Sepharose FF column. The final product was >95% pure by SDS-PAGE and the yield of fusion protein was about 40 mg/L of culture. UT-7 cell proliferation and human cord blood cell colony-forming assays showed that the fusion proteins exhibited more potent activity than recombinant human SCF, suggesting a new strategy to enhance biological activities of growth factors.
干细胞因子（SCF）和促红细胞生成素对于正常红细胞生成至关重要，并且协同诱导红系祖细胞增殖和分化。在此，我们报告了关于构建SCF/促红细胞生成素模拟肽（EMP）融合蛋白基因的工作，其中人SCF cDNA（1 - 165aa）和EMP序列（20aa）使用短（GGGGS）或长（GGGGSGGGGGS）连接子序列连接。将SCF/EMP基因克隆到pBV220载体中，并在大肠杆菌DH5α菌株中表达。融合蛋白的表达水平约占总细胞蛋白的30%。所得包涵体用8 M尿素溶解，随后进行稀释复性。复性后的蛋白随后通过Q - Sepharose FF柱纯化。通过SDS - PAGE分析，最终产物纯度>95%，融合蛋白产量约为40 mg/L培养物。UT - 7细胞增殖和人脐血细胞集落形成试验表明，融合蛋白表现出比重组人SCF更强的活性，提示了一种增强生长因子生物学活性的新策略。
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