Expression, purification, and characterization of a novel recombinant fusion protein, rhTPO/SCF, in Escherichia coli

文献类型: 外文期刊

第一作者: Zang, YH

作者: Zang, YH;Zhang, X;Yuan, DW;Zhang, YM;Zhu, J;Lu, HQ;Chang, C;Qin, JC

作者机构:

关键词: megakaryopoiesis;thrombopoietin;stem cell factor;fusion protein;chaperone;STEM-CELL FACTOR;C-MPL LIGAND;COLONY-STIMULATING FACTOR;MEGAKARYOCYTIC PROGENITORS;INSECT CELLS;IN-VITRO;EX-VIVO;THROMBOPOIETIN;EXPANSION;DIFFERENTIATION

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

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收录情况: SCI

摘要: Thrombopoietin (TPO) is the principal regulatory cytokine of megakaryopoiesis and thrombopoiesis and promotes all aspects of megakaryocyte development. Stem cell factor (SCF) is mainly a pleiotropic cytokine acting on hematopoiesis by promoting the survival and proliferation of hematopoietic stem cells and has a potent synergistic effect on megakaryopoiesis in the presence of TPO. Here, we report the construction, expression, and purification of a novel recombinant human thrombopoietin/stem cell factor (rhTPO/SCF) fusion protein, which consists of a truncated human thrombopoietin (1-157 a.a.) plus a truncated human stem cell factor (1-145 a.a.), linked by a peptide (GGGGSPGGSGGGGSGG). The TPO/SCF gene was cloned into the Escherichia coli expression vector pET28a and expressed in BL21(DE3) strain. The rhTPO/SCF constituted up to 6% of the total bacterial protein. Co-expression with E. coli chaperones, Trigger Factor (TF) and GroES/GroEL, and lowering cultivation temperature cooperatively improved the solubility of expressed rhTPO/SCF, resulting in about fourfold increase in the yield soluble rhTPO/SCF. The rhTPO/SCF was purified to homogeneity using anion exchange followed by metal affinity chromatography. Western blot analysis confirmed the identity of the purified protein. rhTPO/SCF stimulated a dose-dependent cell proliferation in both TF1 and Mo7e cell lines. (c) 2005 Elsevier Inc. All rights reserved.

分类号: Q51

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