文献类型： 外文期刊

作者： Zang, YH ¹ ; Zhang, X ² ; Yuan, DW ² ; Zhang, YM ² ; Zhu, J ² ; Lu, HQ ² ; Chang, C ³ ; Qin, JC;

作者机构： 1.Nanjing Univ, Sch Life Sci, State Key Lab Pharamceut Biotechnol, Nanjing 210093, Peoples R China

2.Nanjing Univ, Sch Life Sci, State Key Lab Pharamceut Biotechnol, Nanjing 210093, Peoples R China; Jiangsu Acad Agr Sci, Inst Agrobiol Genet & Physiol, Nanjing 210014, Peoples R China

3.Nanjin

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 ）

ISSN：

年卷期：

页码：

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.