重组人Flt3配体在大肠杆菌中的高效表达和生物学活性的鉴定

Expression of recombinant fms-like tyrosine kinase 3 ligand in E.coli and identification of its bioactivity

根据天然的人Flt3配体(Flt3ligand,FL)基因和遵循适合大肠杆菌BL21(DE3)表达体系表达条件及不改变FL天然蛋白氨基酸序列的原则,对其进行改造,所获得的FL功能片段(FL134)克隆入PET30a表达载体,在C端融合了6个His,继而在大肠杆菌中诱导表达。结果表明重组蛋白rhFL134以包涵体形式在大肠杆菌(BL21)中可得到较高水平的表达。通过蛋白的变性、复性和HiTrapTM亲和层析获得了纯度达92%以上的rhFL134重组蛋白。体外的活性实验显示,重组蛋白rhFL134具有显著的促小鼠骨髓细胞的集落形成和扩增人脐带血中的CD34+细胞的生物学活性。

Flt3 (fms-like tyrosine kinase 3) ligand (FL) is a most potent hematopoietic cytokine that affects the proliferation and differentiation of progenitor and stem cells both in vivo and in vitro by binding with Flt3 receptor. A synergistic effect with a wide range of colony stimulating factors (CSF), interleukins, and thrombopoietin was observed to promote the production of the lymphoid cells including T, B, NK and dendritic cells. A series of studies have showed its considerable value in anti-cancer and anti-virus immunotherapy. In the present study, some site-specific gene mutations of human FL for the optimal E.coli BL21 (DE3) expression were manipulated with the unchanged amino acid sequence. Then the artificial FL gene encoding function domain was amplified and cloned into expression plasmid PET30a. The protein rhFL134 with His-tag at the C-terminal was effectively expressed in E.coli BL21 (DE3) as inclusion bodies and a denaturation and refolding procedure was performed to obtain the soluble rhFL134. The fusion protein rhFL134 was conveniently purified using HiTrap?affinity column with more than 92% purity. Furthermore, the in vitro bioassays demonstrated that rhFL134 could effectively promote the colony formation of mice bone marrow progenitor cells and augmented the production of CD34+ cells from umbilical blood mononuclear cells in vitro.