人干细胞因子受体膜外区1～3免疫球蛋白样结构域的表达、纯化及活性

目的在原核和真核细胞中表达人干细胞因子受体(c-Kit)配体结合区膜外N端1~3免疫球蛋白样结构域(c-Kit/Ig1~3),研究其配体结合活性。方法采用重叠延伸PCR定点诱变法合成c-Kit/Ig1~3双突变片段(c-Kit/Ig1~3DM),构建pET16b-c-Kit/Ig1~3DM表达质粒,转化E·coliBL21(DE3)诱导表达,稀释复性,镍柱纯化。将C端添加组氨酸标签的c-Kit/Ig1~3基因克隆入真核表达载体pEAK12中,转染HEK293ET细胞构建c-kit/Ig1~3高效表达细胞株,从细胞培养上清中使用镍金属螯合柱收集纯化蛋白。His-tag pull-down和酶联免疫结合实验检测融合蛋白配体结合活性。结果在大肠杆菌中c-Kit/Ig1~3DM以包涵体形式表达,复性后配体结合活性很低。在HEK293ET细胞中筛选到2个高效表达c-Kit/Ig1~3的细胞株,上清表达量为2μg/ml,融合蛋白相对分子质量为58000;受体配体结合实验显示真核表达的c-Kit/Ig1~3有明显的干细胞因子特异结合活性,解离常数Kd值为9·39nmol/L。结论获得了具有较高配体结合活性的c-Kit/Ig1~3融合蛋白。

Expression, purification, and characterization of the first three immunoglobulin-like domains of human stem cell factor receptor

OBJECTIVE: To express the first three immunoglobulin-like domains of human stem cell factor receptor (c-Kit/Ig1-3) in E. coli and HEK293 ET cells and study their binding activity for stem cell factor (SCF). METHODS: In prokaryotic expression system, a double mutant form of c-Kit /Ig1-3 (c-Kit /Ig1-3(DM) was produced by overlap PCR and cloned into pET16b. The recombinant protein was expressed in E. coli BL21 (DE3) and refolded by dilution. In eukaryotic expression system, the gene of c-Kit/Igl13 with eight histidine segments was cloned into pEAK12 and the recombinant plasmid was transfected into HEK293 ET cells. The fusion protein was harvested from the growth medium and purified on Ni-NTA agarose column. The recombinant protein was tested for the receptor binding activity with his-tag pull-down and enzyme-linked immunosorbent binding assay. RESULTS: In E. coli c-Kit /Ig1-3(DM) as produced as an inclusion body and showed low binding activity for SCF after refolding. Two HEK293 ET cell clones that express high levels of c-Kit/Ig1-3 were produced and each clone secreted 2p micro/ml of recombinant protein, whose relative molecular mass was about 58,000. Eukaryotically expressed c-Kit/Ig1-3 had specific binding activity for SCF, and the dissociation constant (Kd) was 9.39 nmol/L. CONCLUSION: c-Kit/Ig1-3 with high receptor binding activity is successfully produced in HEK293 ET cells.