hK1-L-Fc融合蛋白在CHO细胞中的表达及其活性研究

为进一步改造重组人激肽释放酶1(hK1),以期提高其生物活性,制备了通过柔性接头相连接的重组人激肽释放酶1-L-IgG1 Fc融合蛋白(hK1-L-Fc)。采用重叠延伸PCR技术构建hK1-L-Fc融合基因,克隆至表达载体pcDNA3.1,在中国仓鼠卵巢细胞(CHO-S)中表达。利用Protein A 亲和层析柱纯化融合蛋白,SDS-PAGE、Western blotting、飞行时间质谱(MALDI-TOF-MS)、HPLC检测表达产物,底物法检测融合蛋白的体外活性。结果显示:成功构建pcDNA3.1-hK1-L-Fc重组表达载体;获得稳定表达融合蛋白的细胞株;无血清悬浮批式培养的表达量在0.7 mg/L以上;纯化的蛋白其纯度在95%以上,分子量约60 kDa;活性检测显示其比活性在9.2 U/mg以上,较hK1-Fc蛋白提高了18%以上。

Expression of hK1-L-Fc Fusion Protein in CHO Cells and Analysis of its Bioactivity

To further modify the recombinant human kallikrein 1(hK1) for higher activity,new form of recombinant human kallikrein 1(hK1-L-Fc) was constructed by fusion of the human kallikrein1 gene and the coding sequences for Fc fragment of human IgG1 with a flexible linker peptide.The fusion gene hK1-L-Fc was constructed by PCR,then inserted into expression vector pcDNA3.1,and expressed in Chinese Hamster Ovary cells.The chimeric protein was purified by Protein A affinity chromatography,and further analyzed by SDS-P...