人GM－CSF在大肠杆菌中的表达及纯化

Expression and Purification of Human GM CSF in E.coli

通过ＰＣＲ定点突变，对编码人ＧＭ－ＣＳＦ因子成熟蛋白基因的５′端进行改造．将改造的ＧＭ－ＣＳＦ基因克隆入质粒ｐＢＶ２２０，构建成表达质粒ｐＢＶ２２０－ＧＭ－ＣＳＦ，将其转化ＤＨ５ａ感受态菌后，在温度诱导下，ＧＭ－ＣＳＦ非融合基因获得了表达，表达产物以包涵体形式沉积在细胞内，占细胞总蛋白的１３％，并能特异地与抗人ＧＭ－ＣＳＦ单克隆抗体结合．通过对包涵体的提取、裂解，得到变性的ＧＭ－ＣＳＦ，再经疏水柱层析，凝胶过滤，可纯化出ＧＭ－ＣＳＦ，纯度达９９％以上，比活达１．２８×１０７ｕ／ｍｇ。

Six bases mutations were introduced into 5' terminal of mature GM CSF gene by PCR in order to inhance the expression of GM CSF gene in E.coli . The expression product of the modified GM CSF gene in E.coli was accumulated mainly in the form of inclusion bodies and constitute dabout 13% of the total bacterial proteins. Western blotting demonstrated this product could combine specifically with monoclonal antibody against human GM CSF. After solubilization of purified inclusion bodies and renaturation of solubilized polypeptides, the authentic GM CSF with its purity above 99% was obtained by means of hydrophobic chromatography on Phenyl Sepharose CL 4B and gel filtrationon Sephadex G 100. The activity, measured by viable cell MTT staining method, was 1 28×10 7 u/mg. The purified product could maintain the growth of TF 1 cells which dependes on human GM CSF.