重组人粒细胞-巨噬细胞集落刺激因子包涵体提取、复性和纯化的研究

Extraction of Inclusion Bodies,Renaturation and Purification of Recombinant Human Granulocyte macrophage Colony stimulating Factor(rhGM CSF)

由基因工程大肠杆菌表达的重组人粒细胞巨噬细胞集落刺激因子（ｒｈＧＭＣＳＦ）以包涵体的形式存在于细胞中，通过破菌、洗涤获得包涵体，再经过溶解、凝胶过滤、复性、疏水和离子交换柱层析得到了均一的产品，经高压液相和ＳＤＳＰＡＧＥ电泳测定纯度均大于９８％，ｒｈＧＭＣＳＦ的比活为３２×１０７ＩＵ／ｍｇ，纯化获得的ｒｈＧＭＣＳＦ为一酸性蛋白，等电点约为５２，ＮＨ２末端前２０个氨基酸序列测定结果与文献报道一致。ｒｈＧＭＣＳＦ的ＮＨ２末端不均一，其中带Ｍｅｔ的分子约占５９５％，第一个氨基酸为Ａｌａ的分子约占２４６％，为Ｐｒｏ的分子约占１４６％。纯化过程中蛋白的纯化倍数为３９，生物活性总得率为６９１％，工艺重复性好。

Recombinant human granulocyte macrophage colony stimulating factor(rhGM CSF),produced as inclusion bodies in genetically engineered E.coli cells was purified to homogeneity by extraction and solubilization of inclusion bodies,gel filtration,renaturation,hydrophobic interation and ion exchange chromatographic procedure.The purified rhGM CSF has been obtained the criteria of purity level above 98% examined by SDS PAGE and HPLC,has a specific activity of about 3 2×10 7 u/mg.rhGM CSF is an acidic protein(PI=5 2),partial NH 2 terminal sequence(up to twenty residues) are ideatical with those reported for this protein.The results also showed that it is apparently heretogeneous from its NH 2 terminal side as it is composed of three polypeptides in which the intact Met anologue accounts for 59 5%,Ala anologue accounts for 25%,Pro anologue accounts for 14 6%.The overall purification factor obtained was about 3 9,the recovery of purified rhGM CSF was 69 1% by a biological assay method.The adoptied purification procedure is reproducible and well suited for process scale operations.