摘要
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(rhGMCSF)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SDSPAGE电泳测定纯度均大于98%,rhGMCSF的比活为32×107IU/mg,纯化获得的rhGMCSF为一酸性蛋白,等电点约为52,NH2末端前20个氨基酸序列测定结果与文献报道一致。rhGMCSF的NH2末端不均一,其中带Met的分子约占595%,第一个氨基酸为Ala的分子约占246%,为Pro的分子约占146%。纯化过程中蛋白的纯化倍数为39,生物活性总得率为691%,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.
出处
《生物工程学报》
CAS
CSCD
北大核心
1997年第2期142-148,共7页
Chinese Journal of Biotechnology
基金
上海市"八五"攻关课题
关键词
人粒细胞巨噬细胞集落刺激因子
包涵体
复性
纯化
Granulocyte macrophage colony stimulating factor,inclusion body,renaturation,purification