高活性重组hG-CSF的制备  

Overproduction of Recombinant hG CSF With High Activity

作　　者：崔立斌[1] 马清钧[1] 

机构地区：[1]军事医学科学院生物工程研究所

出　　处：《生物工程进展》1997年第3期24-27,共4页Progress in Biotechnology

摘　　要：我们构建了新的硫氧还蛋白（Ｔｈｉｏｒｅｄｏｘｉｎ）融合表达载体ｐＥＴＴｒｘＬ和ｐＥＴＴｒｘ－ＨｉｓＬ，它们可使功能蛋白在大肠杆菌胞质中以可溶性形式高效表达。利用此表达系统成功地获得的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白的高效可溶性表达，表达水平达总细胞可溶蛋白的４１％以上。所表达的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白可通过Ｃｕ２＋－ＩＤＡＳｅｐｈａｒｏｓｅＦＦ固相金属螯合层析柱，方便地从细胞破碎可溶上清中直接纯化。所获得的融合蛋白具有ｈＧ－ＣＳＦ特异的生物活性，其比活性达到０．５－１．３３×１０７ｕ／ｍｇ融合蛋白。这样表达的ｈＧ－ＣＳＦ融合蛋白能被ＩｇＡ蛋白酶特异地切割。A novel set of thioredoxin fusion expression vectors pETTrxL and pETTrxHisL was constructed for overproduction of functional heterologous proteins in soluble form in E.coli cytoplasm.Using this system,hG CSF was succesfully expressed to a level of more than 41% of total cellular proteins in complete soluble form as thioredoxin fusion.To facilate the purification of the fusion protein,a (his) 5 tag coding sequence was inserted in the active site of thioredoxin gene in pETTrxHisL without any detrimental effect on the solubility of thioredoxin fusion,so the fusion expressed by BL21DE3(pETTrxHisG CSF) can be purified directly from the cell lysate using a Cu 2+ IDA Sepharose FF immobilized metal affinity chromatography column.The purified fusion protein retains fully biological activity as nature hG CSF.In addition,the fusion protein can be cleaved efficiently by IgA protease with high specifity,generating mature hG CSF with exactly the same primary structure as natural hG CSF.

关 键 词：药物 粒细胞 集落刺激因子 制备 重组 

分 类 号：R392－33[医药卫生—免疫学] R394.8[医药卫生—基础医学]