重组人尿激酶原的工业化制备与性质研究

将含有 pET2 9a prouk重组质粒的人尿激酶原工程菌经 10L种子罐培养及 10 0L发酵 ,IPTG诱导表达 ,其表达量为占菌体总蛋白的 2 0 % ,表达产物经体外变复性 ,CM 纤维素离子交换层析 ,Superdex 75分子筛层析及Affi preppolymyxinsupport亲和层析去热源 ,每 10 0L发酵液得重组人尿激酶原纯品 6g ,纯度达 95 %以上 ,比活大于 1.2× 10 4 IU /mg ,双链含量低于0 .5 % ,内毒素及热源含量、宿主蛋白残留量、宿主DNA残留量等均达到临床使用标准 .其分子量 (质谱测定 )、氨基酸组成、N、C 端氨基酸序列分析等均与理论值相符 .进行了等电点及肽图等性质研究

Characterization and Large Scale Preparation of Human Recombinant Prourokinase

The engineered E.coli containing pET29a prouk was cultured in a 10 L seeding tank, and then grew in a 100 L fermentor under IPTG induction. The expressed protein accounted for 20% of the bacteria total proteins. After renaturation, the folding solution was applied to a column of CM cellulose, and the fraction containing recombinant prourokinase activity further was purified by superdex 75 gel filtration and depynogened by affi prep polymyxin affinity chromatography. A pilot purification yielded 6 g of purified human recombinant pro urokinase from 100 L medium. The purity of the resulting protein was higher than 95%, and its specific activity was over 120 000 iu/mg. The content of the two chain urokinase was less than 0.5%, and the trace content of pyrogen, the residual protein and DNA from the host cell all met the requirements for clinical use. The molecular weight of the recombinant prourokinase and the amino acid composition were consistent with the theoretical data. The isoelectrophoretic point and peptide mapping of the pro urokinase were also determined.