摘要
抽提人肝组织总RNA,通过RT-PCR方法扩增出纤溶酶原Kringle5片段,将其和分泌表达载体pPIC9K相连,转化毕赤酵母GS115,筛选获得在酵母细胞中遗传稳定表达Kringle5重组蛋白的工程菌。经甲醇诱导表达5d后,发酵液总蛋白分泌量约120mg/L,SDS-PAGE呈现相对分子质量约为15×103特异表达带。经Ni-NTA-Agarose纯化后,重组蛋白对人脐静脉内皮细胞有明显的抑制作用,ID50约为4.0μg/ml。
Total RNA was extracted from human fresh liver.The gene encoding Kringle5of plasminogen was am-plified by RT-PCR.The Kringle5gene was introduced into vector pPIC9K.Yeast cell GS115was transfected by linear recombinant plasmid,then the yeast engineering cells were obtained stably expressing Kringle5.After5days of continuous methanol induction,it was observed that the total secreted proteins in culture supernatants reached up to120mg/L,migrating as the main band of15KD on SDS-PAGE.The protein was purified with Ni-NTA-Agarose and the activity analysis of Kringle5on HUVEC showed that this recombinant protein could obviously block the growth of the cell with ID 50 ≈4.0μg/ml.
出处
《生物技术通讯》
CAS
2002年第2期112-114,117,共4页
Letters in Biotechnology
基金
上海市卫生局课题基金(项目号99425)资助
