人尿激酶原cDNA在大肠杆菌中的高效表达与纯化

High-level expression and purification of human pro-UK cDNA in Escherichia coli.

作者信息

Xue Y, Peng G, Ma Z, Chen Y, Zhu D

机构信息

Department of Biochemistry, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, China.

出版信息

Chin J Biotechnol. 1997;13(4):233-8.

PMID:9631258

Abstract

A chemically synthesized human pro-urokinase (pro-UK) CDNA was cloned into the expression vector PET-11d and expressed in E. coli BL21(DE3) pLysS under the control of the T7 promoter. Using 0.1 mmol/L IPTG induction, the expression level of the recombinant pro-UK attained up to 15% of the total bacterial proteins and existed as inclusion bodies. After denaturation and renaturation in vitro, the expressed pro-UK was purified to be identified by Zn2+ selective precipitation, immuno-affinity chromatography, and benzamidine affinity adsorption. The specific activity of the purified human pro-UK was about 110,000 IU/mg.

摘要

将化学合成的人尿激酶原（pro-UK）cDNA克隆到表达载体PET-11d中，并在T7启动子的控制下在大肠杆菌BL21(DE3)pLysS中表达。使用0.1 mmol/L异丙基-β-D-硫代半乳糖苷（IPTG）诱导，重组人尿激酶原的表达水平达到细菌总蛋白的15%，并以包涵体形式存在。体外变性和复性后，通过Zn2+选择性沉淀、免疫亲和层析和苯甲脒亲和吸附对表达的人尿激酶原进行纯化鉴定。纯化的人尿激酶原的比活性约为110,000 IU/mg。