中国药科大学学报2004，Vol.35Issue(4)：374-378,5.
硫氧还蛋白-(His)6融合的瑞替普酶的表达、纯化和活性检测
A Thioredoxin-(His)6 Fusion Protein of Reteplase:Expression,Purification,and Activity Analysis
摘要
Abstract
AIM:To increase the expression amount of reteplase,a vector was constructed which expressed the fusion protein thioredoxin-(His)6-rPA. The purification of reteplase was simplified by one-step affinity chromatography in this study. METHOD: Reteplase gene was inserted into the prokaryotic expression vector pET32a and was fused to the downstream of the thioredoxin and the (His)6Tag in the same reading frame. The fusion protein was expressed in E. coli BL21 (DE3) induced by lactose. After refolded in vitro by one-step dilution,the fusion protein was simply purified using Ni2+-chelating chromatography. Fibrin plate assay was used to detect the bioactivity of the fusion protein. RESULT:A fusion protein of 56 kD was obtained. The amount of the fusion protein was more than 60% of the total bacterial protein. Comparing with the non-fusion reteplase,the yields of target protein increased about 50%. The purity of the fusion protein reached above 80% after one-step purification using Ni2+-chelating chromatography. The bioactivity of the refolded and purified fusion protein was detected. CONCLUSION: Expression level of the fusion protein increased distinctively compared with that of the non-fusion protein. Bioactivity of reteplase was detected even though a fusion peptide was fused to the N-terminal of reteplase.关键词
硫氧还蛋白/(组氨酸)6标签/瑞替普酶/融合蛋白/表达/纯化/活性Key words
Reteplase(rPA)/Thioredoxin/(His)6Tag/Fusion protein/Expression/Purification/Activity分类
医药卫生引用本文复制引用
张新元,廖建民,孙石静,沈子龙..硫氧还蛋白-(His)6融合的瑞替普酶的表达、纯化和活性检测[J].中国药科大学学报,2004,35(4):374-378,5.基金项目
江苏省科技厅基金资助项目(No.BG2002318) （No.BG2002318）
