人重组载脂蛋白E在大肠杆菌中的表达和纯化

Expression and Purification of Human Recombinant Apolipoprotein E from Escherichia coli

用含有载脂蛋白EcDNA的PET32a原核表达载体转化大肠杆菌BL2 1(DE3) ,使之高效表达载脂蛋白E thioredoxin融合蛋白。利用融合蛋白上的一段组氨酸序列 ,用镍离子亲合层析柱进行分离纯化。由于在载脂蛋白E和thioredoxin之间存在凝血酶的识别位点 ,用凝血酶消化后 ,经SephacrylS 30 0凝胶过滤得到人重组载脂蛋白E。用此方法可以从 1L大肠杆菌培养液中纯化 2 0～ 30mg高纯度的各种载脂蛋白E异构体和非自然存在的突变体 ,方法简便 ,产量高 ,纯度达 95 %以上。

Aim To separate and purify human recombinant apolipoprotein E (Apo E) from Escherichia coli strain BL21(DE3). Methods PET32a Apo E constructs were transformed into BL21 and protein expression was induced. Apo E thioredoxin fusion protein was separated by Ni 2+ affinity column. After digestion of thioredoxin by thrombin, Apo E was purified by Sephacryl S 300 gel filtration column. Results Apo E2, Apo E3 and Apo E4 were produced by this method, with high yield and high purity. Conclusions A new system for separation and purification of human recombinant Apo E was successfully established.