人胰岛素原基因在大肠杆菌中的表达和纯化

将人胰岛素原基因序列(human proinsulin, PI)经密码子优化后与TrxA蛋白融合表达,构建原核表达载体TrxA-PI转入不同大肠杆菌表达菌株BL21(DE3)、TransB(DE3)和Rosetta-gami(DE3)中。SDS-PAGE显示融合蛋白在3种表达菌株中均有表达,在Rosetta-gami(DE3)中表达量最高,是普通大肠杆菌BL21(DE3)表达量的10倍。通过优化诱导温度等发酵条件,可溶性重组融合蛋白TrxA-PI在Rosetta-gami(DE3)菌株中表达量为3.5 g/L,比未优化时提高约10倍。TrxA-PI用肠激酶酶切后,利用HisTrap FF柱分离纯化PI,经Western-blot检测重组PI具有免疫原性。

Expression and purification of human proinsulin gene in Escherichia coli

The human proinsulin (PI) gene sequence was optimized using the Escherichia coli preferred codons. The reformed PI gene was amplified, the fused expression vector (TrxA-PI) was constructed and transformed into three E.coli expression strains BL21(DE3),TransB(DE3) and Rosetta-gami(DE3). SDS-PAGE analysis showed that the expression level of the fused protein was the highest in Rosetta-gami (DE3) strain, which was about 10 times higher than in BL21(DE3) strain. The production of soluble fused protein was 3.5 mg/mL after optimization of the fermentation conditions. The purified recombinant PI was obtained after enterokinase digestion and HisTrap FF column purification, and its antigen activity was measured using Western-blot analysis.