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- 2015
人胰岛素原基因在大肠杆菌中的表达和纯化
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Abstract:
摘要： 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。
Abstract: 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
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