Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fisheries Science Research Institute, Dalian, 116023, China,
Mol Biol Rep. 2013 Dec;40(12):6987-95. doi: 10.1007/s11033-013-2818-6. Epub 2013 Nov 1.
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. The goal of this study was to clone, efficiently express and purify the ecdysteroid-regulated protein (ERP) in the form of a GST fusion protein. The mature peptide-coding cDNA fragment was extracted from Chinese mitten crap (Eriocheir sinensis), and then after using PCR to obtain the open reading frame, a recombinant plasmid designated pGEX-4T-1_ERP was successfully generated and showed to efficiently express the ERP fusion protein as determined by SDS-PAGE. The resulting expressed protein was successfully purified by a combination of affinity and conventional chromatographic methods. After purification, the recombinant protein showed the expected size of 41 kDa on SDS-PAGE gels which was further confirmed by mass spectrometry and western blotting. Purification of recombinant protein was achieved by fast protein liquid chromatography. About 2.4 mg/l recombinant protein with purity more than 80 % was obtained.
谷胱甘肽 S-转移酶（GST）融合蛋白系统广泛用于从细菌中高水平表达和有效纯化重组蛋白。本研究的目的是克隆、高效表达和纯化 GST 融合蛋白形式的蜕皮甾类调节蛋白（ERP）。从中华绒螯蟹（Eriocheir sinensis）中提取成熟肽编码 cDNA 片段，然后通过 PCR 获得开放阅读框，成功构建了命名为 pGEX-4T-1_ERP 的重组质粒，SDS-PAGE 分析表明该质粒能够高效表达 ERP 融合蛋白。通过亲和和常规色谱方法的组合，成功地对表达的蛋白进行了纯化。纯化后，重组蛋白在 SDS-PAGE 凝胶上显示出预期大小的 41 kDa，质谱和 Western blot 进一步证实了这一点。通过快速蛋白液相色谱法实现了重组蛋白的纯化。获得了约 2.4 mg/L、纯度超过 80%的重组蛋白。
