Expression in E.coli and preparation of CP4-EPSPS protein | CP4-EPSPS蛋白在大肠杆菌中的表达与制备
2012
Liu Shupeng, Chinese Academy of Agricultural Sciences, Beijing (China), Biotechnology Research Institute | Li Gangqiang, Chinese Academy of Agricultural Sciences, Beijing (China), Biotechnology Research Institute | Wang Nan, Chinese Academy of Agricultural Sciences, Beijing (China), Biotechnology Research Institute
Chinese. 构建了一个含有CP4-EPSPS外源基因的细菌表达载体并在大肠杆菌Transtta菌株中的高效表达,通过SDS-PAGE检测发现,目标蛋白完全以可溶性方式存在于细胞破碎上清液中,且约占到细菌可溶性蛋白的40%左右。收集该上清液并进而通过连续2次镍柱亲和层析,CP4-EPSPS重组蛋白纯度可达99.9%以上,对其进行N端序列分析,与预期的结果完全一致。以其作为标准品,通过ELISA,成功检测出三株CP4-EPSPS转基因烟草中目标蛋白含量分别为0.292 μg/g、0.477 μg/g和0.703 μg/g。该蛋白表达和纯化体系可为CP4-EPSPS转基因植物蛋白量值溯源传递的标准物质研制提供稳定物质基础。
Show more [+] Less [-]English. The CP4-EPSPS gene was cloned into a bacterial expression vector and transformed into E. coli strain Transtta. The expressed protein was detected by SDS-PAGE. The results showed that target protein accounted for about 40% of the total soluble protein. After the nickel affinity column purification for 2 times, the purity of the CP4-EPSPS recombinant protein can reach up to 99.9%, and its N terminal sequence analysis was consistent with the expected result. Using the purified CP4-EPSPS protein as standard substance, we successfully detected the hetero-protein level in 3 CP4-EPSPS transgenic tobacco lines through ELISA. The CP4EPSPS expression level in the GMO plant was 0.292 μg/g, 0.477 μg/g and 0.703 μg/g, respectively. We conclude that the CP4-EPSPS expression and purification system can provide a steady source for tracing and inspecting the CP4-EPSPS transgenic plant.
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