四环素-绿色荧光蛋白融合蛋白的构建及其活性测定

将来源于水母的绿色荧光蛋白基因 (gfp)和来源于E .coli转座子Tn10的四环素阻遏蛋白基因 (tetR)共同构建到E .coli表达载体pET_30a +上 ,获得TetRC_端与GFPN_端融合蛋白。对经诱导表达并纯化后的融合蛋白 (TR∷GFP)进行荧光发射光谱分析表明 ,该融合蛋白保留了GFP的荧光特性 ,即在 395nm激发下 ,可在 5 10nm附近有特征发射峰。在加入四环素后 ,融合蛋白在 395nm激发下 ,在400nm～700nm范围内的发射光谱发生明显变化 ,荧光强度普遍增加 ,且以 510nm处最大发射峰增幅最大 ,由原来 1132增至 2214 ,而四环素对相同浓度的GFP与TetR荧光影响不大 ,结果表明该融合蛋白 ,能感受外界四环素 ,并产生一定的荧光变化。

Construction and Characterization of TetR and GFP Fusion Protein

Tetracycline repressor gene (tetR) from E. coli transposon Tn10 was fused in frame with green fluorescent protein gene (gfp) from jellyfish Aequorea Victoria on an E. coli expression vector and the fusion protein (TR::GFP) was purified. The binding of TR::GFP with tetracycline (tc) was demonstrated by nitrocellulose filter binding assay. TR::GFP also maintained the fluorescence property of GFP. Most significantly, fluorescence emission intensity of TR::GFP increased by 2-fold in the presence of tc, from 1.132 to 2.214, while those of GFP and TetR showed little change under similar conditions. The results indicated TR::GFP possesses characteristics of a tetracycline biosensor.