Objective: To establish and optimize a method for site-specific biotinylation, purification and detection of enhanced green fluorescent protein (eCFP) in human embryonic kidney cells based on Avi-tag technology. Methods: We respectively construct the Avi-tagged eGFP eukaryotic expression vector plenti-Avi-eGFP and BirA enzyme over-expression eukaryotitc vector pQCXIH-BirA, then plenti-Avi-eGFP and pQCXIH-BirA were co-trans-fected into human embryonic kidney cells 293T. After 12 h, we observed that whether the Avi-tag have an impact on the location of eGFP in the cells. After 48 h the 293T cells were cracked and biotinylated eGFP was purified from lysis supernatant by using streptavidin beads. SDS-PAGE was used to observe the situation of purification and enrichment and then the condition of detecting biotinylated eGFP based on Western blotting was optimized. Results: The Avi-tag had no effect on the location of eGFP in the cells. At the same time the Avi-tagged eGFP was successfully biotinylated by BirA in 293T cells, and the biotinylated eGFP was purified and enriched specifically by streptavidin beads, and the purity of eGFP reached to 95%. The ultimate terms for detecting biotinylated protein based on Western blotting were determinated as 5% BSA as the blocking reagent and 100 ng/mL streptavidin -HRP as working solution. Conclusion: The Avi-tag method for site-specific biotinylation, purification and detection of the eGFP in human embryonic kidney cells is set up in our lab.%目的:建立并优化基于Avi-tag标签技术的人胚肾细胞增强型绿色荧光蛋白(eGFP)的定点生物素化标记、纯化和检测方法.方法:分别构建具有Avi-tag标签的eGFP真核表达载体plenti-Avi-eGFP和BirA酶真核过表达载体pQCXIH-BirA,将plenti-Avi-eGFP和pQCXIH-BirA共转染人胚肾293T细胞,12 h后观察Avi-tag标签对eGFP蛋白在细胞内定位的影响；48 h后裂解细胞,用链霉亲和素珠子纯化生物素标记的eGFP,SDS-PAGE观察eGFP纯化和富集情况,并优化基于Western印迹的生物素化eGFP检测方法.结果:Avi-tag标签对eGFP在细胞内的定位无影响,同时BirA酶在293T细胞内可将带Avi-tag标签的eGFP标记上生物素；生物素化的eGFP可特异性地被链霉亲和素珠子纯化和富集,纯度可达95%；Western印迹检测生物素化蛋白的最终条件为5%的BSA作为封闭液和终浓度为100 ng/mL的链霉亲和素-HRP.结论:建立了基于Avi-tag技术的人胚肾细胞内增强型绿色荧光蛋白的生物素化标记、纯化与检测方法,为该方法的广泛应用奠定了前期技术基础.
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