Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems:: toward functional protein arrays
Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems:: toward functional protein arrays
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DOI：
10.1038/nprot.2006.338
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Yao, Shao Q.
中科院分区：
文献类型：
--
作者：
Chattopadhaya, Souvik;Tan, Lay Pheng;Yao, Shao Q.
该协议详细介绍了使用体外、体内和无细胞系统对蛋白质进行位点特异性生物素化的方法，以制造功能性蛋白质阵列。重组蛋白的体外和体内生物素化依赖于半胱氨酸-生物素和通过内含肽介导的切割产生的蛋白质 C 末端的反应性硫酯基团之间的化学选择性反应。无细胞系统利用低浓度的生物素结合的嘌呤霉素。与其他需要繁琐且昂贵的下游蛋白质纯化步骤的方法不同，C 端生物素化蛋白质可以从其他细胞蛋白质的混合物中直接捕获到抗生物素蛋白功能化的载玻片上，以生成相应的蛋白质阵列。这些方法旨在以微阵列形式维持蛋白质的完整性和活性，这可能允许同时对数千种蛋白质进行功能测定。假设目标蛋白已被克隆到表达载体中，菌株的转化和起始培养物的生长将需要大约 2 天的时间。表达、体外蛋白质纯化和生物素化大约需要 3 天，而体内方法大约需要 2 天。无细胞蛋白质生物素化策略仅需要6-8小时。
This protocol details methodologies for the site-specific biotinylation of proteins using in vitro, in vivo and cell-free systems for the purpose of fabricating functional protein arrays. Biotinylation of recombinant proteins, in vitro as well as in vivo, relies on the chemoselective reaction between cysteine-biotin and a reactive thioester group at the C-terminus of a protein generated via intein-mediated cleavage. The cell-free system utilizes low concentrations of biotin-conjugated puromycin. Unlike other approaches that require tedious and costly downstream steps of protein purification, C-terminal biotinylated proteins can be captured directly onto avidin-functionalized slides from a mixture of other cellular proteins to generate the corresponding protein array. These methods were designed to maintain the integrity and activity of proteins in a microarray format, which potentially allows simultaneous functional assays of thousands of proteins. Assuming that the target proteins have been cloned into the expression vector, transformation of bacterial strain and growth of starter culture would take similar to 2 days. Expression and in vitro protein purification and biotinylation will take similar to 3 days whereas the in vivo method would take similar to 2 days. The cell-free protein biotinylation strategy requires only 6-8 h.