Chattopadhaya Souvik, Tan Lay Pheng, Yao Shao Q
Department of Biological Science, National University of Singapore, 3 Science Drive 3, Singapore 117543, Republic of Singapore.
Nat Protoc. 2006;1(5):2386-98. doi: 10.1038/nprot.2006.338.
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 approximately 2 days. Expression and in vitro protein purification and biotinylation will take approximately 3 days whereas the in vivo method would take approximately 2 days. The cell-free protein biotinylation strategy requires only 6-8 h.
本方案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，目的是构建功能性蛋白质阵列。重组蛋白的体外和体内生物素化依赖于半胱氨酸 - 生物素与通过内含肽介导的切割在蛋白质C末端产生的反应性硫酯基团之间的化学选择性反应。无细胞系统使用低浓度的生物素偶联嘌呤霉素。与其他需要繁琐且昂贵的蛋白质纯化下游步骤的方法不同，C末端生物素化的蛋白质可以直接从其他细胞蛋白质的混合物中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保持蛋白质的完整性和活性，这有可能允许对数千种蛋白质同时进行功能测定。假设目标蛋白质已克隆到表达载体中，细菌菌株的转化和起始培养物的生长大约需要2天。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6 - 8小时。
