Department of Chemistry, Sogang University, Seoul 121-742, Republic of Korea.
Bioconjug Chem. 2012 Nov 21;23(11):2256-61. doi: 10.1021/bc300364z. Epub 2012 Oct 17.
There are currently many methods available for labeling proteins in order to study their structure and function. However, the utility of these methods is hampered by low efficiency, slow reaction rates, nonbiocompatible reaction conditions, large-sized labeling groups, and the requirement of specific side chains such as cysteine or lysine. In this study, a simple and efficient method for protein labeling was developed, in which an azide-containing amino acid was introduced into a protein and conjugated to a labeling reagent by strain-promoted azide-alkyne cycloaddition (SPAAC). This method allowed us to label proteins by simply mixing a protein and a labeling reagent in physiological conditions with a labeling yield of approximately 80% in 120 min. In addition, the specificity of SPAAC made it possible to analyze the expression level of a protein quantitatively by simple mixing and SDS-PAGE analysis with no need for antibodies or multistep incubations. Because the genetic incorporation of the azide-containing amino acid can be generally applied to any protein and the SPAAC reaction is highly specific, this method should prove useful for labeling and analyzing proteins.
目前有许多方法可用于标记蛋白质，以研究其结构和功能。然而，这些方法的实用性受到低效率、反应速率慢、非生物相容的反应条件、大尺寸的标记基团以及对半胱氨酸或赖氨酸等特定侧链的要求的限制。在这项研究中，开发了一种简单有效的蛋白质标记方法，其中将含叠氮化物的氨基酸引入蛋白质中，并通过应变促进的叠氮化物-炔烃环加成（SPAAC）与标记试剂结合。这种方法允许我们在生理条件下通过简单地混合蛋白质和标记试剂来标记蛋白质，在 120 分钟内标记产率约为 80%。此外，SPAAC 的特异性使得通过简单混合和 SDS-PAGE 分析即可定量分析蛋白质的表达水平，而无需使用抗体或多步孵育。由于含叠氮化物的氨基酸的遗传掺入可普遍应用于任何蛋白质，并且 SPAAC 反应具有高度特异性，因此该方法应有助于标记和分析蛋白质。
