Site-specific protein modification on living cells catalyzed by Sortase
Site-specific protein modification on living cells catalyzed by Sortase
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DOI：
10.1002/cbic.200700614
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发表时间：
2008-03-25
期刊：
影响因子：
3.2
通讯作者：
Nagamune, Teruyuki
中科院分区：
文献类型：
--
作者：
Tanaka, Tsutomu;Yamamoto, Teruyasu;Nagamune, Teruyuki
由于酶的底物特异性，使用酶是对活细胞进行位点特异性蛋白质修饰的一种有前景的方法。在此，我们描述了使用合成分子对细胞表面蛋白进行位点特异性修饰的一般策略，其中使用 Sortase（一种来自金黄色葡萄球菌的转肽酶）。短肽标签 LPETGG 通过基因技术引入到细胞表面表达的目标蛋白的 C 末端。随后添加分选酶和含有 N 末端三甘氨酸的探针，从而对标记蛋白进行位点特异性标记。我们成功地在活细胞表面用含有生物素或荧光团的短肽对破骨细胞分化因子 (ODF) 进行 C 端特异性标记。标记反应在含血清培养基以及无血清培养基或 PBS 中有效发生。孵育5分钟后检测标记产物。此外，通过分选酶催化反应，在活细胞表面成功地证明了位点特异性蛋白质-蛋白质缀合。该策略为细胞生物学和细胞表面工程提供了6种强大的工具。
The use of enzymes is a promising approach for site-specific protein modification on living cells owing to their substrate specificity. Herein we describe a general strategy for the site-specific modification of cell surface proteins with synthetic molecules by using Sortase, a transpeptidase from Staphylococcus aureus. The short peptide tag LPETGG is genetically introduced to the C terminus of the target protein, expressed on the cell surface. Subsequent addition of Sortase and an N-terminal triglycine-containing probe results in the site-specific labeling of the tagged protein. We were successful in the C-terminal-specific labeling of osteoclast differentiation factor (ODF) with a biotin- or fluorophore-containing short peptide on the living cell surface. The labeling reaction occurred efficiently in serum-containing medium, as well as serum-free medium or PBS. The labeled products were detected after incabation for 5 min. In addition, site-specific protein-protein conjugation was successfully demonstrated on a living cell surface by the Sortase-catalyzed reaction. This strategy provides 6 powerful tool for cell biology and cell surface engineering.