Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation
Sortase A as a novel molecular "stapler" for sequence-specific protein conjugation
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DOI：
10.1021/bc060339w
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发表时间：
2007-03-01
影响因子：
4.7
通讯作者：
Boder, Eric T.
中科院分区：
文献类型：
--
作者：
Parthasarathy, Ranganath;Subramanian, Shyamsundar;Boder, Eric T.
转肽酶的分选酶家族催化蛋白质与革兰氏阳性细菌的细胞壁的序列特异性连接。在这里，我们描述了重组金黄色葡萄球菌分选酶A的应用程序，以附加标记的模型蛋白质底物（绿色荧光蛋白）的聚苯乙烯珠化学修饰的烷基胺或在体内分选酶A配体，甘氨酸-甘氨酸-甘氨酸，在其表面上。此外，我们表明，分选酶A可用于序列特异性连接eGFP的氨基封端的聚（乙二醇），并产生蛋白质寡聚体和环化单体使用适当标记的eGFP。我们发现，烷基胺可以取代天然Gly（3）底物，这表明在材料应用中使用酶的可能性。基于不太可能干扰蛋白质表达的小识别标签的高度特异性和温和的分选酶A催化的反应因此代表了对蛋白质固定和修饰工具试剂盒的有用添加。
The Sortase family of transpeptidase enzymes catalyzes sequence-specific ligation of proteins to the cell wall of Gram-positive bacteria. Here, we describe the application of recombinant Staphylococcus aureus Sortase A to attach a tagged model protein substrate (green fluorescent protein) to polystyrene beads chemically modified with either alkylamine or the in vivo Sortase A ligand, Gly-Gly-Gly, on their surfaces. Furthermore, we show that Sortase A can be used to sequence-specifically ligate eGFP to amino-terminated poly(ethylene glycol) and to generate protein oligomers and cyclized monomers using suitably tagged eGFP. We find that an alkylamine can substitute for the natural Gly(3) substrate, which suggests the possibility of using the enzyme in materials applications. The highly specific and mild Sortase A-catalyzed reaction, based on small recognition tags unlikely to interfere with protein expression, thus represents a useful addition to the protein immobilization and modification tool kit.