Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.
Quantifying the Kinetics of Pilus-Specific Sortase-Catalyzed Crosslinking Using High-Performance Liquid Chromatography.
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使用高效液相色谱定量菌毛特异性分选酶催化交联的动力学。
DOI：
10.1007/978-1-0716-3491-2_11
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发表时间：
2024
期刊：
影响因子：
--
通讯作者：
Clubb,RobertT
中科院分区：
文献类型：
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作者：
Cheung,NicoleA;Song,Mabel;Sue,ChristopherK;Clubb,RobertT
革兰氏阳性细菌显示菌毛的蛋白质成分(菌毛蛋白)由菌毛特有的分类酶共价交联。这些半胱氨酸转肽酶催化转肽反应，通过赖氨酸异肽键将菌丝连接在一起。白喉棒状杆菌SPAA菌毛形成的交联反应是由SrtA排序酶(CDSrtA)介导的，并已在体外重组。在这里，我们提出了一种方法，可以用来用高效液相色谱(HPLC)测量CdSrtA催化的交联剂的动力学。原则上，这种生化程序可以用来测量任何菌毛专一性的索糖酶的体外交联性。
Gram-positive bacteria display pili whose protein components (pilins) are covalently crosslinked by pilus-specific sortase enzymes. These cysteine transpeptidase enzymes catalyze a transpeptidation reaction that joins the pilins together via lysine isopeptide bonds. The crosslinking reaction that builds the SpaA pilus inCorynebacterium diphtheriaeis mediated by the SrtA sortase (CdSrtA) and has been reconstituted in vitro. Here, we present a protocol that can be used to measure the kinetics ofCdSrtA-catalyzed crosslinking using high-performance liquid chromatography (HPLC). In principle, this biochemical procedure can be used to measure the in vitro crosslinking activity of any pilus-specific sortase.