State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering , Hunan University , Changsha , 410082 , P. R. China.
Anal Chem. 2018 Mar 6;90(5):3245-3252. doi: 10.1021/acs.analchem.7b04756. Epub 2018 Feb 22.
Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen, and probing sortase activity and screening its inhibitors are of great significance to fundamental biological research and pharmaceutical development, especially novel antivirulence drug design. Herein, we developed a novel fluorescent biosensor to detect sortase activity based on a transpeptidation-triggered assembly of tripartite split green fluorescent protein (split GFP). Peptide P, composed the 10th β-sheet of GFP (GFP10) and the sortase A (SrtA) recognition sequence (LPETX), and peptide P, the 11th β-sheet of GFP (GFP11) with oligoglycine at N-terminal, were designed and synthesized, respectively. Existence of SrtA enables P and P to ligate into one peptide, which could spontaneously bind to GFP1-9 (the 1st to 9th β-sheets of GFP) and assemble into functional GFP. Thus, the sortase-catalyzed transpeptidation can switch on the fluorescence signal of GFP. The method was successfully applied to detect SrtA activity with a low detection limit of 0.16 nM and for its inhibition measurement. Moreover, the feasibility of the proposed assay was further expanded to detect SrtA in human blood and further Gram-positive pathogens analysis in frozen food. Our method, using tripartite split GFP as a readout, is facile, label-free, and sensitive and exhibits great potential as a promising platform for sortase detection and inhibitor screening.
表面蛋白的转肽作用由转肽酶 sortase 催化，在革兰氏阳性病原体的感染过程中起着关键作用，探测 sortase 的活性并筛选其抑制剂对于基础生物学研究和药物开发具有重要意义，尤其是新型抗毒力药物的设计。在此，我们开发了一种基于三聚体分裂绿色荧光蛋白（split GFP）的转肽反应触发组装的新型荧光生物传感器来检测 sortase 活性。肽 P 由 GFP 的第 10 个β-折叠（GFP10）和 sortase A（SrtA）识别序列（LPETX）组成，肽 P 由 GFP 的第 11 个β-折叠（GFP11）和 N 端的寡甘氨酸组成，分别进行了设计和合成。存在 SrtA 时，P 和 P 能够连接成一个肽，该肽可以自发与 GFP1-9（GFP 的第 1 到 9 个β-折叠）结合并组装成功能性 GFP。因此，sortase 催化的转肽反应可以打开 GFP 的荧光信号。该方法成功地用于检测 SrtA 活性，检测限低至 0.16 nM，并可用于其抑制测量。此外，还进一步扩展了该方法用于检测人血液中的 SrtA 和冷冻食品中进一步的革兰氏阳性病原体分析。我们的方法使用三聚体分裂 GFP 作为读出，简便、无标记且灵敏，有望成为一种有前途的 sortase 检测和抑制剂筛选平台。
