Development of a general bioluminescent activity assay for peptide ligases
Development of a general bioluminescent activity assay for peptide ligases
复制标题
DOI：
10.1111/febs.16416
复制
发表时间：
2022-03
期刊：
影响因子：
--
通讯作者：
Cong-Hui Zhang;X. Shao;Xin-Bo Wang;Li-Li Shou-Li;Ya‐Li Liu;Zeng-guang Xu;Zhan-Yun Guo
中科院分区：
文献类型：
--
作者：
Cong-Hui Zhang;X. Shao;Xin-Bo Wang;Li-Li Shou-Li;Ya‐Li Liu;Zeng-guang Xu;Zhan-Yun Guo
近年来，一些肽连接酶已被鉴定出来，如细菌分解酶和某些植物天冬酰胺或脯氨酰内肽酶。多肽连接酶在蛋白质标记和环肽合成中有着广泛的应用。为了表征各种已知的多肽连接酶或鉴定新的生物发光活性，我们提出了一种通用的生物发光活性检测方法，方法是将多肽连接酶的识别基序(S)与非活性的大片段纳米Luc片段(LgBiT)的C末端进行遗传融合，并在低亲和力SmBiT互补标签的N端化学引入多肽连接酶(S)偏好的亲核基序。用预期的肽连接酶(S)将失活连接的LgBiT蛋白与低亲和力连接的SmBiT Tag连接后，其荧光素酶活性将恢复，并可根据测定的生物发光进行灵敏的定量。在本研究中，我们首先验证了细菌分类酶A和植物来源的丁基酶-1的生物发光活性测定。随后，我们使用两个LgBiT-SmBiT连接对从所选植物的粗提物中筛选出新的多肽连接酶。在80种常见高等植物中，5种可能表达天冬酰胺内肽酶类型的肽连接酶，4种可能表达Pro-内肽酶类型的肽连接酶，这表明肽连接酶在高等植物中并不罕见，更多的肽连接酶还有待于发现。本发明的生物发光活性测定方法具有超灵敏、使用方便、不受蛋白酶干扰等优点，在今后的研究中将广泛应用于已知肽连接酶的鉴定或从不同来源筛选新的连接酶。
In recent years, some peptide ligases have been identified, such as bacterial sortases and certain plant asparaginyl or prolyl endopeptidases. Peptide ligases have wide applications in protein labelling and cyclic peptide synthesis. To characterize various known peptide ligases or identify new ones, we propose a general bioluminescent activity assay via the genetic fusion of a recognition motif of peptide ligase(s) to the C‐terminus of an inactive large NanoLuc fragment (LgBiT) and the chemical introduction of a nucleophilic motif preferred by the peptide ligase(s) to the N‐terminus of the low‐affinity SmBiT complementation tag. After the inactive ligation version LgBiT protein was ligated with the low‐affinity ligation version SmBiT tag by the expected peptide ligase(s), its luciferase activity would be restored and could be quantified sensitively according to the measured bioluminescence. In the present study, we first validated the bioluminescent activity assay using bacterial sortase A and plant‐derived butelase‐1. Subsequently, we screened novel peptide ligases from crude extracts of selected plants using two LgBiT‐SmBiT ligation pairs. Among 80 common higher plants, we identified that five of them likely express asparaginyl endopeptidase‐type peptide ligase and four of them likely express prolyl endopeptidase‐type peptide ligase, suggesting that peptide ligases are not so rare in higher plants and more of them await discovery. The present bioluminescent activity assay is ultrasensitive, convenient for use, and resistant to protease interference, and thus would have wide applications for characterizing known peptide ligases or screening new ones from various sources in future studies.