Development of a general bioluminescent activity assay for peptide ligases

Abstract

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.

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

“…The expression construct for the ligation version LgBiT (LgBiT-NHV) was generated in our previous study [39]. The expression constructs for the ligation version NanoLucs (NanoLuc-NHV and NanoLuc-NAL) were generated by site-directed mutagenesis via a QuikChange approach based on our previous construct pET/6×His-NanoLuc-Cys [40], and their nucleotide and amino acid sequences are shown in Fig. S1.…”

“…S1. After overexpression in the E. coli strain BL21(DE3) as soluble proteins, the ligation version LgBiT and NanoLucs were purified by an immobilized metal ion affinity chromatography (Ni 2+ column) and an ion-exchange chromatography (DEAE column), analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and bioluminescence measurement, and stored at −80 ºC according to our previous procedures [39,40]. 3) if not mentioned otherwise, and plotted using SigmaPlot10.0 software.…”

“…The NanoBiT-based peptide ligase activity assays were conducted according to our previous procedure [39]. Briefly, the pre-warmed crude or purified peptide ligase was mixed with the recombinant LgBiT-NHV and the synthetic GI-SmBiT at the indicated concentrations.…”

“…To prepare the crude extract, 1.0 g of the fresh bamboo leaves was cut to small pieces and ground in a mortar with 4.0 ml of cold extraction buffer [PBS plus 1.0 mM ethylene glycol tetraacetic acid (EGTA) and 5.0 mM β-mercaptoethanol]. After centrifugation (10000 g, 5 min), the supernatant was used for peptide ligase activity assay according to our previous procedure [39].…”

“…To identify more AEP-type peptide ligases, we recently developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay via the introduction of a peptide ligase recognition motif to the C-terminus of an inactive large NanoLuc fragment (LgBiT) and the introduction of a peptide ligase-preferred nucleophilic motif to the N-terminus of a synthetic low-affinity SmBiT complementation tag [39].…”