An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

Wang, X.-B.; Zhang, C.-H.; Zhang, T.; Li, H.-Z.; Liu, Y.-L.; Xu, Z.-G.; Lei, G.; Cai, C.-J.; Guo, Z.-Y.

doi:10.1101/2023.09.07.556767

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bioRxiv · 10.1101/2023.09.07.556767

An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase

Wang, X.-B.·Zhang, C.-H.·Zhang, T.·Li, H.-Z.·Liu, Y.-L.·Xu, Z.-G.·Lei, G.·Cai, C.-J.·Guo, Z.-Y.

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Abstract

In recent years, a few asparaginyl endopeptidases (AEPs) from certain higher plants have been identified as efficient peptide ligases with wide applications in protein labeling and cyclic peptide synthesis. Recently, we developed a NanoLuc Binary Technology (NanoBiT)-based peptide ligase activity assay to identify more AEP-type peptide ligases. Herein, we screened 61 bamboo species from 16 genera using this assay and detected AEP-type peptide ligase activity in the crude extract of all tested bamboo leaves. From a popular bamboo species, Bambusa multiplex, we identified a full-length AEP-type peptide ligase candidate (BmAEP1) via transcriptomic sequencing. After its zymogen was overexpressed in Escherichia coli and self-activated in vitro, BmAEP1 displayed high peptide ligase activity, but with considerable hydrolysis activity. After site-directed mutagenesis of its ligase activity determinants, the mutant zymogen of [G238V]BmAEP1 was normally overexpressed in E. coli, but failed to activate itself. To solve this problem, we developed a novel protease-assisted activation approach in which trypsin was used to cleave the mutant zymogen and was then conveniently removed via an ion-exchange chromatography. After the non-covalently bound cap domain was dissociated from the catalytic core domain under acidic conditions, the recombinant [G238V]BmAEP1 displayed high peptide ligase activity with much lower hydrolysis activity, and could efficiently catalyze inter-molecular protein ligation and intra-molecular peptide cyclization. Thus, the engineered bamboo-derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis.

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Wang, X.-B., Zhang, C.-H., Zhang, T., Li, H.-Z., Liu, Y.-L., Xu, Z.-G., Lei, G., Cai, C.-J., Guo, Z.-Y.. 2023-09-08. An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase. https://doi.org/10.1101/2023.09.07.556767

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