An efficient peptide ligase engineered from a bamboo asparaginyl endopeptidase.

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 hydrolytic 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 resolve 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 ion‐exchange chromatography. After the noncovalently 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 hydrolytic activity and could efficiently catalyze inter‐molecular protein ligation and intramolecular peptide cyclization. Thus, the engineered bamboo‐derived peptide ligase represents a novel tool for protein labeling and cyclic peptide synthesis. [ABSTRACT FROM AUTHOR]