Discovery, engineering and functional study of asparagine-specific ligases from plants

Abstract

Asparaginyl endopeptidases (AEPs) are cysteine proteases cutting peptide bonds after Asx (Asn/Asp). Peptide Asx-specific ligases (PALs) are AEP-like peptide ligases reversing the proteolysis of AEPs and making peptide bonds at the Asx sites. Peptide ligases have considerable potentials in biotechnology, material sciences, and precision biomanufacturing of biologics. The objective of my thesis comprises two parts. The first part focuses on the discovery of novel PALs through screening ligases from seeds and data mining search. The second part focuses on the engineering of AEP on the ligase activity determinants (LADs), which reside in the substrate-binding pockets flanking at the active pocket. Together, my thesis provides further insights to understand the occurrence and molecular determinants of peptide ligases which are useful tools for site-specific labeling proteins and targeted drug development.