Protein engineering using asparaginyl peptide ligases

• Main Author: Wang, Zhen
• Other Authors: Liu Chuan Fa
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2023
• Subjects: Science::Biological sciences::Biochemistry
• Online Access: https://hdl.handle.net/10356/167030
• Institution: Nanyang Technological University

Peptidyl Asx-specific ligases (PALs) function as molecular gluing machines to join peptide and proteins together at Asn or Asp junctions. Because of their highly specific and efficient catalytic activity, PALs continue to attract the attention of researchers from the biotech industry and biomedical research communities. In this thesis, I first report a bio-orthogonal scheme using two asparaginyl peptide ligases with differential substrate specificities – butelase-1 and VyPAL2. This scheme allows for tandem ligation on the same protein in either the N-to-C or C-to-N direction, making it possible to prepare dually labelled proteins as potential theranostic agents. Second, my study shows PALs can use non-canonical nucleophilic compounds – such as hydrazides – as acyl acceptor substrates. This expanded substrate scope is explored to engineer multi-functional proteins, including bi-specific protein engagers capable of mediating the killing of cancer cells by Car-NK cells. Finally, I report the design of an auto-processing mechanism of a C-terminal Cys-Asn dipeptide for traceless protein ligation by combining PAL ligation with native chemical ligation or subtiligation. My work further demonstrates PALs as powerful tools of biotechnology for protein engineering.