Protein engineering using asparaginyl peptide ligases

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...

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Main Author: Wang, Zhen
Other Authors: Liu Chuan Fa
Format: Thesis-Doctor of Philosophy
Language:English
Published: Nanyang Technological University 2023
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Online Access:https://hdl.handle.net/10356/167030
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Institution: Nanyang Technological University
Language: English
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spelling sg-ntu-dr.10356-1670302023-06-01T08:00:48Z Protein engineering using asparaginyl peptide ligases Wang, Zhen Liu Chuan Fa School of Biological Sciences CFLiu@ntu.edu.sg Science::Biological sciences::Biochemistry 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. Doctor of Philosophy 2023-05-21T05:32:33Z 2023-05-21T05:32:33Z 2023 Thesis-Doctor of Philosophy Wang, Z. (2023). Protein engineering using asparaginyl peptide ligases. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/167030 https://hdl.handle.net/10356/167030 10.32657/10356/167030 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences::Biochemistry
spellingShingle Science::Biological sciences::Biochemistry
Wang, Zhen
Protein engineering using asparaginyl peptide ligases
description 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.
author2 Liu Chuan Fa
author_facet Liu Chuan Fa
Wang, Zhen
format Thesis-Doctor of Philosophy
author Wang, Zhen
author_sort Wang, Zhen
title Protein engineering using asparaginyl peptide ligases
title_short Protein engineering using asparaginyl peptide ligases
title_full Protein engineering using asparaginyl peptide ligases
title_fullStr Protein engineering using asparaginyl peptide ligases
title_full_unstemmed Protein engineering using asparaginyl peptide ligases
title_sort protein engineering using asparaginyl peptide ligases
publisher Nanyang Technological University
publishDate 2023
url https://hdl.handle.net/10356/167030
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