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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2023
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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 |
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Science::Biological sciences::Biochemistry Wang, Zhen Protein engineering using asparaginyl peptide ligases |
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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. |
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Liu Chuan Fa |
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Liu Chuan Fa Wang, Zhen |
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Thesis-Doctor of Philosophy |
author |
Wang, Zhen |
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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 |
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Protein engineering using asparaginyl peptide ligases |
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Protein engineering using asparaginyl peptide ligases |
title_sort |
protein engineering using asparaginyl peptide ligases |
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Nanyang Technological University |
publishDate |
2023 |
url |
https://hdl.handle.net/10356/167030 |
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