Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs)
Asparaginyl endopeptidases (AEPs) are thiol proteases that cleave peptide bonds after Asn/Asp(Asx) residues. However, certain AEPs also make Asx bonds and which we named peptide asparaginyl ligases (PALs). PALs, ATP-independent and stand-alone, are ideal for site-specific modifications of proteins,...
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Format: | Thesis-Master by Research |
Language: | English |
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Nanyang Technological University
2023
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Online Access: | https://hdl.handle.net/10356/169289 |
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author | Tan, Shaun Jun Hao |
author2 | James P Tam |
author_facet | James P Tam Tan, Shaun Jun Hao |
author_sort | Tan, Shaun Jun Hao |
collection | NTU |
description | Asparaginyl endopeptidases (AEPs) are thiol proteases that cleave peptide bonds after Asn/Asp(Asx) residues. However, certain AEPs also make Asx bonds and which we named peptide asparaginyl ligases (PALs). PALs, ATP-independent and stand-alone, are ideal for site-specific modifications of proteins, live cells, and biopolymers. Expanding the availability and diversity of PALs will expand tools for biotechnological and biochemical applications. In my thesis, I studied the improved production of the recombinant butelase-1, a prototype PAL, and engineering of PAL from an AEP, PeAEP. Butelase-1 was initially reported for its poor recombinant expression, and I used consensus-based engineering approach successfully to increase its recombinant expression three-fold. The second part of my thesis focuses on engineering of PeAEP into a PAL based on a hypothesis of ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. I used data mining to identify amino acids important for LADs which are located at the substrate binding pockets. They were then mutated in PeAEP to convert it successfully into a PAL. Taken together, PALs can be expanded through consensus-based engineering approach or exploiting our understanding of molecular ligase determinants of PALs for expanding the repertoire of Asx-specific ligases. |
first_indexed | 2024-10-01T03:13:45Z |
format | Thesis-Master by Research |
id | ntu-10356/169289 |
institution | Nanyang Technological University |
language | English |
last_indexed | 2024-10-01T03:13:45Z |
publishDate | 2023 |
publisher | Nanyang Technological University |
record_format | dspace |
spelling | ntu-10356/1692892023-08-01T07:08:34Z Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) Tan, Shaun Jun Hao James P Tam School of Biological Sciences JPTam@ntu.edu.sg Science::Biological sciences Asparaginyl endopeptidases (AEPs) are thiol proteases that cleave peptide bonds after Asn/Asp(Asx) residues. However, certain AEPs also make Asx bonds and which we named peptide asparaginyl ligases (PALs). PALs, ATP-independent and stand-alone, are ideal for site-specific modifications of proteins, live cells, and biopolymers. Expanding the availability and diversity of PALs will expand tools for biotechnological and biochemical applications. In my thesis, I studied the improved production of the recombinant butelase-1, a prototype PAL, and engineering of PAL from an AEP, PeAEP. Butelase-1 was initially reported for its poor recombinant expression, and I used consensus-based engineering approach successfully to increase its recombinant expression three-fold. The second part of my thesis focuses on engineering of PeAEP into a PAL based on a hypothesis of ligase activity determinants (LADs) that control the catalytic directionality of these enzymes. I used data mining to identify amino acids important for LADs which are located at the substrate binding pockets. They were then mutated in PeAEP to convert it successfully into a PAL. Taken together, PALs can be expanded through consensus-based engineering approach or exploiting our understanding of molecular ligase determinants of PALs for expanding the repertoire of Asx-specific ligases. Master of Science 2023-07-11T07:08:42Z 2023-07-11T07:08:42Z 2022 Thesis-Master by Research Tan, S. J. H. (2022). Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs). Master's thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/169289 https://hdl.handle.net/10356/169289 10.32657/10356/169289 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 |
spellingShingle | Science::Biological sciences Tan, Shaun Jun Hao Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title | Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title_full | Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title_fullStr | Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title_full_unstemmed | Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title_short | Expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases (PALs) |
title_sort | expanding the ligation toolbox through protein engineering of peptide asparaginyl ligases pals |
topic | Science::Biological sciences |
url | https://hdl.handle.net/10356/169289 |
work_keys_str_mv | AT tanshaunjunhao expandingtheligationtoolboxthroughproteinengineeringofpeptideasparaginylligasespals |