Expanding the scope of substrate specificity for peptide asparaginyl ligases
Asparaginyl endopeptidases (AEPs) are Asn/Asp (Asx)-specific proteases. Peptide asparaginyl ligases (PALs) belong to AEPs that exhibit dominant ligase activity, making them valuable stand-alone biotechnological and biochemical tools for precision biomanufacturing of proteins. Structure-guided mut...
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| Format: | Thesis-Doctor of Philosophy |
| Language: | English |
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Nanyang Technological University
2023
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| Online Access: | https://hdl.handle.net/10356/165142 |
| _version_ | 1826113039971123200 |
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| author | Lim, Ching Koon |
| author2 | James P Tam |
| author_facet | James P Tam Lim, Ching Koon |
| author_sort | Lim, Ching Koon |
| collection | NTU |
| description | Asparaginyl endopeptidases (AEPs) are Asn/Asp (Asx)-specific proteases. Peptide asparaginyl
ligases (PALs) belong to AEPs that exhibit dominant ligase activity, making them valuable
stand-alone biotechnological and biochemical tools for precision biomanufacturing of proteins.
Structure-guided mutagenesis in AEP has been the gold standard for protein engineering, but
it remains laborious and time-consuming. Meanwhile, AEP substrates are also an equally
important parameter that affects ligation efficiency and often neglected, as their dipeptide
leaving groups following AEP catalysis exist as competitive nucleophiles that reverse ligation
reaction, thus requiring substantial amount of incoming labels to achieve desirable product
yield, particularly in intermolecular ligation. My thesis showed that ligation product yield can
be improved via alteration of substrate incoming (P1'' – P2'') and leaving groups (P1' – P2').
Besides, we have characterized lysine side chain-to-tail cyclization and showed its potential in
multicyclic peptide drug design. Taken together, my thesis provides further insights into
understanding the potential of peptide substrates, which serve as a vital subject for engineering
to enhance ligation product yield in the development of biologics. |
| first_indexed | 2024-10-01T03:16:40Z |
| format | Thesis-Doctor of Philosophy |
| id | ntu-10356/165142 |
| institution | Nanyang Technological University |
| language | English |
| last_indexed | 2024-10-01T03:16:40Z |
| publishDate | 2023 |
| publisher | Nanyang Technological University |
| record_format | dspace |
| spelling | ntu-10356/1651422023-04-04T02:58:00Z Expanding the scope of substrate specificity for peptide asparaginyl ligases Lim, Ching Koon James P Tam School of Biological Sciences JPTam@ntu.edu.sg Science::Biological sciences::Biochemistry Asparaginyl endopeptidases (AEPs) are Asn/Asp (Asx)-specific proteases. Peptide asparaginyl ligases (PALs) belong to AEPs that exhibit dominant ligase activity, making them valuable stand-alone biotechnological and biochemical tools for precision biomanufacturing of proteins. Structure-guided mutagenesis in AEP has been the gold standard for protein engineering, but it remains laborious and time-consuming. Meanwhile, AEP substrates are also an equally important parameter that affects ligation efficiency and often neglected, as their dipeptide leaving groups following AEP catalysis exist as competitive nucleophiles that reverse ligation reaction, thus requiring substantial amount of incoming labels to achieve desirable product yield, particularly in intermolecular ligation. My thesis showed that ligation product yield can be improved via alteration of substrate incoming (P1'' – P2'') and leaving groups (P1' – P2'). Besides, we have characterized lysine side chain-to-tail cyclization and showed its potential in multicyclic peptide drug design. Taken together, my thesis provides further insights into understanding the potential of peptide substrates, which serve as a vital subject for engineering to enhance ligation product yield in the development of biologics. Doctor of Philosophy 2023-03-15T01:36:22Z 2023-03-15T01:36:22Z 2022 Thesis-Doctor of Philosophy Lim, C. K. (2022). Expanding the scope of substrate specificity for peptide asparaginyl ligases. Doctoral thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/165142 https://hdl.handle.net/10356/165142 10.32657/10356/165142 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::Biochemistry Lim, Ching Koon Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title | Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title_full | Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title_fullStr | Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title_full_unstemmed | Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title_short | Expanding the scope of substrate specificity for peptide asparaginyl ligases |
| title_sort | expanding the scope of substrate specificity for peptide asparaginyl ligases |
| topic | Science::Biological sciences::Biochemistry |
| url | https://hdl.handle.net/10356/165142 |
| work_keys_str_mv | AT limchingkoon expandingthescopeofsubstratespecificityforpeptideasparaginylligases |