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Discovery of human-like L-asparaginases with potential clinical use by directed evolution
L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K(M) for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of ba...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Nature Publishing Group UK
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579231/ https://www.ncbi.nlm.nih.gov/pubmed/28860480 http://dx.doi.org/10.1038/s41598-017-10758-4 |
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author | Rigouin, Coraline Nguyen, Hien Anh Schalk, Amanda M. Lavie, Arnon |
author_facet | Rigouin, Coraline Nguyen, Hien Anh Schalk, Amanda M. Lavie, Arnon |
author_sort | Rigouin, Coraline |
collection | PubMed |
description | L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K(M) for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar K(M) for asparagine. We recently identified the low K(M) guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low K(M) chimeras we developed a suitable bacterial selection system (E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low K(M) clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63(N)-h(C) and 65(N)-h(C), share respectively 85.7% and 87.1% identity with the hASNase1 but have a K(M) similar to gpASNase1. These clones possess 100–140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential. |
format | Online Article Text |
id | pubmed-5579231 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-55792312017-09-06 Discovery of human-like L-asparaginases with potential clinical use by directed evolution Rigouin, Coraline Nguyen, Hien Anh Schalk, Amanda M. Lavie, Arnon Sci Rep Article L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K(M) for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar K(M) for asparagine. We recently identified the low K(M) guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low K(M) chimeras we developed a suitable bacterial selection system (E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low K(M) clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63(N)-h(C) and 65(N)-h(C), share respectively 85.7% and 87.1% identity with the hASNase1 but have a K(M) similar to gpASNase1. These clones possess 100–140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential. Nature Publishing Group UK 2017-08-31 /pmc/articles/PMC5579231/ /pubmed/28860480 http://dx.doi.org/10.1038/s41598-017-10758-4 Text en © The Author(s) 2017 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Rigouin, Coraline Nguyen, Hien Anh Schalk, Amanda M. Lavie, Arnon Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title | Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title_full | Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title_fullStr | Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title_full_unstemmed | Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title_short | Discovery of human-like L-asparaginases with potential clinical use by directed evolution |
title_sort | discovery of human-like l-asparaginases with potential clinical use by directed evolution |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579231/ https://www.ncbi.nlm.nih.gov/pubmed/28860480 http://dx.doi.org/10.1038/s41598-017-10758-4 |
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