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Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli

PURPOSE: Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicte...

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Autores principales: Dastmalchi, Mahrokh, Alizadeh, Mahdiyeh, Jamshidi-Kandjan, Omid, Rezazadeh, Hassan, Hamzeh-Mivehroud, Maryam, Farajollahi, Mohammad M, Dastmalchi, Siavoush
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Tabriz University of Medical Sciences 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10676546/
https://www.ncbi.nlm.nih.gov/pubmed/38022803
http://dx.doi.org/10.34172/apb.2023.085
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author Dastmalchi, Mahrokh
Alizadeh, Mahdiyeh
Jamshidi-Kandjan, Omid
Rezazadeh, Hassan
Hamzeh-Mivehroud, Maryam
Farajollahi, Mohammad M
Dastmalchi, Siavoush
author_facet Dastmalchi, Mahrokh
Alizadeh, Mahdiyeh
Jamshidi-Kandjan, Omid
Rezazadeh, Hassan
Hamzeh-Mivehroud, Maryam
Farajollahi, Mohammad M
Dastmalchi, Siavoush
author_sort Dastmalchi, Mahrokh
collection PubMed
description PURPOSE: Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. METHODS: Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. RESULTS: Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). CONCLUSION: A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications.
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spelling pubmed-106765462023-06-12 Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli Dastmalchi, Mahrokh Alizadeh, Mahdiyeh Jamshidi-Kandjan, Omid Rezazadeh, Hassan Hamzeh-Mivehroud, Maryam Farajollahi, Mohammad M Dastmalchi, Siavoush Adv Pharm Bull Original Article PURPOSE: Medical usage of L-asparaginase (ASNase), the first-line of acute lymphoblastic leukemia treatment, is linked to allergic responses and toxicities, which necessitates the development of new bio-better ASNases. The aim of the current study was in silico design of a novel ASNase with predicted improved enzymatic properties using strategies encompassing sequence-function analysis of known ASNase mutants. Additionally, current study aimed to show that the new enzyme is active. METHODS: Based on 21 experimentally reported mutations for ASNase, a virtual library of mutated enzymes with all 7546 possible combinations of up to 4 mutations was generated. Three-dimensional models of proposed mutant enzymes were built and their in silico stabilities were calculated. The most promising mutant was selected for preparing a genetic construct suitable for expression of the designed ASNase in bacterial cells. RESULTS: Computational study predicted that Y176F/S241C double mutation of Escherichia coli ASNase may increase its folding stability. The designed ASNase was expressed in two different E. coli strains (Origami B(DE3) and BL21(DE3)pLysS) and then the soluble fractions prepared from the cell lysates of the host cells were used in enzyme activity assay. Results showed that enzyme activity of soluble fraction from Origami (95.4 ± 7.5 IU/0.1 mL) was four times higher than that of soluble fraction from pLysS (25.8 ± 2.5 IU/0.1 mL). CONCLUSION: A novel functional double mutant ASNase with predicted improved enzymatic properties was designed and produced in E. coli. The results of the current study suggest a great commercial potential for the identified enzyme in pharmaceutical and industrial applications. Tabriz University of Medical Sciences 2023-11 2023-06-12 /pmc/articles/PMC10676546/ /pubmed/38022803 http://dx.doi.org/10.34172/apb.2023.085 Text en ©2023 The Authors. https://creativecommons.org/licenses/by/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers.
spellingShingle Original Article
Dastmalchi, Mahrokh
Alizadeh, Mahdiyeh
Jamshidi-Kandjan, Omid
Rezazadeh, Hassan
Hamzeh-Mivehroud, Maryam
Farajollahi, Mohammad M
Dastmalchi, Siavoush
Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title_full Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title_fullStr Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title_full_unstemmed Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title_short Expression and Biological Evaluation of an Engineered Recombinant L-asparaginase Designed by In Silico Method Based on Sequence of the Enzyme from Escherichia coli
title_sort expression and biological evaluation of an engineered recombinant l-asparaginase designed by in silico method based on sequence of the enzyme from escherichia coli
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10676546/
https://www.ncbi.nlm.nih.gov/pubmed/38022803
http://dx.doi.org/10.34172/apb.2023.085
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