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Cell-Free Expression of a Therapeutic Protein Serratiopeptidase

Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious...

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Autores principales: Meng, Yaru, Yang, Miaomiao, Liu, Wanqiu, Li, Jian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095615/
https://www.ncbi.nlm.nih.gov/pubmed/37049893
http://dx.doi.org/10.3390/molecules28073132
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author Meng, Yaru
Yang, Miaomiao
Liu, Wanqiu
Li, Jian
author_facet Meng, Yaru
Yang, Miaomiao
Liu, Wanqiu
Li, Jian
author_sort Meng, Yaru
collection PubMed
description Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca(2+) ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications.
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spelling pubmed-100956152023-04-13 Cell-Free Expression of a Therapeutic Protein Serratiopeptidase Meng, Yaru Yang, Miaomiao Liu, Wanqiu Li, Jian Molecules Article Serratiopeptidase is a clinical therapeutic protein for the treatment of human diseases such as arthritis, bronchitis, and thrombosis. Yet production of this protein in a heterologous host (e.g., Escherichia coli) is difficult due to the issue of protein insolubility and the requirement of laborious refolding procedures. Cell-free protein synthesis (CFPS) systems, derived from crude cell extracts, are effective platforms for the expression of recombinant proteins in vitro. Here, we report a new method to produce serratiopeptidase by using an E. coli-based CFPS system. After rational selection of cell extracts and construction of expression vectors, soluble expression of serratiopeptidase was achieved and the enzyme activity could be readily tested in the cell-free reaction mixture. By further optimizing the key parameters, optimum conditions for the enzyme activity assay were obtained, including the pH value at 5, reaction temperature at 45 °C, substrate concentration at 10 mg/mL, and supplementing Ca(2+) ions at 5 mM. Moreover, the CFPS mixture was freeze-dried and the activity of serratiopeptidase could be regenerated by hydration without losing activity. Overall, the CFPS system enabled soluble expression of serratiopeptidase with catalytic activity, providing a new and promising approach for this enzyme production. Our work extends the utility of the cell-free platform to produce therapeutic proteins with clinical applications. MDPI 2023-03-31 /pmc/articles/PMC10095615/ /pubmed/37049893 http://dx.doi.org/10.3390/molecules28073132 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Meng, Yaru
Yang, Miaomiao
Liu, Wanqiu
Li, Jian
Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title_full Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title_fullStr Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title_full_unstemmed Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title_short Cell-Free Expression of a Therapeutic Protein Serratiopeptidase
title_sort cell-free expression of a therapeutic protein serratiopeptidase
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10095615/
https://www.ncbi.nlm.nih.gov/pubmed/37049893
http://dx.doi.org/10.3390/molecules28073132
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