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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...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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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. |
format | Online Article Text |
id | pubmed-10095615 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
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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