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Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator
Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent alt...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632163/ http://dx.doi.org/10.3390/mps2020052 |
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author | Yang, Seung-Ook Nielsen, Gregory H. Wilding, Kristen M. Cooper, Merideth A. Wood, David W. Bundy, Bradley C. |
author_facet | Yang, Seung-Ook Nielsen, Gregory H. Wilding, Kristen M. Cooper, Merideth A. Wood, David W. Bundy, Bradley C. |
author_sort | Yang, Seung-Ook |
collection | PubMed |
description | Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours. |
format | Online Article Text |
id | pubmed-6632163 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-66321632019-08-19 Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator Yang, Seung-Ook Nielsen, Gregory H. Wilding, Kristen M. Cooper, Merideth A. Wood, David W. Bundy, Bradley C. Methods Protoc Benchmark Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours. MDPI 2019-06-21 /pmc/articles/PMC6632163/ http://dx.doi.org/10.3390/mps2020052 Text en © 2019 by the authors. 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 (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Benchmark Yang, Seung-Ook Nielsen, Gregory H. Wilding, Kristen M. Cooper, Merideth A. Wood, David W. Bundy, Bradley C. Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title | Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title_full | Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title_fullStr | Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title_full_unstemmed | Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title_short | Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator |
title_sort | towards on-demand e. coli-based cell-free protein synthesis of tissue plasminogen activator |
topic | Benchmark |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6632163/ http://dx.doi.org/10.3390/mps2020052 |
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