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Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

1
Department of Chemical Engineering, Brigham Young University, Provo, UT 84602, USA
2
Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, OH 43210, USA
*
Author to whom correspondence should be addressed.
Methods Protoc. 2019, 2(2), 52; https://doi.org/10.3390/mps2020052
Received: 2 March 2019 / Revised: 16 April 2019 / Accepted: 18 April 2019 / Published: 21 June 2019
(This article belongs to the Special Issue Cell-Free Synthetic Biology)
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. View Full-Text
Keywords: cell-free protein synthesis; CFPS; tPa; tissue plasminogen activator; ischemic stroke cell-free protein synthesis; CFPS; tPa; tissue plasminogen activator; ischemic stroke
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Yang, S.-O.; Nielsen, G.H.; Wilding, K.M.; Cooper, M.A.; Wood, D.W.; Bundy, B.C. Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator. Methods Protoc. 2019, 2, 52.

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