Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production
| Autor: | Joshua W. Wilkerson, Rebecca L. Swensen, Michael L. Christian, Bradley C. Bundy, John Hunt, Parker J. Funk, William C. Carver, Kristen M. Wilding |
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| Rok vydání: | 2018 |
| Předmět: |
0106 biological sciences
Lysis medicine.disease_cause 01 natural sciences Applied Microbiology and Biotechnology Chromatography Affinity chemistry.chemical_compound Affinity chromatography Neoplasms 010608 biotechnology Escherichia coli medicine Protein biosynthesis Humans Cell-free protein synthesis Downstream processing Septic shock 010401 analytical chemistry Proteins General Medicine medicine.disease 0104 chemical sciences Endotoxins chemistry Biochemistry Protein Biosynthesis Polylysine Molecular Medicine |
| Zdroj: | Biotechnology Journal. 14:1800271 |
| ISSN: | 1860-6768 |
| DOI: | 10.1002/biot.201800271 |
| Popis: | Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated. |
| Databáze: | OpenAIRE |
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