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Endotoxin-free E. coli-based cell-free protein synthesis: Pre-expression endotoxin removal approaches for on-demand cancer therapeutic production.

Approximately one third of protein therapeutics are produced in E. 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. Here we evaluate three strategies for removing endotoxins from E. coli cell lysate: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi® cells. We demonstrate that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, we demonstrate the first adaptation of ClearColi® cells to prepare cell-free extract with high protein synthesis capability. Finally, we demonstrate production of the lymphocytic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi® extract.

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