Endotoxin‐Free E. coli‐Based Cell‐Free Protein Synthesis: Pre‐Expression Endotoxin Removal Approaches for on‐Demand Cancer Therapeutic Production. Issue 3 (20th September 2018)

• Record Type: Journal Article
• Authors: Wilding, Kristen M.; Hunt, John P.; Wilkerson, Joshua W.; Funk, Parker J.; Swensen, Rebecca L.; Carver, William C.; Christian, Michael L.; Bundy, Bradley C.
• Is Part Of: Biotechnology journal. Volume 14:Issue 3(2019)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2018-09-20
• Subjects: cell‐free protein synthesis -- endotoxin‐free -- endotoxin removal -- phase separation -- polylysine affinity; Biotechnology -- Periodicals; 660.605
• DOI: 10.1002/biot.201800271
• Languages: English
• ISSNs: 1860-6768

Abstract

Abstract : 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. Abstract : Endotoxic lipopolysaccharides must be removed from traditional E. coli ‐based cell‐free protein synthesis systems in order to enable on‐demand therapeutic production. In this study, cell‐free protein synthesis extract preparation methods are successfully adapted to reduce or eliminate endotoxin content prior to protein synthesis via extract treatment or modified extract preparation from ClearColi. High yields of endotoxin‐free therapeutic crisantaspase are produced.