title: Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.
abstract: BACKGROUND
  Lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product.
  
  
  RESULTS
  As an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels.
  
  
  CONCLUSIONS
  This paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.
authors.affiliation: Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany. umamat@fz-borstel.de.
authors.forename: Uwe
authors.initials: U
authors.lastname: Mamat
authors.affiliation: Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany. kathleen_wilke@gmx.de.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Wilke
authors.affiliation: Research Corporation Technologies, Inc, 5210 East Williams Circle, Suite 240, Tucson, AZ, 85711-4410, USA. dbramhill@gmail.com.|Present address: Bramhill Biological Consulting, LLC, 8240 East Moonstone Drive, Tucson, AZ, 85750, USA. dbramhill@gmail.com.
authors.forename: David
authors.initials: D
authors.lastname: Bramhill
authors.affiliation: Division of Immunobiophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany. aschromm@fz-borstel.de.
authors.forename: Andra Beate
authors.initials: AB
authors.lastname: Schromm
authors.affiliation: Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany. blindner@fz-borstel.de.
authors.forename: Buko
authors.initials: B
authors.lastname: Lindner
authors.affiliation: Division of Molecular Mycobacteriology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany. tkohl@fz-borstel.de.
authors.forename: Thomas Andreas
authors.initials: TA
authors.lastname: Kohl
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain. jlcorchero1967@gmail.com.|Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. jlcorchero1967@gmail.com.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. jlcorchero1967@gmail.com.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Corchero
authors.affiliation: CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.es.|Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.es.|Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain. antoni.villaverde@uab.es.
authors.forename: Antonio
authors.initials: A
authors.lastname: Villaverde
authors.affiliation: NGS and Microarray Core Facility, The Scripps Research Institute, 10550 North, Pines Road, La Jolla, Torrey, CA, 92037, USA. schaffer@scripps.edu.
authors.forename: Lana
authors.initials: L
authors.lastname: Schaffer
authors.affiliation: NGS and Microarray Core Facility, The Scripps Research Institute, 10550 North, Pines Road, La Jolla, Torrey, CA, 92037, USA. shead@scripps.edu.
authors.forename: Steven Robert
authors.initials: SR
authors.lastname: Head
authors.affiliation: Research Corporation Technologies, Inc, 5210 East Williams Circle, Suite 240, Tucson, AZ, 85711-4410, USA. csouvignier@rctech.com.
authors.forename: Chad
authors.initials: C
authors.lastname: Souvignier
authors.affiliation: Department of Biochemistry and Molecular Biology, 206 South Frear, Pennsylvania State University, University Park, PA, 16802, USA. tcmered@gmail.com.
authors.forename: Timothy Charles
authors.initials: TC
authors.lastname: Meredith
authors.affiliation: Department of Medicinal Chemistry, University of Michigan, 428 Church Street, Ann Arbor, MI, 48109-1065, USA. rww@med.umich.edu.
authors.forename: Ronald Wesley
authors.initials: RW
authors.lastname: Woodard
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D006017:Glycolipids / Q000096:biosynthesis; D008050:Lipid A / Q000031:analogs & derivatives / Q000096:biosynthesis; D008070:Lipopolysaccharides / Q000096:biosynthesis; D013058:Mass Spectrometry; D060847:Metabolic Engineering / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013400:Sugar Acids / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D004731:Endotoxins; D029968:Escherichia coli Proteins; D006017:Glycolipids; D008050:Lipid A; D008070:Lipopolysaccharides; C079726:MsbA protein, Bacteria; D011994:Recombinant Proteins; D013400:Sugar Acids; C040142:lipid A precursors, bacterial; D019747:Aldose-Ketose Isomerases; C477520:D-arabinose 5-phosphate isomerase, E coli; C120356:3-deoxy-manno-oct-2-ulopyranosonic acid