Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

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 di...

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Bibliographic Details
Published in:	Microbial cell factories 2015-04, Vol.14 (1), p.57-57, Article 57
Main Authors:	Mamat, Uwe, Wilke, Kathleen, Bramhill, David, Schromm, Andra Beate, Lindner, Buko, Kohl, Thomas Andreas, Corchero, José Luis, Villaverde, Antonio, Schaffer, Lana, Head, Steven Robert, Souvignier, Chad, Meredith, Timothy Charles, Woodard, Ronald Wesley
Format:	Article
Language:	eng
Subjects:	Aldose-Ketose Isomerases - genetics Aldose-Ketose Isomerases - metabolism ATP-Binding Cassette Transporters - genetics ATP-Binding Cassette Transporters - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism Biosynthetic Pathways - genetics Carbohydrate Sequence Consulting services Electrophoresis, Polyacrylamide Gel Endotoxins - biosynthesis Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Gene Deletion Glycolipids - biosynthesis Health aspects Lipid A - analogs & derivatives Lipid A - biosynthesis Lipopolysaccharides - biosynthesis Mass Spectrometry Metabolic Engineering - methods Mitogens Molecular Sequence Data Mutation Production processes Recombinant Proteins - biosynthesis Recombinant Proteins - isolation & purification Reproducibility of Results Sugar Acids - metabolism
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Summary:	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. 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. 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.
ISSN:	1475-2859 1475-2859