Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins
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...
| Authors: | , , , , , , , , , , , , |
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| Format: | article |
| Publication Date: | 2015 |
| Country: | España |
| Institution: | Universitat Autònoma de Barcelona |
| Repository: | Dipòsit Digital de Documents de la UAB |
| Language: | English |
| OAI Identifier: | oai:ddd.uab.cat:132806 |
| Online Access: | https://ddd.uab.cat/record/132806 https://dx.doi.org/urn:doi:10.1186/s12934-015-0241-5 |
| Access Level: | Open access |
| Keyword: | Escherichia coli Lipopolisacàrid LPS Lípid A Activitat endotòxica Proteïna recombinant Activació TLR4/MD-2 Lipopolysaccharide Lipid A Endotoxic activity Recombinant protein TLR4/MD-2 activation |
| Summary: | 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. |
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