Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins
Mamat, Uwe (Forschungszentrum Borstel)
Wilke, Kathleen (Forschungszentrum Borstel)
Bramhill, David (Research Corporation Technologies (Tucson))
Schromm, Andra Beate (Forschungszentrum Borstel)
Lindner, Buko (Forschungszentrum Borstel)
Kohl, Thomas Andreas (Forschungszentrum Borstel)
Corchero Nieto, José Luis (Universitat Autònoma de Barcelona. Institut de Biotecnologia i de Biomedicina "Vicent Villar Palasí")
Villaverde Corrales, Antonio (Universitat Autònoma de Barcelona. Departament de Genètica i de Microbiologia)
Schaffer, Lana (Scripps Research Institute)
Head, Steven Robert (Scripps Research Institute)
Souvignier, Chad (Research Corporation Technologies (Tucson))
Meredith, Timothy Charles (Pennsylvania State University. Department of Biochemistry and Molecular Biology)
Woodard, Ronald Wesley (Bramhill Biological Consulting)

Data:	2015
Resum:	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.
Ajuts:	European Commission 278864
Nota:	Es va publicar un treball amb esmenes a aquest article que es pot consultar a https://ddd.uab.cat/record/185349 (DOI 10.1186/s12934-015-0265-x)
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	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
Publicat a:	Microbial cell factories, Vol. 14, Issue 57 (2015) , ISSN 1475-2859

DOI: 10.1186/s12934-015-0241-5
PMID: 25890161

15 p, 3.4 MB

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