Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

Microbial Cell Factories

Research

José Luis Corchero¹  Timothy Charles Meredith²  Ronald Wesley Woodard³  Buko Lindner⁴  Andra Beate Schromm⁵  Thomas Andreas Kohl⁶  Uwe Mamat⁷  Kathleen Wilke⁷  Antonio Villaverde⁸  Lana Schaffer⁹  Steven Robert Head⁹  Chad Souvignier^1,10  David Bramhill^1,11 

[1] CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain;Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain;Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain;Department of Biochemistry and Molecular Biology, 206 South Frear, Pennsylvania State University, 16802, University Park, PA, USA;Department of Medicinal Chemistry, University of Michigan, 428 Church Street, 48109-1065, Ann Arbor, MI, USA;Division of Bioanalytical Chemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany;Division of Immunobiophysics, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany;Division of Molecular Mycobacteriology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany;Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Parkallee 1-40, D-23845, Borstel, Germany;Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain;Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193, Cerdanyola del Vallès, Spain;CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Bellaterra, 08193, Cerdanyola del Vallès, Spain;NGS and Microarray Core Facility, The Scripps Research Institute, 10550 North, Pines Road, La Jolla, 92037, Torrey, CA, USA;Research Corporation Technologies, Inc, 5210 East Williams Circle, Suite 240, 85711-4410, Tucson, AZ, USA;Research Corporation Technologies, Inc, 5210 East Williams Circle, Suite 240, 85711-4410, Tucson, AZ, USA;Present address: Bramhill Biological Consulting, LLC, 8240 East Moonstone Drive, 85750, Tucson, AZ, USA;

关键词: Escherichia coli;    Lipopolysaccharide;    Lipid A;    Endotoxic activity;    Recombinant protein;    TLR4/MD-2 activation;   

DOI  :  10.1186/s12934-015-0241-5

 received in 2014-12-23, accepted in 2015-04-07,  发布年份 2015

来源: Springer

【 摘 要 】

BackgroundLipopolysaccharide (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.ResultsAs 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.ConclusionsThis paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination.

【 授权许可】

CC BY   
© Mamat et al.; licensee BioMed Central. 2015