Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins.

Uwe Mamat,Buko Lindner,Timothy Charles Meredith,Ronald Wesley Woodard,Lana Schaffer,Antonio Villaverde,Chad Souvignier,David Bramhill,Thomas Andreas Kohl,Kathleen Wilke,Andra Beate Schromm,Steven Robert Head,José Luis Corchero

doi:10.1186/s12934-015-0241-5

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0241-5) contains supplementary material, which is available to authorized users.

Highlights

Lipopolysaccharide (LPS), referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria
Research relating the structure of LPS to the activation of Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) has demonstrated that lipid A is the component of LPS that is responsible for its TLR4/MD-2-dependent endotoxic activity [8]
This strain contains deletions of kdsD and gutQ, which encode Darabinose 5-phosphate isomerases essential for the biosynthesis of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) [13,14], and a C:G to T:A transition at position 52 of msbA, which acts as a suppressor of the normally lethal ΔKdo phenotype [12]

Summary

Introduction

Lipopolysaccharide (LPS), 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, represents the endotoxic principle of LPS. One third of the unique recombinant protein therapeutics [2] and approximately one half of all products approved [3] are produced using an Escherichia coli based expression platform. The outer membrane of E. coli, like that of most Gram-negative bacteria, contains the potent immunostimulatory molecule lipopolysaccharide (LPS). LPS-mediated activation of a cell-surface receptor, consisting of Toll-like receptor 4 (TLR4) complexed with myeloid differentiation factor 2 (MD-2), results in the production of pro-inflammatory cytokines and type-1 interferons that are the chief effectors of the endotoxic response [7]. When all the secondary acyl chains are removed, the under-acylated lipid A precursor lipid IVA lacks endotoxic activity in human immune cells, and becomes a hTLR4/ MD-2 receptor antagonist [8]

Methods

Results

Conclusion