Redefining the Minimal Lipopolysaccharide Structure in Escherichia coli: Development of Endotoxin-free E.Coli Strains

Synopsis

Recombinant proteins, commonly manufactured in Escherichia coli, are inherently contaminated with constituents of the bacterial cell such as lipopolysaccharide (LPS). LPS, also known as endotoxin, is a potent agonist for hTLR4/MD-2-mediated proinflammatory activity in human immune cells. To prevent toxicity, biological material prepared from E. coli must be essentially free of endotoxin.

However, efficient and cost-effective elimination of endotoxin is still a challenging task, and none of the downstream applications described thus far remove endotoxin entirely. As an alternative strategy for the production of endotoxin-free biomolecules, a set of non-conditional E. coli derivatives was constructed that lack all outer membrane agonists for hTLR4/MD-2 activation.

The mutant strains entirely lack LPS, yet remain viable despite exclusively elaborating the tetra-acylated, endotoxically inactive lipid A precursor lipid IVA. Viability requires a suppressor mutation that enables the strains to translocate lipid IVA across the inner membrane to support outer membrane biogenesis. Consistent with the results that activation of hTLR4/MD-2 signalling by the mutant cells was of several orders of magnitude lower compared with cells of the E. coli wild-type strain, heterologous proteins prepared from different mutant strains were free of endotoxic activity. This new series of genetically engineered E. coli strains lacking LPS holds promise for the manufacture of endotoxin-free products.

Programme

Day 1 - 03 June 2016

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Speaker

Dr Uwe Mamat, Division of Structural Biochemistry, Research Center Borstel, Leibniz-Center for Medicine and Biosciences