Abstract
Lipopolysaccharide (LPS, endotoxin, pyrogen) constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol). During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU) in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 103 and 105 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 103-105 EU/109 PFU (plaque forming units) down to an average of 2.8 EU/109 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli) and F8 (P. aeruginosa).
Highlights
During the last decades antibiotic resistance among pathogenic bacteria has increased and forms a serious threat for humans and animals
The amount of endotoxins is defined by an endotoxic unit (EU) which corresponds to activity of 100 pg of E. coli lipopolysaccharide
The purification procedure begun with the removal of debris from the T4 bacteriophage lysate of E. coli by filtration, and for smaller scales prior centrifugation (8000×g, 30 min)
Summary
During the last decades antibiotic resistance among pathogenic bacteria has increased and forms a serious threat for humans and animals. While it is difficult to assign hydrophilicity to complex molecular assemblies such as bacteriophages, our experiments with ion-exchange chromatography and electrodynamic measurements of phages suggested that extraction methods could be employed for their purification. This was tempting, since preparative isolation of LPS entails a step, in which lipopolysaccharide is extracted into phenol [30] and butanol [31]. Removal of LPS from biomolecules by extraction was previously accomplished with Triton X-114 [32,33,34] and tetra(ethyleneoxide) decyl ether [35] In these methods the detergent-water system is maintained outside the miscibility area by adjusting temperature or concentration. We demonstrate that endotoxin can be efficiently removed from a bacteriophage lysate by extraction with water immiscible solvents such as butanol and octanol, which are removed
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