title: Endotoxin depletion of recombinant protein preparations through their preferential binding to histidine tags.
abstract: The presence of endotoxins in preparations of recombinantly produced therapeutic proteins poses serious problems for patients. Endotoxins can cause fever, respiratory distress syndromes, intravascular coagulation, or endotoxic shock. A number of methods have been devised to remove endotoxins from protein preparations using separation procedures based on molecular mass or charge properties. Most of the methods are limited in their endotoxin removal capacities and lack general applicability. We are describing a biotechnological approach for endotoxin removal. This strategy exploits the observation that endotoxins form micelles that expose negative charges on their surface, leading to preferential binding of endotoxins to cationic surfaces, allowing the separation from their resident protein. Endotoxins exhibit high affinity to stretches of histidines, which are widely used tools to facilitate the purification of recombinant proteins. They bind to nickel ions and are the basis for protein purification from cellular extracts by immobilized metal affinity chromatography. We show that the thrombin-mediated cleavage of two histidine tags from the purified recombinant protein and the adsorption of these histidine tags and their associated endotoxins to a nickel affinity column result in an appreciable depletion of the endotoxins in the purified protein fraction.
authors.affiliation: Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt am Main, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Mack
authors.affiliation: Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt am Main, Germany.
authors.forename: Boris
authors.initials: B
authors.lastname: Brill
authors.affiliation: Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt am Main, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Delis
authors.affiliation: Georg-Speyer-Haus, Institute for Tumor Biology and Experimental Therapy, 60596 Frankfurt am Main, Germany. Electronic address: groner@em.uni-frankfurt.de.
authors.forename: Bernd
authors.initials: B
authors.lastname: Groner
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002626:Chemistry, Pharmaceutical / Q000379:methods* / Q000592:standards / Q000639:trends; D004731:Endotoxins / Q000302:isolation & purification*; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008070:Lipopolysaccharides / Q000302:isolation & purification; D051379:Mice; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D004731:Endotoxins; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D006639:Histidine
keywords: Affinity chromatography; Histidine tags; Lipopolysaccharides; Recombinant protein purification; Thrombin cleavage