Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages

摘要: Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor α (TNFα) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 Toll-like receptor 4-mediated signal transduction pathway. In current study, we demonstrated highly purified preparation (designated as rhHsp70–1) with LPS content 1.4 pg/μg was unable TNFα release RAW264.7 murine at concentrations up 5 μg/ml. contrast, less rhHsp70–2) 1 μg/ml 0.2 ng/μg able same extent induced ng/ml LPS. Failure rhHsp70–1 not because defective physical properties since rhHsp70–2 contained identical hsp70 determined SDS gels stained Coomassie Blue Western blots probed an anti-rhHsp70 antibody. Both preparations also had enzymatic activities judged their ability remove clathrin from clathrin-coated vesicles. Removal polymyxin B-agarose column or direct addition B incubation medium essentially eliminated TNFα-inducing activity rhHsp70–2. The concentration found resulted observed rhHsp-2, alone, plus were all equally sensitive inactivation. These results suggest rhHsp-70 does is entirely due contaminating