title: Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.
abstract: METHODS
  Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for β-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays.
  
  
  RESULTS
  Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination.
  
  
  CONCLUSIONS
  The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.
authors.affiliation: Department of Cell Biology and Immunology, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Malgorzata
authors.identifier: http://orcid.org/0000-0002-4602-285X
authors.initials: M
authors.lastname: Teodorowicz
authors.affiliation: Department of Cell Biology and Immunology, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Olaf
authors.initials: O
authors.lastname: Perdijk
authors.affiliation: Nanobiophysics Group, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands.
authors.forename: Iris
authors.initials: I
authors.lastname: Verhoek
authors.affiliation: Food and Biobased Research, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Coen
authors.initials: C
authors.lastname: Govers
authors.affiliation: Department of Cell Biology and Immunology, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Huub F J
authors.initials: HF
authors.lastname: Savelkoul
authors.affiliation: Food and Biobased Research, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Yongfu
authors.initials: Y
authors.lastname: Tang
authors.affiliation: Food and Biobased Research, Wageningen University and Research, Wageningen, the Netherlands.
authors.forename: Harry
authors.initials: H
authors.lastname: Wichers
authors.affiliation: Nanobiophysics Group, Faculty of Science and Technology, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, the Netherlands.
authors.forename: Kerensa
authors.initials: K
authors.lastname: Broersen
mesh_terms: D000818:Animals; D002417:Cattle; D002460:Cell Line; D003902:Detergents / Q000737:chemistry* / Q000302:isolation & purification; D005504:Food Analysis; D015870:Gene Expression / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D053583:Interleukin-1beta / Q000235:genetics / Q000276:immunology; D015850:Interleukin-6 / Q000235:genetics / Q000276:immunology; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology; D007782:Lactoglobulins / Q000737:chemistry / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000302:isolation & purification* / Q000494:pharmacology; D059625:Liquid-Liquid Extraction / Q000379:methods*; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000276:immunology
chemical_list: D003902:Detergents; C508585:IL1B protein, human; C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; D016209:Interleukin-8; D007782:Lactoglobulins; D008070:Lipopolysaccharides; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40