Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins

PLoS One. 2017 Mar 29;12(3):e0173778. doi: 10.1371/journal.pone.0173778. eCollection 2017.


Scope: 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.

Methods and 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.

Conclusion: 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.

MeSH terms

  • Animals
  • Cattle
  • Cell Line
  • Detergents / chemistry*
  • Detergents / isolation & purification
  • Food Analysis
  • Gene Expression / drug effects
  • HEK293 Cells
  • Humans
  • Interleukin-1beta / genetics
  • Interleukin-1beta / immunology
  • Interleukin-6 / genetics
  • Interleukin-6 / immunology
  • Interleukin-8 / genetics
  • Interleukin-8 / immunology
  • Lactoglobulins / chemistry
  • Lactoglobulins / pharmacology*
  • Lipopolysaccharides / isolation & purification*
  • Lipopolysaccharides / pharmacology
  • Liquid-Liquid Extraction / methods*
  • Macrophage Activation / drug effects
  • Macrophages / cytology
  • Macrophages / drug effects*
  • Macrophages / immunology
  • Octoxynol
  • Polyethylene Glycols / chemistry*
  • Polyethylene Glycols / isolation & purification
  • Toll-Like Receptor 2 / genetics
  • Toll-Like Receptor 2 / immunology
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / immunology
  • Tumor Necrosis Factor-alpha / genetics
  • Tumor Necrosis Factor-alpha / immunology


  • Detergents
  • IL1B protein, human
  • IL6 protein, human
  • Interleukin-1beta
  • Interleukin-6
  • Interleukin-8
  • Lactoglobulins
  • Lipopolysaccharides
  • TLR2 protein, human
  • TLR4 protein, human
  • Toll-Like Receptor 2
  • Toll-Like Receptor 4
  • Tumor Necrosis Factor-alpha
  • Polyethylene Glycols
  • Octoxynol
  • Nonidet P-40

Grants and funding

Malgorzata Teodorowicz was supported by European Seventh Framework Program FP7-PEOPLE-2011-IEF, grant number PIEF-GA-2011-302295. Presented work was a part of the FibeBiotics EU project supported by the European Community’s Seventh Framework Programme [FP7/2007-2013]’ and ‘Nutricia Research Foundation’ project number 2012-35. Kerensa Broersen is supported by a UTWIST fellowship.