Abstract
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
Substances
-
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.