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

Malgorzata Teodorowicz, Olaf Perdijk, Iris Verhoek, Coen Govers, Huub F.J. Savelkoul, Yongfu Tang, Harry Wichers, Kerensa Broersen

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18 Citations (Scopus)
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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 nontreated 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.

Original languageEnglish
Article number0173778
JournalPLoS ONE
Volume12
Issue number3
DOIs
Publication statusPublished - 29 Mar 2017

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lipopolysaccharides
protein sources
Lipopolysaccharides
Food
Proteins
Macrophages
proteins
methodology
macrophages
Contamination
HEK293 Cells
Assays
lactoglobulins
Nonidet P-40
cells
Lactoglobulins
Soybean Proteins
structural proteins
Masks
Cell Extracts

Cite this

Teodorowicz, Malgorzata ; Perdijk, Olaf ; Verhoek, Iris ; Govers, Coen ; Savelkoul, Huub F.J. ; Tang, Yongfu ; Wichers, Harry ; Broersen, Kerensa. / Optimized triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. In: PLoS ONE. 2017 ; Vol. 12, No. 3.
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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 nontreated 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.",
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Optimized triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins. / Teodorowicz, Malgorzata; Perdijk, Olaf; Verhoek, Iris; Govers, Coen; Savelkoul, Huub F.J.; Tang, Yongfu; Wichers, Harry; Broersen, Kerensa.

In: PLoS ONE, Vol. 12, No. 3, 0173778, 29.03.2017.

Research output: Contribution to journalArticleAcademicpeer-review

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AU - Teodorowicz, Malgorzata

AU - Perdijk, Olaf

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AU - Govers, Coen

AU - Savelkoul, Huub F.J.

AU - Tang, Yongfu

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AU - Broersen, Kerensa

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N2 - 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 nontreated 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.

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