Fig 1.
Optimization of TX-114 removal method from beta-lactoglobulin (BLG) and soy protein extract (SPE).
(A) Triton concentrations can be quantified in protein-free solutions using spectroscopic absorbance at 280 nm. Absorbance spectrum of 0.005% (v/v) TX-114 solution in PBS was determined by a NanoDrop ND1000 spectrophotometer. (B) TX-114 in PBS dose-dependent absorption at 280 nm. Results are corrected for PBS background and represent an average of three independent measurements. (C) Concentration of TX-114 in PBS spiked with LPS (0.45 EU/l) after applying the TX-114 treatment described in Materials and Methods measured after one TX-114 cycle, three TX-114 cycles or after one TX-114 cycle followed with Bio-Beads treatment. (D) TX-114 reduces LPS detection with EndoZyme recombinant factor C assay in a dose-dependent manner. LPS concentration was measured in PBS spiked with 0.45 EU/l of LPS and decreasing concentration of TX-114. (E) Concentration of TX-114 in the medium equal or higher than 0.006% (v/v) decreases the viability of THP-1 derived macrophages. Viability of THP-1 macrophages cultured for 24 h in the presence of TX-114 in the medium expressed as relative to cells grown in TX-114 free medium = 1).
Fig 2.
Extraction of remaining TX-114 from protein extract with high-affinity Bio-Beads results in most effective lowering of detergent concentration to non-toxic levels.
Comparison of different TX-114 extraction methods. Starting from an initial TX-114 concentration of ~ 2% (v/v), the concentration of this detergent is effectively lowered by the application of dialysis, various centrifugation conditions, spin-X column and Bio-Bead assisted purification. Application of Bio-Beads results in most efficient TX-114 extraction down to 0.005% (v/v).
Fig 3.
Protein yield and structure are retained upon application of the TX-114 assisted LPS extraction procedure.
(A) BLG and SPE were dissolved in PBS at an initial concentration of ~ 10 mg/ml and protein concentrations were determined again after application of the LPS extraction procedure. (B) Non-reducing SDS-PAGE of not purified BLG, SPE and LPS- and Triton-extracted (TX BLG, TX SPE), M—molecular weight marker. (C, D) Intrinsic tryptophan fluorescence and far-UV CD spectra (E, F) of BLG and SPE. BLG concentrations used for far-UV CD were determined spectroscopically using absorbance at 280 nm and resulting CD spectra were normalized for molar ellipticity. SPE CD spectra were distorted at wavelength values below 205 nm as a result of high (>800) voltage.
Fig 4.
TX-114 assisted LPS extraction reduce LPS levels in BLG and SPE extracts to the levels below 1 EU/ml.
(A) LPS concentration in protein preparations before (BLG, SPE) and after purification (pBLG, pSPE) with TX-114 method. (B) Repeated TX-114-assisted LPS extraction does not affect LPS extraction efficiency. BLG or PBS spiked with 0.45 EU/l of LPS were treated once, twice or three times with a 2% (v/v) TX-114 solution and subjected to incubation at 4°C for 30 min followed by a 10 min incubation at 37°C and centrifugation.
Fig 5.
Reduced TLR4 (A,B) and TLR2 (C) activation upon incubation of HEK-Blue 293 with LPS purified protein preparations. HEK-Blue 293 cells were incubated 24 h with non-treated BLG (BLG, black bars A, C), BLG purified with TX-114 method (TX BLG, white bars A, C), non-treated SPE (SPE, black bars B), SPE purified with TX-114 method (TX SPE, white bars B). The samples were tested in a range of dilutions and the corresponding concentration of protein (mg/ml) and LPS (EU/ml) in each dilution is presented on the x axis. The results are expressed as the relative to unstimulated cells (medium control = 1). Statistically significant differences between corresponding dilutions of LPS-purified and non-treated preparations are shown (p<0.05). LPS standard curve, MQ water and MQ water spiked with 0.005% of TX-114 (MQ/TX-114) were used as controls.
Fig 6.
THP-1 derived macrophages as a model to study immunomodulatory potential of non-purified and LPS-purified BLG preparation.
(A) LPS dose-dependence and differential gene expression of inflammatory cytokines and NF-κB in THP-1 macrophages. The data obtained for 3 repetitions is normalized to GAPDH and relative to unstimulated macrophages cultured in medium. Statistically significant differences relative to non-stimulated THP-1 macrophages were calculated with Student’s t-tests: * = P<0.05. (B) LPS dose-dependent secretion of IL-8 by THP-1 macrophages. THP-1 cells were incubated 24h with increasing concentration of LPS and IL-8 concentration in supernatant was determined with flow cytometry. The results and statistically significant differences are related to unstimulated cells medium control = 1). (C-E) Dose dependent secretion of pro-inflammatory cytokines from THP-1 macrophages incubated 24h with non-purified BLG (BLG) and BLG purified with TX-114 method (TX BLG). The results are expressed as the relative to unstimulated cells (medium control = 1). Statistically significant differences between corresponding dilutions of purified and non-purified BLG preparations are shown (p<0.05).