TX-114 assisted LPS extraction reduce LPS levels in BLG and SPE extracts to the levels below 1 EU/ml.

<p><i>(A)</i> LPS concentration in protein preparations before (BLG, SPE) and after purification (pBLG, pSPE) with TX-114 method. <i>(B)</i> 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.</p

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.

<p>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).</p

THP-1 derived macrophages as a model to study immunomodulatory potential of non-purified and LPS-purified BLG preparation.

<p><i>(A)</i> 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 <i>t</i>-tests: * = P<0.05. <i>(B)</i> 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). <i>(C-E)</i> 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).</p

Protein yield and structure are retained upon application of the TX-114 assisted LPS extraction procedure.

<p><i>(A)</i> 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. <i>(C</i>, <i>D)</i> Intrinsic tryptophan fluorescence and far-UV CD spectra <i>(E</i>, <i>F)</i> 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.</p

Optimization of TX-114 removal method from beta-lactoglobulin (BLG) and soy protein extract (SPE).

<p><i>(A)</i> 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. <i>(B)</i> TX-114 in PBS dose-dependent absorption at 280 nm. Results are corrected for PBS background and represent an average of three independent measurements. <i>(C)</i> 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. <i>(D)</i> 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. <i>(E)</i> 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).</p