Figure 1.
Optimization of anion-exchange chromatography method.
(A) Elution of lunasin using a linear NaCl gradient. White flake was mixed with extraction buffer (75.5 mM sodium phosphate, 68.4 mM NaCl, 10 mM sodium metabisulfite, 20 mM ascorbic acid, pH 7.4) at a 12.5∶1 buffer to biomass ratio and mixed for one hour at 4°C. The mixture was filtered through four layers of cheesecloth and one layer of miracloth and then centrifuged at 10,000× g for 10 minutes at 4°C. The supernatant was collected, filtered through a 0.2 µm filter, and the clarified extract stored at 4°C until used. For chromatography, a 5 mL Q-Fast Flow HiTrap pre-packed anion-exchange column was equilibrated with ten CV of Buffer A (75.5 mM Sodium Phosphate, 68.4 mM NaCl, pH 7.4) prior to loading 100 mL of soy clarified extract. The column was washed with five CV of Buffer A to remove unbound proteins. A 25 CV linear salt gradient beginning at 68.4 mM and ending at 1000 mM NaCl at a flow rate of 5 mL/min was used to elute proteins from the column. Five mL fractions were collected during the elution. Fractions were pooled based on the A280 profile and analyzed for lunasin by ELISA and SDS-PAGE. The largest amount of lunasin was detected in fractions 7–11 which corresponds to NaCl concentrations of between 290 mM and 480 mM. (B) Development of a step-gradient elution method. Anion-exchange chromatography was performed using the same 5 mL Q-Fast Flow HiTrap column used in (A). After the column was stripped and re-equilibrated in Buffer A, 100 mL of clarified extract was applied, followed by eight CV washes with Buffer A. A step gradient consisting of six steps (10, 20, 30, 40, 50, and 100% Buffer B) of five CV each at of flow rate of 5 mL/min was used to elute lunasin. Each step fraction was analyzed for the presence of lunasin by ELISA and SDS-PAGE. Lunasin was detected in both the 20% and 30% B elution fractions which correspond to NaCl concentrations of between 255 mM and 348 mM. (C) Optimized step-gradient purification of lunasin using anion-exchange chromatography. A final step gradient chromatography run was performed as described in (B), except that a ten CV wash was done prior to the step elution. Two elution steps of 30% (5 CV) and 100% Buffer B (10 CV) which corresponded to NaCl concentrations of 348 mM and 1000 mM, respectively, were done. The presence of lunasin in each sample was determined by ELISA and SDS-PAGE. Lunasin was detected only within the 30% B elution fraction. Chromatograms show the A280 (solid line ______), percent Buffer B (_ _ _ _ _), and the percent maximum lunasin content as determined by ELISA (-------).
Figure 2.
SDS-PAGE and immunoblot analysis of anion-exchanged purified lunasin.
Aliquots of samples corresponding to the bench-scale anion-exchange chromatography method where lunasin was eluted using a step gradient (Figure 1C) were subjected to SDS-PAGE and immunoblot analysis. (A) SDS-PAGE of the clarified extract, column flow through (Q flow through), and the 30% Buffer B elution (Q 30% B Fraction). Clarified extract, Q flow through, and Q-30%B fraction were prepared at dilutions of 1∶8, 1∶8, and 1∶10, respectively, and electrophoresed using 15% Tris-glycine gels. Molecular weight standards (MW Std) are shown in the first lane. (B) Immunoblot analysis of the clarified extract, Q flow through, and the Q 30% B Fraction. Proteins were separated by SDS-PAGE as described in (A), transferred to a PVDF membrane, and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, clarified extract, Q flow through, and Q-30% B fraction were prepared at dilutions of 1∶20, 1∶20, and 1∶40, respectively. Molecular weight standards (MW Std) are shown in the first lane.
Figure 3.
Detection of lunasin within a 14 kDa protein complex using SDS-PAGE and immunoblot analyses.
Coomassie-stained SDS-PAGE gel (A), and corresponding immunoblot (B) of purified lunasin-containing complex under reducing and non-reducing conditions. The first two lanes represent lunasin (5.1 kDa) and lunasin-containing complex (14.1 kDa) under standard reducing conditions while the 4th and 5th lanes represent equivalent samples under non-reducing conditions (without BME in the sample buffer). Lanes with lunasin contain 300 ng of synthetic lunasin as a reference, while lanes with complex contain 3 µg of lunasin-containing complex. Identification of the lunasin-containing complex by immunoblot analysis was accomplished using a 1∶5000 dilution of rabbit polyclonal anti-lunasin as the primary antibody and a 1∶100,000 dilution of HRP-conjugated goat anti-rabbit as the secondary antibody. Molecular weight standards (MW Std) are shown in the 3rd lane.
Figure 4.
Quantitation of lunasin and lunasin complex in clarified extracts.
White flake (120 g) was extracted with 1.5 L of 75.5 mM sodium phosphate/150 mM NaCl/20 mM ascorbic acid/10 mM sodium metabisulfite, pH 7.4 for one hour. A clarified extract was produced by treating the initial extract with Celpure P100 and filtration using one micron M-503 filter pads. (A) SDS-PAGE analysis of reduced and non-reduced clarified extract. The clarified extract was diluted 1∶10 and analyzed by SDS-PAGE using a 15% Tris-glycine gel under standard reducing and non-reducing (without BME in the sample buffer) conditions. Molecular weight standards (MW Std) are shown in the first lane. The arrow indicates a ∼5 kDa band that corresponds to lunasin. The lack of a clear lunasin band in the sample analyzed under non-reducing conditions indicates that most of the lunasin present in the clarified extract is in protein complexes stabilized by disulfide bridges. (B) Immunoblot analysis of reduced and non-reduced clarified extract. Clarified extract was diluted 1∶10 and subjected to SDS-PAGE along with a series of synthetic lunasin standards as described in (A). The separated proteins were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody diluted 1∶100,000. The amount of lunasin present was determined by image analysis using the synthetic lunasin band intensities to generate a standard curve. This analysis demonstrated that <20% of the extractable lunasin is present in the ∼5 kDa form.
Figure 5.
Reversed-phase chromatography (RPC) method development.
Bench-scale RPC was performed using a 1.6×8.0 cm Source15RPC column that was sanitized with 1 N NaOH and equilibrated with ten CV of Buffer A (75.5 mM sodium phosphate/68.4 mM NaCl, pH 7.4) prior to sample load. The ultrafiltration (UF) permeate was brought to a final concentration of 1 M ammonium sulfate and then applied to the column followed by a five CV wash with Buffer A. Bound proteins were eluted using a five-step gradient consisting of 20%, 40%, 60%, 80%, and 100% Buffer B (64.2 mM sodium phosphate/58 mM NaCl/15% n-propanol (v/v)). Each gradient step was approximately five CV except the 100% B step which was ten CV. The column was then stripped using 65% n-propanol. (A) RPC of the UF permeate. Letters with arrows represent beginning of (a) sample load, (b) column wash, and (c) column strip. The presence of lunasin in each sample was determined by ELISA and SDS-PAGE. Lunasin was detected primarily within the 100% B elution fraction. Chromatogram shows the A280 (solid line ______), the A215 (.__. __.), percent Buffer B (_ _ _ _ _), and the percent maximum lunasin content as determined by ELISA (-------). (B) Coomassie-stained SDS-PAGE gel of RPC fractions. SDS-PAGE using a 15% Tris-glycine gel was performed on 1∶10 dilution and 1∶4 dilutions of the UF permeate and column strip, respectively, and undiluted samples from the column flow through and Buffer B step gradient fractions. Molecular weight standards (MW Std) are shown in the first lane. The majority of lunasin (>95%) was detected in the 100% B eluate, with minor amounts detected in the 80% B eluate and in the column strip. The major contaminating ∼9 kDa protein was detected exclusively in the column strip. (C) Immunoblot analysis of the UF permeate, column flow through, step gradient fractions, and column strip. Proteins separated by SDS-PAGE were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. For SDS-PAGE, dilutions of the UF permeate (1∶10), 80% B eluate (1∶4), 100% B eluate (1∶4), and column strip (1∶4) were made. All other samples were undiluted. The position of the 4 kDa and 6 kDa molecular weight standards (MW Std) are shown in the first lane.
Figure 6.
Mass spectrometry of the purified lunasin.
(A, top panel) Deconvoluted MS Spectra of purified lunasin. The monoisotopic mass of the purified lunasin was found to be 5139.25 Da, which is 114.02 Da higher than the expected monoisotopic mass (5025.23 Da) for the 43 amino-acid form of lunasin described in the literature. The mass difference suggests that the predominant form of our purified lunasin contains 44 amino acids and that it contains an additional asparagine residue. (A, middle panel) Deconvoluted spectrum of lunasin reduced with DTT. Reduction with DTT did not cause a mass shift, indicating there is no disulfide bond present in the purified lunasin. (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The monoisotopic mass of lunasin shifted to 5253.29 Da after alkylation with IAA, which is 114.04 Da higher than unalkylated lunasin. This mass shift confirmed that lunasin has two free cysteine residues as expected. (B) MS/MS spectrum of C-terminal peptide of lunasin. Calculated b and Y ions for the peptide GDDDDDDDDDN are shown in the table inset. The matched b (red) and Y (blue) ions detected match very well the expected fragment ion values for this peptide. Signals corresponding to the loss of one (green) or more H2O molecules, which are expected in MS/MS spectra of peptides with multiple acidic residues, are also evident in the spectrum. These [b – H2O] signals are consistent with the presence of the GDDDDDDDDDN peptide. This analysis confirmed that the residue at the C-terminus of lunasin purified from soybean is asparagine rather than aspartic acid.
Table 1.
Peptides identified from lunasin complex subunits by LC-MS/MS analysis.
Figure 7.
Mass spectrometry of the purified lunasin-containing complex.
(A, top panel) Deconvoluted spectrum of purified lunasin complex. The most abundant isotopic mass in the spectrum is at 14109.3 Da. The mass signal adjacent to lunasin complex (14207.3 Da) is the adduct of lunasin complex with phosphoric acid (plus 98 Da). (A, middle panel) Deconvoluted spectrum of reduced lunasin complex. The most abundant isotopic masses shown in the spectrum are lunasin (5141.3 Da) and soybean albumin long chain (8975.1 Da). (A, bottom panel) Deconvoluted spectrum of lunasin complex treated with DTT and IAA. The most abundant masses shown in the spectrum are lunasin (5256.3 Da) and soybean albumin long chain (9317.2 Da). The monoisotopic masses are 5139.28 Da and 5253.33 Da for lunasin and lunasin treated with DTT and IAA respectively. The monoisotopic masses of lunasin complex and soybean albumin long chain were too low to be detected. (B) Sequence of 2S albumin preproprotein. Sequence in red is corresponds to our purified lunasin and its monoisotopic and average molecular weights are 5139.27 and 5142.43 Da, respectively. Sequence in blue corresponds to soybean albumin long chain and its monoisotopic and average molecular weights are 8969.05 and 8975.17 Da, respectively.
Figure 8.
Pilot scale lunasin purification.
A) Flow diagram of the optimized lunasin purification method. (B) Coomassie-stained SDS-PAGE gel of protein samples representing each stage of the pilot-scale purification. SDS-PAGE using a 15% Tris-glycine gel and diluted samples of clarified extract (1∶20), Q anion-exchange fraction (1∶40), UF permeate (1∶20), and RPC fraction (1∶40). Synthetic lunasin (500 ng) was loaded as a positive control. Molecular weight standards (M) are shown in the first lane. (C) Immunoblot analysis of protein samples representing each stage of pilot-scale purification. Proteins separated by SDS-PAGE as described for (B) were transferred to a PVDF membrane and probed with a lunasin-specific mouse monoclonal antibody. Lunasin was detected in all the samples as a band with an apparent molecular weight of ∼5 kDa. (D) Coomassie-stained SDS-PAGE gel of final RPC-purified lunasin product. SDS-PAGE was performed on a 15% gel using 10 µg of RPC-purified lunasin. Molecular weight standards (M) are shown in the first lane.
Figure 9.
In vitro histone-binding of synthetic and purified lunasin to core histones H3 and H4.
In vitro binding of synthetic and purified lunasin (0.1, 1, 10 and 100 µM) to human recombinant core histones H3 and H4 was assessed utilizing a modified ELISA format using 500 ng of either H3 or H4 as the capture protein. Both synthetic and purified lunasin bound to both histones H3 and H4 in a dose dependant manner. However, while both synthetic and purified lunasin bound to histone H3 with similar affinity, the purified lunasin exhibited a higher affinity for histone H4 when compared to its synthetic counterpart. Error bars represent +/− SD of 5 independent experiments were each sample was assayed in triplicate. Similar results were obtained when either 100 ng or 300 ng of H3 or H4 were used (data not shown).