Conceived and designed the experiments: BWB SDH KRD. Performed the experiments: LES BWB EJM SDH JC KB. Analyzed the data: LES EJM SDH JC KB KRD. Wrote the paper: KRD LES EJM JC KB.
Current address: BASF Plant Science, L.P., Research Triangle Park, North Carolina, United States of America
Portions of this research were funded by Owensboro Grain Company, LLC. Owensboro Grain Company was not involved in the performance or reporting of the described research. SDH is an employee of Kentucky BioProcessing, LLC, and KRD serves on the Board of Directors of Kentucky BioProcessing; KRD does not receive any compensation for this service. LES, BWB, and KRD are listed as inventors on a patent application that includes some of the data presented in this manuscript. These authors may benefit financially if the technology described in this patent is licensed or sold. There are no further products in development or marketed products to declare. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.
Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin
Lunasin has been described as a 43 amino-acid peptide that is encoded within the soybean GM2S-1 gene and was first identified as a novel peptide found in soybean seed extracts
More recent studies have demonstrated that lunasin can inhibit the growth of some cancer cells in culture and in a mouse xenograft model
Although the potential anticancer effect of lunasin has been known for over a decade, little progress has been made to test
Previous reports describing the partial purification of lunasin utilized extraction of soy flour with water and phosphate buffered saline (PBS)
Previously published results
(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×
Aliquots of samples corresponding to the bench-scale anion-exchange chromatography method where lunasin was eluted using a step gradient (
During our analysis of the Q-Sepharose FF purified lunasin fractions by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), we observed that the ∼5 kDa lunasin peptide always co-purified with a ∼9 kDa protein. Analysis of the Q-Sepharose FF purified lunasin by SDS-PAGE under standard reducing conditions or in the absence of beta-mercaptoethanol (BME) in conjunction with immunoblotting revealed that the majority of lunasin present in the partially purified preparation was in a ∼14 kDa protein complex (
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.
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.
To test the ability of various reducing agents to disrupt the lunasin-containing complex, we generated a highly purified lunasin-containing complex using Q-Sepharose FF chromatography followed by ultrafiltration using a 50 kDa molecular weight cut-off (MWCO) membrane. The purified complex was treated with varying concentrations of BME, tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT), then analyzing the resulting protein profiles by SDS-PAGE (
Since many of the contaminating proteins still present in the Q-Sepharose FF purified lunasin preparation were >20 kDa, ultrafiltration using a 30 kDa MWCO membrane was performed. In this application of ultrafiltration, lunasin accumulates in the permeate. This ultrafiltration step removed most of the contaminating proteins except for the ∼9 kDa protein that is a component of the ∼14 kDa lunasin-containing complex. To further purify lunasin, we tested the ability of reversed-phase chromatography to remove the remaining contaminating proteins (
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.
To confirm the identity of our purified ∼5 kDa protein as lunasin and to attempt to determine the identity of the ∼9 kDa protein that is present in the lunasin complex, we performed electrospray ionization mass spectrometry (ESI-MS) on the purified lunasin and the lunasin complex. The monoisotopic mass of the most abundant peptide in the purified lunasin sample was found to be 5139.25 Da, which is 114.02 Da higher than the expected monoisotopic mass of 5025.23 Da for the 43 amino-acid form of lunasin described in the literature (
(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.
Subunit | Peptide | MH+ | ΔM (ppm) | z | P |
|
QLQGVNLTPCEK | 1386.7046 | −6.12 | 2 | 9.67E-09 |
QLQGVNLTPCEK | 1386.7046 | −7.07 | 3 | 6.97E-04 | |
KQLQGVNLTPCEK | 1514.7995 | −7.45 | 2 | 2.73E-08 | |
KQLQGVNLTPCEK | 1514.7995 | −5.42 | 3 | 1.63E-03 | |
WQHQQDSCR | 1244.5225 | −7.37 | 2 | 4.71E-08 | |
GDDDDDDDDDN | 1225.3247 | −0.01 | 1 | 5.15E-03 | |
|
ELINLATMCR | 1220.6126 | −6.17 | 2 | 6.36E-08 |
ELINLATM*CR | 1236.6075 | −6.54 | 2 | 1.31E-07 | |
IMENQSEELEEK | 1478.6679 | −4.47 | 2 | 1.85E-07 | |
IM*ENQSEELEEK | 1494.6628 | −4.29 | 2 | 1.25E-05 | |
CCTEMSELR | 1185.4697 | −5.80 | 2 | 4.21E-07 | |
CCTEM*SELR | 1201.4646 | −6.67 | 2 | 4.70E-05 | |
FGPMIQCDLSSDD | 1484.6032 | −6.26 | 2 | 2.10E-06 | |
FGPM*IQCDLSSDD | 1500.5981 | −5.53 | 2 | 5.27E-06 |
M* indicates oxidized methionine; MH+ is the monoisotopic mass calculated from peptide sequence; ΔM is the error of detected MH+ in ppm; z is the charge state of the ion from which MS/MS spectrum was generated; and P is the probability that MS/MS spectrum matched the sequence randomly. Note that alkylation of cysteine by IAA increases the mass of cysteine by 57 Da.
ESI-MS analysis of the purified ∼14 kDa lunasin complex revealed that the most abundant isotopic mass of the complex was 14109.3 Da (
(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.
The mass of the 8975.1 Da subunit of the lunasin complex matched very well with the expected mass of the soybean 2S albumin large subunit (GenBank AAB71140.1). Analysis of peptides in a tryptic digest of the 8975.1 Da subunit by LC/MS/MS confirmed that this subunit does correspond to the 2S albumin large subunit (
To test the scalability of our purification scheme and determine the amount of lunasin that can be obtained with these methods, we utilized the pilot-scale facility available at Kentucky BioProcessing (Owensboro, KY, USA). For this study we began with 20.8 kg of soybean white flake and followed the process outlined in
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.
Previous studies have demonstrated that a key component of the biological activity of lunasin is its ability to bind to the core histones H3 and H4 and modulate histone acetylation
The recent advances in studies of the biological activities of lunasin and the potential use of lunasin as an anti-inflammatory or anticancer agent provide a strong rational for developing a robust and scalable purification method for lunasin that would generate large quantities of highly purified peptide suitable for large-scale animal and human trials. Although partially purified lunasin preparations may be adequate for oral administration as a nutraceutical, highly purified lunasin will be required to develop therapeutics. Previous studies have described methods that produce lunasin preparations that are ∼80–85% pure
We have developed an alternative method for purifying lunasin that utilizes an initial anion-exchange step followed by ultrafiltration using 30 kDa MWCO membranes and reversed-phase chromatography that represents a significant improvement over existing methods. This method yields highly purified lunasin (>99%) that is fully biologically active as assessed by histone binding. During the development of this method, we determined that the majority of the ∼5 kDa lunasin peptide in the initial white flake extract was found in a ∼14 kDa protein complex. This observation suggests that de Mejia and colleagues
We investigated the precise nature of the lunasin peptide obtained using our purification method by conducting an extensive MS analysis of the purified lunasin and the purified ∼14 kDa protein complex. Characterization of the purified lunasin by ESI-MS revealed that the major peptide in this preparation has a monoisotopic mass of 5139.25. This corresponds to the sequence SKWQHQQDSCRKQLQGVNLTPCEKHIMEKIQGRGDDDDDDDDDN and indicates that soy-derived lunasin has an asparagine residue at the C-terminus. LC-MS/MS analysis of tryptic peptides derived from purified lunasin confirmed the presence of the C-terminal asparagine. This is consistent with a previous analysis of soybean 2S albumin encoded by the AL3 gene
MS analysis of the purified ∼14 kDa complex and the gel-purified ∼9 kDa protein isolated from the complex after reduction with DTT demonstrated that the complex corresponds to the processed GM2S albumin protein. This is consistent with reports that lunasin copurifies with the large subunit of the GM2S protein
To test the scalability of our lunasin purification method, we conducted a pilot scale production trial at Kentucky BioProcessing. This trial started with 20.8 kg of soybean white flake and utilized an up-scaled version of our optimized method. A total of 9.2 g of lunasin at a purity of >99% was recovered, representing a 20% yield from the initial amount of lunasin present in the white flake extract. This yield was very similar to the yields we obtained performing the purification method at the bench scale. Given the fact that the pilot facility at Kentucky BioProcessing replicates efficiently to their commercial-scale facility, it will be possible to further upscale this method to produce kilogram quantities of highly purified lunasin for use in the develop of new lunasin-based therapeutics for the treatment of cancer and inflammatory diseases.
All chemicals were ACS grade or better and were purchased from Sigma-Aldrich (St. Louis, MO, USA) except sodium phosphate (dibasic anhydrous and monobasic monohydrate) were from EMD Chemicals (Gibbstown, NJ, USA) and sodium chloride, Tris-base, glycine, and bovine serum albumin (BSA) were from Fisher Scientific (Pittsburgh, PA, USA). Defatted soy flour (white flake) was prepared and provided by Owensboro Grain (Owensboro, KY, USA). Briefly, de-hulled soybeans were processed in a flaking roll and then further processed by conveying the flake through an expander to form a collet. The collet was transferred to a solvent extractor where the oil was removed by extensive washing with hexane. The defatted flake was then air-dried under fans at ambient temperature to remove the hexane. The white flake was stored at ambient temperature until used. Synthetic lunasin peptide along with a lunasin-specific mouse monoclonal lunasin antibody were from GenScript Corporation (Piscataway, NJ, USA). The lunasin-specific mouse monoclonal antibody was raised against the synthetic peptide CEKHIMEKIQGRGDD (98.7% pure) conjugated to keyhole limpet hemocyanin. Most of our studies were done using the lunasin-specific monoclonal antibody that was raised using the peptide CEKHIMEKIQGRGDD as the antigen. Initial studies were performed using a lunasin-specific rabbit polyclonal primary antibody and synthetic lunasin provided by Dr. Ben O. de Lumen (University of California-Berkeley, USA). Horse-radish peroxidase (HRP)-conjugated sheep anti-mouse and HRP-conjugated goat anti-rabbit antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Human, recombinant histones were purchased from New England BioLabs (Ipswich, MA, USA). All chromatography columns and resins were obtained from GE Healthcare (Piscataway, NJ, USA). Ultrapure water was by generated using a Milli-Q Synthesis system (Millipore, Billerica, MA, USA).
Protein concentrations were determined using a bicinchoninic acid-based assay (BCA Protein Assay Reagent, Thermo Scientific, Rockford, IL, USA). BSA was used as a standard for crude and partially purified lunasin samples whereas synthetic lunasin was used as a standard for the highly purified lunasin samples.
SDS-PAGE was performed using 15% PAGEr Gold Tris-Glycine PreCast gels (Lonza, Rockland, ME, USA) according to the manufacturer's recommendations. Molecular weight standards correspond to SeeBlue® Plus2pre-stained proteins (Invitrogen/Life Technologies, Grand Island, NY, USA). Gels were fixed in 40% ethanol/10% acetic acid, stained with Coomassie Brilliant Blue 250 (Fluka/Sigma-Aldrich, St. Louis, MO, USA), and destained with a 7% isopropanol/5% acetic acid solution. Gels were imaged using a Kodak Image Station 4000R Pro (Carestream, Rochester, NY, USA) or an ImageQuant-RT ECL (GE Healthcare, Piscataway, NJ, USA) and individual protein bands quantified using Carestream Molecular Imaging Software version 5.0.
SDS-PAGE gels were run as previously described to perform immunoblot analysis. Proteins were transferred to Immobilon-P 0.45 um PVDF membranes (Millipore, Billerica, MA, USA) at 20 V for 90 min at 4°C. Five percent (w/v) instant non-fat dry milk in Tris-Tween buffered saline (TTBS; 16.1 mM Tris-HCl/3.88 mM Tris-base/150 mM NaCl/0.5% Tween 20, pH 7.5) was used as a blocking reagent. Two washes of TTBS were performed prior to incubation with primary antibody for 90 minutes. The lunasin mouse monoclonal primary antibody was used at a 1∶75,000 or 1∶100,000 dilution into primary antibody solution (TTBS/0.5% BSA/0.04% NaN3). The lunasin polyclonal rabbit primary antibody was used at a 1∶5000 dilution. Three washes with TTBS were performed prior to incubation with the appropriate secondary antibody for 60 minutes. A 1∶100,000 dilution of the HRP-conjugated sheep anti-mouse secondary antibody or HRP-conjugated goat anti-rabbit secondary antibody in 1% (w/v) instant non-fat dry milk in TTBS was used. Three washes with TTBS were performed before incubating with the chemiluminescent detection solution (ECL Advance™ Western Blotting Detection Kit, GE Healthcare, Piscataway, NJ, USA) and imaging using a Kodak Image Station 4000R Pro and Carestream Molecular Imaging Software version 5.0 (Carestream, Rochester, NY, USA). The image shown in
A direct ELISA was performed for quantitative measurements of lunasin concentration in partially-purified preparations. Samples were diluted into coating buffer (15 mM Na2CO3/35 mM NaHCO3/3 mM NaN3, pH 9.6), 50 µL aliquots of sample were added to wells of a 96-well plate (Nunc MaxiSorp™, Nalge Nunc International, Rochester, NY), and the plates were incubated for 60 minutes at 37°C. Wells were washed two times with PBST (137 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, 0.05% Tween 20, pH 7.4) and then blocked with 150 µL per well of PBSTM 5% (PBST containing 5% w/v instant non-fat dry milk) for 60 minutes at room temperature or overnight at 4°C. The wells were then washed two times with ultrapure water. Lunasin primary antibody was prepared in PBSTM 1% (PBST containing 1% w/v instant non-fat dry milk) at a 1∶50,000 dilution. A 50 µL aliquot of diluted primary antibody was added to each well and incubated for 60 minutes at 37°C. The wells were then washed three times with ultrapure water. The HRP-conjugated secondary antibody was diluted to 1∶5000 in PBSTM 1% and 50 µL aliquots were added to each well prior to incubating for 60 minutes at 37°C. The wells were then washed three times with ultrapure water. The plate was developed using 50 µL per well of a tetramethyl benzidine-based reagent (TMBW; BioFX One Component HRP Microwell Substrate, SurModics, Eden Prairie, MN, USA) and an incubation time of four minutes at room temperature. The reaction was stopped with 50 µL per well of stop solution (0.6 N H2SO4/1 N HCl). The absorbance at 450 nm for each well was measured using a DTX 880 Multimode Detector (Beckman Coulter, Indianapolis, IN, USA). This ELISA format was useful for accurately measuring between 7 and 26 ng lunasin.
For the large-scale purification of lunasin from soybean white flake, a 12.5∶1 extraction buffer (20 mM sodium phosphate/150 mM NaCl/20 mM ascorbic acid/10 mM sodium metabisulfite, pH 7.4) to white flake soy flour ratio was used. The white flake was suspended in extraction buffer and mixed for one hour. After mixing, a diatomite filter aid, Celpure 300 (Advanced Minerals Corporation, Santa Barbara, CA, USA), was added to the extract (33 g/L). The extract was passed through a filter press fitted with 1 micron M-503 filter pads (ErtelAlsop, Kingston, NY, USA) to produce a clarified extract. After filtering, the filter cake was blown dry with compressed air and a wash was performed using extraction buffer (wash volume: 50% of total extract). The wash was combined with the initial filtered extract to generate the final clarified extract.
All chromatography procedures were performed in clean room suites at Kentucky BioProcessing to ensure sterility of final product. Anion-exchange chromatography was performed using a 20.0×13.0 cm Q-Sepharose FF column on a Pharmacia 10 mm Bioprocess System Skid. The skid and column were both sanitized with 1 N NaOH and then pre-conditioned with 10 CV of equilibration buffer (Buffer A: 20 mM sodium phosphate/150 mM NaCl, pH 7.4) prior to applying samples. Clarified extract was applied onto the column through the sample inlet at a residence time between 2 and 2.77 minutes. The column was washed with 14.8 CV of equilibration buffer and the lunasin eluted using a linear gradient of NaCl in the elution buffer (Buffer B: 20 mM sodium phosphate/1 M NaCl, pH 7.4). Lunasin eluted from the column between 0.26 M and 0.50 M NaCl. The fractions containing lunasin were filtered through an inline 0.2 µm capsule filter and combined.
The lunasin-containing fraction obtained by Q-Sepharose FF chromatography was brought to a final concentration of 2 mM DTT and stirred with an overhead mixer at room temperature for one hour. The DTT-treated fraction was subjected to ultrafiltration using five, 0.1 sq. meters each, 30 kDa MWCO polyethersulfone membranes using a Sartorius Sartocon Slice unit (Sartorius Stedium Biotech, Bohemia, NY, USA). Lunasin accumulates in the permeate fraction during this procedure. Ultrafiltration was continued until the retentate remaining in the sample reservoir reached a volume of ∼1 L. The retentate was then washed with five volumes of buffer (20 mM sodium phosphate/300 mM NaCl, pH 7.4) with each wash being reduced to a final volume of ∼1 L. Permeates generated from these washes were combined with the initial permeate for further purification.
RPC was used as the final step in the purification process using a 10.0×9.2 cm Source 15RPC column on an AKTApilot™ system (GE Healthcare, Piscataway, NJ, USA). Prior to chromatography, the column was sanitized with 1 N NaOH and equilibrated with ten CV of equilibration buffer (20 mM sodium phosphate/150 mM NaCl, pH 7.4). The lunasin fraction was applied onto the column with a residence time of 2.5 minutes. A five CV wash with equilibration buffer was performed, followed by a step elution using 20%, 40%, 60%, 80%, and 100% elution buffer (Buffer B: 17 mM sodium phosphate/127.5 mM NaCl/15% n-propanol, pH 7.4). Fractions were collected at each gradient step; as expected, the 100% B gradient step was the lunasin-containing fraction.
Next, the lunasin-containing fraction obtained by RPC was concentrated using a 0.5 m2 2 kDa cellulose cassette (Sartorius Stedium Biotech, Bohemia, NY, USA). Difiltration was performed to exchange the RPC elution buffer with 50 mM sodium phosphate, pH 7.4. The retentate and wash were collected and filtered through a 0.2 µm filter. The amount of lunasin present in the concentrated sample was determined using a BCA protein assay with synthetic lunasin as a standard. The lunasin concentrate was then diluted with 50 mM sodium phosphate, pH 7.4 to a final concentration of 4.65 mg/mL. Sterile, glass vials were each filled with 5.5 mL of final product and stored at 4°C.
Purified lunasin complex was desalted with C18 ZipTip (Millipore, Billerica, MA) and ESI spectra of lunasin complex was obtained using an Orbitrap XL mass spectrometer (Thermo Scientific, San Jose, CA) equipped with TriVersa NanoMate system (Advion BioSciences, Ithaca, NY). The MS spectra were deconvoluted with Xtract (Thermo Scientific, San Jose, CA). To analyze subunits of lunasin complex, purified lunasin complex was reduced with 5 mM DTT at 70°C for 15 minutes, followed by alkylation with 15 mM iodoacetamide (IAA) at room temperature in the dark for 15 min. Reduced lunasin complex samples, with or without further alkylation, were desalted with C18 ZipTip and analyzed using an Orbitrap XL mass spectrometer.
Gel-purified lunasin subunits were desalted with PepClean C18 spin column (Pierce/Thermo Scientific, Rockford, IL), reduced with DTT, alkylated with IAA, and incubated with sequencing grade modified trypsin (Promega, Madison, WI) at 37°C overnight. Incubation was stopped by adding 5% formic acid to the samples and the digests were loaded on to a Hypersil Gold C18 column and separated using an Accela HPLC system (Thermo Scientific, Waltham, MA, USA) with an aqueous acetonitrile/0.1% formic acid gradient. The eluted peptides were directed to an Orbitrap XL mass spectrometer and MS/MS spectra of the peptides were acquired in data dependent scan mode.
Aliquots of recombinant human histones H3 or H4 stock solutions where the histone concentration was determined by measuring the absorbance at 280 nm were plated in 96 well MaxiSorp™ plates in coating buffer (15 mM Na2CO3/35 mM NaHCO3/0.02% NaN3/1 mM freshly prepared DTT, pH 9.6) to generate final quantities of 100, 300 and 500 ng/well. DTT was added at the time of plating to allow for even distribution and prevention of oligomerization of the histones. Negative controls consisted of wells containing coating buffer alone with no histones. Plates were sealed with adhesive plastic and incubated for 60 minutes at 37°C. Plates were then washed twice with PBST and the remaining protein-binding sites blocked for 60 minutes at room temperature in PBST containing 5% non-fat dry milk. Following two washes with ultrapure water, either synthetic or purified lunasin diluted in PBSTM1% was added to the wells at final concentrations of 0.1, 1, 10, 100 µM. Plates were incubated at 37°C for 60 minutes, washed three times with PBST and incubated for another 60 minutes at 37°C with the primary monoclonal mouse anti-lunasin antibody diluted 1∶5000 in PBSTM1%. After three washes with ultrapure water, the secondary HRP-conjugated sheep anti-mouse antibody diluted 1∶5000 in PBSTM1% was added and the plates incubated at 37°C for 60 minutes. Plates were then washed three times with ultrapure water. Detection of lunasin bound to the histones was accomplished by the addition of the HRP substrate SureBlue™ (KPL, Gaithersburg, MD, USA) followed by six minutes incubation in the dark at room temperature. The reaction was terminated by the addition of an equal volume of 1 N HCl and the absorbance at 450 nm for each well was measured using a DTX 880 Multimode Detector (Beckman Coulter, Indianapolis, IN, USA).
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The authors thank Dr. Ben O. de Lumen, (University of California, Berkeley) for helpful discussions prior to the initiation of these studies and for generously providing samples of synthetic lunasin and a lunasin polyclonal antibody. The authors also thank Mr. John Wright at Owensboro Grain for providing the white flake used in these studies and Josh Morton, Travis Newton, Eric Simon and Cara Working at Kentucky BioProcessing for technical assistance during the pilot-scale purification.