Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography
- Eva Sewekow†1,
- Lars Christian Kessler†2,
- Andreas Seidel-Morgenstern2, 3 and
- Hermann-Josef Rothkötter1Email author
© Sewekow et al; licensee BioMed Central Ltd. 2008
Received: 10 August 2007
Accepted: 11 March 2008
Published: 11 March 2008
Soybeans play a prominent role in allergologic research due to the high incidence of allergic reactions. For detailed studies on specific proteins it is necessary to have access to a large amount of pure substance.
In this contribution, a method for purifying soybean (Glycine max) protein P34 (also called Gly m Bd 30 K or Gly m 1) using hydrophobic interaction chromatography is presented. After screening experiments using 1 mL HiTrap columns, Butyl Sepharose 4 FF was selected for further systematic investigations. With this stationary phase, suitable operation conditions for two-step gradient elution using ammonium sulphate were determined experimentally. The separation conditions obtained in a small column could be scaled up successfully to column volumes of 7.5 and 75 mL, allowing for high product purities of almost 100% with a yield of 27% for the chromatographic separation step. Conditions could be simplified further using a onestep gradient, which gave comparable purification in a shorter process time. The identity of the purified protein was verified using in-gel digestion and mass spectrometry as well as immunological techniques.
With the technique presented it is possible to produce, within a short timeframe, pure P34, suitable for further studies where an example antigen is needed.
In recent years, soybeans were identified as one of the main sources of allergic reactions in humans . The incidence of adverse reactions to food antigens is especially high in children (2–8%, compared to adults with 1–2%) . Since the spectrum varies from clinical to systemic anaphylactic symptoms , there is a need to develop models analysing how food antigens reach the immune cells eliciting these allergenic reactions. Especially food proteins which cause adverse reactions only in some patients are of interest. As an increasing number of food products is enriched with plant proteins due to their emulsifying properties, simple production and good digestibility, detailed studies on their allergenic potential are necessary.
In this contribution, a new purification procedure for P34 (also called Gly m Bd 30 K or Gly m 1) was developed. The protein was discovered to be the main allergen for soybean sensitive humans . Soybean protein P34 is a monomeric insoluble glycoprotein with an isoelectric point of 4.5 [4, 5] and an amino acid based calculated mass of 28.643 Da according to the Informall database , representing 2–3% of total soybean protein . In its glycosylated form, the mass will be slightly larger, resulting in a band of ~32 kDa in non-reduced SDS PAGE gels . As a thiol protease, it belongs to the papain superfamily. Due to an absence of catalytic cysteine it exhibits no enzymatic function . In disrupted plant cells, P34 associates to soybean oil bodies, but has no membrane insertion region and is stored in storage vacuoles of soybean cotyledons [7, 8]. After translation, P34 loses the pre- and pro-protein region containing one glycosylation and during seedling growth a basic decapeptide is removed [7–9]. P34 attaches the 7S globulin fraction due to disulfide bridges  and exists as a dimer of 58 kDa in non-reduced SDS PAGE gels .
The protein can be extracted from washed soybean oil body pads with 0.1 M sodium carbonate, thus allowing for a simple pre-purification [8, 11]. Alternatively, P34 could be produced as a recombinant protein in E. coli . A subsequent purification, however, is in any case necessary.
A different approach to purifying P34, based on globulin fractionation [13, 14], was proposed by the group of Ogawa in 1993 . There, protein P34 was isolated from the 7S globulin fraction with a multi-step process, including a chromatographic separation using Con A Sepharose.
Results and Discussion
Preparation of soybean protein feedstock for chromatographic separations
Selection of suitable chromatographic conditions
During process development, a wide range of possible purification strategies was tested. However, with neither size exclusion, lectin-based affinity, cation exchange nor anion exchange chromatography was it possible to meet purity requirements. Due to the lipophilic binding of P34 to oil bodies, hydrophobic interaction chromatography (HIC) was tested subsequently as a possible, alternative mode of interaction.
Scale-up of purification 1: From 1 mL to 7.5 mL
Scale-up of purification 2: From 7.5 mL to 75 mL
Estimation of yields for protein P34
In order to compare process performance in terms of yields, it has to be distinguished between total process efficiency, including the preparation of the soybean feedstock according to  (see Fig. 1) and starting with the soybean itself, and the efficiency of HIC purification as a stand-alone operation, starting with the prepared soybean feedstock.
For evaluation of total process performance, the amount of pure P34 obtained has to be related to the P34 content in the soybeans used. As stated already in the Background section, previous studies have shown that P34 represents 2–3% of total soybean protein . Based on the manufacturer s specifications, 39% of the cultivars total mass are proteins, leading to a theoretical maximum amount of 7.8 mg of pure P34 per gram soybean for a P34 content of the total soybean protein of 2%. This also gives the highest possible yield to be obtained during purification. It has to be stressed that these values are only estimates, as both the P34 content as well as the protein mass percentage of the total soybean mass might vary in the beans used for this study. However, these numbers are useful to give at least some measure for the yield's order of magnitude for this type of process. For the case of the 7.5 mL column, where a solution extracted from 1 g soybeans was used, the best result obtained was 173 μg of pure P34. This corresponds to a total yield of ~2%. For column 2 (75 mL) using both two-step and one-step elution, an average amount of 250 μg of pure P34 could be obtained per gram soybean, increasing the overall yield slightly to ~3%. At first glance, this low yield seems disappointing. However, a more detailed analysis of P34 concentrations in the course of separation showed that the majority of protein loss occurred already during feedstock preparation. From 200 g soybeans only 192 mg of P34 could be found in the soybean feedstock used for chromatography, resulting in a yield of only 12%. A modified preparation protocol could help to increase the total process performance significantly.
Consequently, in order to evaluate the chromatographic performance itself, the amount of protein introduced with the column feed, the soybean feedstock has to be taken as reference, resulting in a yield of 18% for P34 for column 1 and 27% for column 2. This is comparable to other HIC processes reported for the isolation of a single protein from a multicomponent mixture , but still low when compared to other chromatographic techniques. For the present example, however, the relatively low yields are somewhat compensated by the low costs and high availability of soybeans.
Confirmation of protein identity and properties
Based on a well-known purification protocol used for feedstock preparation , an effective and easy method for isolating the important allergen P34 using hydrophobic interaction chromatography was presented. A broad screening study led to the selection of Butyl Sepharose 4 FF as suitable stationary phase. For this resin, appropriate (NH4)2SO4 concentrations for binding and for two-step elution were determined experimentally using prepacked 1 mL columns. Based on these results, the process was scaled up twice to 7.5 and 75 mL of column volume, respectively. It was found that the conditions obtained with the small screening columns could be transferred to the larger columns satisfactorily. However, performance could be increased further, after several empiric optimisation steps were employed to obtain operating conditions for at first a two-step and secondly a one-step gradient elution process. Using one-step elution, process time could be reduced while the yield remained constant.
When considering total process yield, from the P34 content in the beans to the amount recovered in the final, purified solution, only 3% of available P34 were recovered. A systematic analysis of protein concentrations during the complete process showed that almost 88% of P34 were lost during the feedstock preparation process, indicating a need for process optimisation even before chromatography. When relating the amount of obtained, pure P34 to the P34 concentration in the feedstock, the HIC process itself performed comparable to other reported HIC applications, resulting in a yield of 27%. Due to the empirical optimisation, separation performance can probably be increased further using a model-based approach.
With the method presented was possible to obtain a sufficient amount of pure P34 in a simple way, compared to the possible alternatives. The use of step changes in gradient conditions allows the application of a simple column-syringe set-up.
Materials and equipment
All chemicals and biochemicals, unless indicated otherwise, were purchased from Roth (Karlsruhe, Germany) and were of analytical grade. Ovalbumin was purchased from Sigma Aldrich (Steinheim, Germany). Dried soybeans (according to manufacturer s specifications cultivars Heinong 40 und Dongnong 42) were purchased from Hensel Soja-Kost (Magstadt, Germany). Centrifugation was carried out in an Avanti J-25 High Performance centrifuge (Beckman Coulter, Krefeld, Germany). Prepacked HiTrap-columns (1 mL, 7 × 25 mm) of Phenyl Sepharose High Performance, Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 Fast Flow (high sub), Butyl-S Sepharose 6 Fast Flow, Butyl Sepharose 4 Fast Flow (FF) and Octyl Sepharose 4 Fast Flow as well as Butyl Sepharose 4 Fast Flow as bulk material (Lot number: for 7.5 mL column 10000941, for 75 mL column 10003753) were purchased from GE Healthcare (Uppsala, Sweden). Both a Merck Superformance column (10 × 150 mm, Merck, Darmstadt, Germany) and an Omnifit glass column (25 × 250 mm, Omnifit, Cambridge, U.K.) were used. The smaller column is referred to as column 1, the larger as column 2.
Chromatographic screening experiments were performed using either a syringe or an ÄKTA prime unit (GE Healthcare, Uppsala, Sweden). Preparative separations were carried out in the ÄKTA prime unit. As binding buffer 1 M (NH4)2SO4 in 0.05 M phosphate buffer (Na2HPO4 and NaH2PO4) at pH 6.5 was used. Buffer solutions were prepared using high purity water from a Millipore System (EASYpure RF, Barnstead, Germany). Fractions were concentrated using centrifugal devices with a molecular cut-off of 3 kDa (Roth, Karlsruhe, Germany). All percentages are given as volume percent unless stated otherwise.
Preparation of soybean protein solution
Soybean solution was prepared according to the methods described in [8, 11]. The feedstock preparation procedure was shown schematically already in Fig. 1. The soybeans were soaked overnight in distilled water, homogenised in 0.1 M Tris-HCl (pH 8.6) using a hand blender and pushed through a layer of gaze. The homogenate which passes the gaze was centrifuged for 20 min (53000 × g, 4°C) (Fig. 1, steps 1–3). The recovered oil body again was washed in 0.1 M Tris-HCl (pH 8.6) and centrifuged. Afterwards, the oil body was homogenised twice in 0.1 M Tris-HCl, 0.5 M NaCl (pH 8.6) and centrifuged (Fig. 1, step 4). To recover the protein, the oil body was finally homogenised in 0.1 M sodium carbonate and centrifuged as described before. The supernatant was concentrated in PEG (20 g/100 mL) using dialysis membranes and dialysed into the binding buffer afterwards (Fig. 1, step 5).
Screening of hydrophobic interaction resins
Each column was equilibrated with binding buffer before 200 μL of protein solution (1 g/L) were applied to the column with a syringe. After washing the column with 15 column volumes (cv) of binding buffer, proteins were eluted with a successive multi-step gradient of 9 cv of 0.8 M, 0.6 M, 0.4 M, 0.2 M and 0.0 M (NH4)2SO4, respectively, in 0.05 M phosphate buffer at pH 6.5.
The collected fractions of 1.5 mL were precipitated with trichloric acid (10 g/100 mL), the pellets washed with acetone and analysed using SDS-PAGE (10% gels) and Coomassie-staining.
Chromatographic separation and scale-up
Two different types of glass columns were used in this study. In column 1, 7.5 mL of Butyl Sepharose 4 FF Sepharose were packed hydrodynamically. Before separation, the column was equilibrated with at least 10 cv of binding buffer. Subsequently, 500 μl of soybean feedstock, containing 705 μg of total protein, of which 541 μg were P34, were loaded onto the column using a sample loop. For P34 separation, a two step gradient of 0.6 M (NH4)2SO4 and 0.4 M (NH4)2SO4 as well as a washing step at 0.0 M (NH4)2SO4 in 0.05 M phosphate buffer (pH 6.5) was used. Each gradient step was held until a constant UV signal could be observed. After each run, the column was washed with a minimum of 10 cv of 0.05 M phosphate buffer and a minimum of 10 cv of 20% ethanol to remove strongly bound contaminants.
In a subsequent experiment, column 2 was packed with 75 mL Butyl Sepharose 4 FF Sepharose. Before separation, it was equilibrated with 4 cv of binding buffer (0.6 M (NH4)2SO4 in 0.05 M phosphate buffer, pH 6.5), before 5 mL of protein solution in binding buffer, containing at least 13 mg of total protein, 77% of which are P34, were applied to the column with a sample loop. Proteins were eluted using either a two or a one step gradient of 0.4 M (NH4)2SO4 and 0.25 M (NH4)2SO4 or only 0.25 M (NH4)2SO4 in 0.05 M phosphate buffer (pH 6.5). Each gradient step was held until a constant UV signal was observed. The same cleaning procedure was used as described for column 1.
Electrophoretic analysis of eluted fractions
For the analysis of the fractions for Fig. 4, 10 mL (fractions for lane 1–19) to 15 mL (fractions for lane 20–29), collected during chromatography with column 1 for subsequent analysis, were concentrated to ~30 μL and analysed using SDS-PAGE (10% gels) and Coomassie-staining. A Bradford assay with 2–10 μg BSA-standards was used to determine total protein concentration in each fraction . For Fig. 7 and 8, 4 mL of each fraction were concentrated and analysed using SDS-PAGE (10% gels) and Coomassie-staining. Densitometric analysis of gels was conducted using an AlphaEase FC Imaging System (Alpha Innotech Corp., San Leandro, CA., U.S.A.).
Detection of glycosylation
After electrophoresis, the separated proteins were fixed in the gel using 70% methanol and 10% acetic acid for 30 minutes. Afterwards, the gel was washed in water for 5 minutes. Subsequently, the gel was incubated with periodic acid (1 g/100 mL)/30% acetic acid for 30 minutes and treated with Schiffs reagent over night. All glycosylated proteins give violet bands . P34 was compared to the reference proteins BSA (no glycosylation) and ovalbumin (glycosylation).
Immunodetection of P34
For immunodetection of P34 with monoclonal antibody F5 (kindly provided by T. Ogawa, Koyoto University, Osaka, Japan), protein samples were separated electrophoretically on 10% SDS-polyacrylamide gels, and blotted semi-dry onto nitrocellulose membranes. The membrane was then stained with F5 and a BM Chemiluminescence Western Blotting Kit (mouse/rabbit, Roche, Grenzach-Wyhlen, Germany) following the manufacturer s instructions. Finally, the blots were analysed on an Alpha Ease FC Imaging System.
This work was supported by the 6th Framework Programme „Feed for Pig Health“ (FOOD-CT-2004-506144). The authors would like to thank PD Dr. Thilo Kähne (Institute for Experimental Medicine, Otto-von-Guericke University Magdeburg, Germany) for protein identification using in gel digestion and mass spectrometry analysis and Prof. T. Ogawa (Koyoto University, Osaka, Japan) for providing the monoclonal antibody F5.
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