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Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method

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Abstract

To detect the soybean allergen P34 (Gly m Bd 30K) from soybean products, the full-length cDNA sequence of P34 was synthesized and inserted into the prokaryotic expression vector pET-28a. The P34 protein was expressed in Escherichia coli BL21 (DE3) as an inclusion body under the induction of 0.8 mmol/L isopropyl β-d-1-thiogalactopyranoside. After purification with His-Bind affinity chromatography, the purity quotient of the recombinant protein was over 92 %, and its molecular weight (approximately 33 kDa) was very close to that of the native soybean P34. The polyclonal antibody (pAB) against P34 was prepared with the purified recombinant P34. The generated pAB, named as pAB-P34, exhibited high specificity to the P34 protein of the soybean meal. The indirect enzyme-linked immunosorbent assay (iELISA) based on pAB-P34 was established to determine the P34 content of soybean products. The CVs of the recovery tests of P34 were less than 7.77 %, which indicated that iELISA had high reproducibility and accuracy. Therefore, the recombinant P34 produced in the E. coli expression system, the prepared pAB-P34, and the developed iELISA could provide a valuable tool for sensitive detection of P34 in various soybean products and for future studies on allergies related to soybean P34.

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References

  • Amnuaycheewa P, de Mejia EG (2010) Purification, characterisation, and quantification of the soy allergen profilin (Gly m 3) in soy products. Food Chem 119(4):1671–1680

    Article  CAS  Google Scholar 

  • Asensio L, Gonz Lez I, Garc AT, Mart NR (2008) Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control 19(1):1–8

    Article  CAS  Google Scholar 

  • Babiker EE, Azakami H, Ogawa T, Kato A (2000) Immunological characterization of recombinant soy protein allergen produced by Escherichia coli expression system. J Agric Food Chem 48(2):571–575

    Article  CAS  Google Scholar 

  • Bando N, Tsuji H, Hiemori M, Yoshizumi K, Yamanishi R, Kimoto M, Ogawa T (1998) Quantitative analysis of Gly m Bd 28K in soybean products by a sandwich enzyme-linked immunosorbent assay. J Nutr Sci Vitaminol (Tokyo) 44(5):655–664

    Article  CAS  Google Scholar 

  • Batista R, Martins I, Jeno P, Ricardo CP, Oliveira MM (2007) A proteomic study to identify soya allergens—the human response to transgenic versus non-transgenic soya samples. Int Arch Allergy Immunol 144(1):29–38

    Article  CAS  Google Scholar 

  • Bohle B, Vieths S (2004) Improving diagnostic tests for food allergy with recombinant allergens. Methods 32(3):292–299

    Article  CAS  Google Scholar 

  • Bu G, Luo Y, Zhang Y, Chen F (2010) Effects of fermentation by lactic acid bacteria on the antigenicity of bovine whey proteins. J Sci Food Agric 90(12):2015–2020

    CAS  Google Scholar 

  • Clavijo A, Zhou E, Hole K, Galic B, Kitching P (2004) Development and use of a biotinylated 3ABC recombinant protein in a solid-phase competitive ELISA for the detection of antibodies against foot-and-mouth disease virus. J Virol Methods 120(2):217–227

    Article  CAS  Google Scholar 

  • Egounlety M, Aworh OC (2003) Effect of soaking, dehulling, cooking and fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soybean (Glycine max Merr.), cowpea (Vigna unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Harms). J Food Eng 56(2–3):249–254

    Article  Google Scholar 

  • Friedman M, Brandon DL (2001) Nutritional and health benefits of soy proteins. J Agric Food Chem 49(3):1069–1086

    Article  CAS  Google Scholar 

  • Guo P, Piao X, Cao Y, Ou D, Li D (2008) Recombinant soybean protein beta-conglycinin alpha′-subunit expression and induced hypersensitivity reaction in rats. Int Arch Allergy Immunol 145(2):102–110

    Article  CAS  Google Scholar 

  • Helm RM, Cockrell G, Connaughton C, West CM, Herman E, Sampson HA, Bannon GA, Burks AW (2000) Mutational analysis of the IgE-binding epitopes of P34/Gly m Bd 30K. J Allergy Clin Immunol 105(2 Pt 1):378–384

    Article  CAS  Google Scholar 

  • Herman EM, Helm RM, Jung R, Kinney AJ (2003) Genetic modification removes an immunodominant allergen from soybean. Plant Physiol 132(1):36–43

    Article  CAS  Google Scholar 

  • Hoffmann-Sommergruber K, Mills EN, Vieths S (2008) Coordinated and standardized production, purification and characterization of natural and recombinant food allergens to establish a food allergen library. Mol Nutr Food Res 52(Suppl 2):S159–S165

    Article  Google Scholar 

  • Hotz C, Gibson RS (2007) Traditional food-processing and preparation practices to enhance the bioavailability of micronutrients in plant-based diets. J Nutr 137(4):1097–1100

    CAS  Google Scholar 

  • Kleine T, Bartsch S, Blaser J, Schnierer S, Triebel S, Valentin M, Gote T, Tschesche H (1993) Preparation of active recombinant TIMP-1 from Escherichia coli inclusion bodies and complex formation with the recombinant catalytic domain of PMNL-collagenase. Biochem 32:14125–14131

    Article  CAS  Google Scholar 

  • L'Hocine L, Boye JI (2007) Allergenicity of soybean: new developments in identification of allergenic proteins, cross-reactivities and hypoallergenization technologies. Crit Rev Food Sci Nutr 47(2):127–143

    Article  Google Scholar 

  • Liu B, Teng D, Yang L, Wang X, Wang J (2012) Development of a competitive ELISA for the detection of soybean α subunit of β-conglycinin. Process Biochem 47(2):280–287

    Article  CAS  Google Scholar 

  • Lorenz A, Scheurer S, Haustein D, Vieths S (2001) Recombinant food allergens. J Chromatogr B Biomed Sci Appl 756(1–2):255–279

    Article  CAS  Google Scholar 

  • Ma X, Sun P, He P, Han P, Wang J, Qiao S, Li D (2010) Development of monoclonal antibodies and a competitive ELISA detection method for glycinin, an allergen in soybean. Food Chem 121(2):546–551

    Article  CAS  Google Scholar 

  • Mills ENC, Breiteneder H (2005) Food allergy and its relevance to industrial food proteins. Biotechnol Adv 23(6):409–414

    Article  CAS  Google Scholar 

  • Mills EN, Jenkins JA, Alcocer MJ, Shewry PR (2004) Structural, biological, and evolutionary relationships of plant food allergens sensitizing via the gastrointestinal tract. Crit Rev Food Sci Nutr 44(5):379–407

    Article  CAS  Google Scholar 

  • Mujoo R, Trinh DT, Ng PKW (2003) Characterization of storage proteins in different soybean varieties and their relationship to tofu yield and texture. Food Chem 82(2):265–273

    Article  CAS  Google Scholar 

  • Ogawa T, Bando N, Tsuji H, Okajima H, Nishikawa K, Sasaoka K (1991) Investigation of the IgE-binding proteins in soybeans by immunoblotting with the sera of the soybean-sensitive patients with atopic dermatitis. J Nutr Sci Vitaminol (Tokyo) 37(6):555–565

    Article  CAS  Google Scholar 

  • Ogawa A, Samoto M, Takahashi K (2000) Soybean allergens and hypoallergenic soybean products. J Nutr Sci Vitaminol (Tokyo) 46(6):271–279

    Article  CAS  Google Scholar 

  • Pedersen MH, Holzhauser T, Bisson C, Conti A, Jensen LB, Skov PS, Bindslev-Jensen C, Brinch DS, Poulsen LK (2008) Soybean allergen detection methods—a comparison study. Mol Nutr Food Res 52(12):1486–1496

    Article  CAS  Google Scholar 

  • Poms RE, Klein CL, Anklam E (2004) Methods for allergen analysis in food: a review. Food Addit Contam 21(1):1–31

    Google Scholar 

  • Sathe SK, Sharma GM (2009) Effects of food processing on food allergens. Mol Nutr Food Res 53(8):970–978

    Article  CAS  Google Scholar 

  • Sealey WM, Barrows FT, Smith CE, Overturf K, LaPatra SE (2009) Soybean meal level and probiotics in first feeding fry diets alter the ability of rainbow trout Oncorhynchus mykiss to utilize high levels of soybean meal during grow-out. Aquaculture 293(3–4):195–203

    Article  Google Scholar 

  • Sewekow E, Kessler LC, Seidel-Morgenstern A, Rothkotter HJ (2008) Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography. BMC Biotechnol 8:27

    Article  Google Scholar 

  • Song YS, Martinez-Villaluenga C, De Meijia EG (2008a) Quantification of human IgE immunoreactive soybean proteins in commercial soy ingredients and products. J Food Sci 73(6):T90–T99

    Article  CAS  Google Scholar 

  • Song YS, Frias J, Martinez-Villaluenga C, Vidal-Valdeverde C, Gonzalez de Mejia E (2008b) Immunoreactivity reduction of soybean meal by fermentation effect on amino acid composition and antigenicity of commercial soy products. Food Chem 108:571–581

    Article  CAS  Google Scholar 

  • Song YS, Perez VG, Pettigrew JE, Martinez-Villaluenga C, de Mejia EG (2010) Fermentation of soybean meal and its inclusion in diets for newly weaned pigs reduced diarrhea and measures of immunoreactivity in the plasma. Anim Feed Sci Tech 159(1–2):41–49

    Article  CAS  Google Scholar 

  • Teng D, Gao M, Yang Y, Liu B, Tian Z, Wang J (2012) Bio-modification of soybean meal with Bacillus subtilis or Aspergillus oryzae. Biocatalysis Agric Biotechnol 1(1):32–38

    Article  CAS  Google Scholar 

  • Tsuji H, Bando N, Kimoto M, Okada N, Ogawa T (1993) Preparation and application of monoclonal antibodies for a sandwich enzyme-linked immunosorbent assay of the major soybean allergen, Gly m Bd 30K. J Nutr Sci Vitaminol 39(4):389–397

    Article  CAS  Google Scholar 

  • Tsuji H, Okada N, Yamanishi R, Bando N, Kimoto M, Ogawa T (1995) Measurement of Gly m Bd 30K, a major soybean allergen, in soybean products by a sandwich enzyme-linked immunosorbent assay. Biosci Biotechnol Biochem 59(1):150–151

    Article  CAS  Google Scholar 

  • Utsumi S, Maruyama N, Satoh R, Adachi M (2002) Structure–function relationships of soybean proteins revealed by using recombinant systems. Enzyme Microb Tech 30(3):284–288

    Article  CAS  Google Scholar 

  • van Boekel M, Fogliano V, Pellegrini N, Stanton C, Scholz G, Lalljie S, Somoza V, Knorr D, Jasti PR, Eisenbrand G (2010) A review on the beneficial aspects of food processing. Mol Nutr Food Res 54(9):1215–1247

    Article  Google Scholar 

  • van Boxtel EL, van den Broek LA, Koppelman SJ, Gruppen H (2008) Legumin allergens from peanuts and soybeans: effects of denaturation and aggregation on allergenicity. Mol Nutr Food Res 52(6):674–682

    Article  Google Scholar 

  • Wang TL, Domoney C, Hedley CL, Casey R, Grusak MA (2003) Can we improve the nutritional quality of legume seeds? Plant Physiol 131(3):886–891

    Article  CAS  Google Scholar 

  • Wilson S, Blaschek K, de Mejia E (2005) Allergenic proteins in soybean: processing and reduction of P34 allergenicity. Nutr Rev 63(2):47–58

    Google Scholar 

  • Wilson S, Martinez-Villaluenga C, De Mejia EG (2008) Purification, thermal stability, and antigenicity of the immunodominant soybean allergen P34 in soy cultivars, ingredients, and products. J Food Sci 73(6):T106–T114

    Article  CAS  Google Scholar 

  • Yaklich R, Helm R, Herman E (1999) Analysis of the distribution of the major soybean allergen in a core collection of Glycine max accessions. Crop Sci 39:1444–1447

    Article  CAS  Google Scholar 

  • You J, Li D, Qiao S, Wang Z, He P, Ou D, Dong B (2008) Development of a monoclonal antibody-based competitive ELISA for detection of [beta]-conglycinin, an allergen from soybean. Food Chem 106(1):352–360

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This research was funded by the Chinese National Support Plan of Science and Technology (no. 2006BAD12B04-06) and the Chinese National ‘863’ Hi-Tech R&D Plan (no. 2004AA246040).

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The authors have declared no conflict of interest.

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Authors and Affiliations

  1. Key Laboratory of Feed Biotechnology, Ministry of Agriculture, No. 12 Zhongguancun Nandajie Street, Beijing, 100081, People’s Republic China

    Bin Liu, Da Teng, Xiumin Wang, Yalin Yang & Jianhua Wang

  2. Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, Zhongguancun Nandajie St., Haidian District, Beijing, 100081, People’s Republic China

    Bin Liu, Da Teng, Xiumin Wang, Yalin Yang & Jianhua Wang

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  1. Bin Liu
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  2. Da Teng
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  3. Xiumin Wang
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  4. Yalin Yang
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  5. Jianhua Wang
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Corresponding author

Correspondence to Jianhua Wang.

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Liu Bin and Teng Da contributed equally to this paper.

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Liu, B., Teng, D., Wang, X. et al. Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method. Appl Microbiol Biotechnol 94, 1337–1345 (2012). https://doi.org/10.1007/s00253-012-4006-3

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  • DOI: https://doi.org/10.1007/s00253-012-4006-3

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