Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis

J. Glenting, Lars K. Poulsen, K. Kato, S.M. Madsen, Hanne Frøkiær, C. Wendt, H.W. Sorensen

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Background: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/ or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high- level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/ L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N- terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.
    Original languageEnglish
    JournalMicrobial Cell Factories
    Volume6
    ISSN1475-2859
    DOIs
    Publication statusPublished - 2007

    Cite this

    Glenting, J., Poulsen, L. K., Kato, K., Madsen, S. M., Frøkiær, H., Wendt, C., & Sorensen, H. W. (2007). Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis. Microbial Cell Factories, 6. https://doi.org/10.1186/1475-2859-6-28
    Glenting, J. ; Poulsen, Lars K. ; Kato, K. ; Madsen, S.M. ; Frøkiær, Hanne ; Wendt, C. ; Sorensen, H.W. / Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis. In: Microbial Cell Factories. 2007 ; Vol. 6.
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    title = "Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis",
    abstract = "Background: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/ or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high- level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/ L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N- terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.",
    author = "J. Glenting and Poulsen, {Lars K.} and K. Kato and S.M. Madsen and Hanne Fr{\o}ki{\ae}r and C. Wendt and H.W. Sorensen",
    year = "2007",
    doi = "10.1186/1475-2859-6-28",
    language = "English",
    volume = "6",
    journal = "Microbial Cell Factories",
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    Glenting, J, Poulsen, LK, Kato, K, Madsen, SM, Frøkiær, H, Wendt, C & Sorensen, HW 2007, 'Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis', Microbial Cell Factories, vol. 6. https://doi.org/10.1186/1475-2859-6-28

    Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis. / Glenting, J.; Poulsen, Lars K.; Kato, K.; Madsen, S.M.; Frøkiær, Hanne; Wendt, C.; Sorensen, H.W.

    In: Microbial Cell Factories, Vol. 6, 2007.

    Research output: Contribution to journalJournal articleResearchpeer-review

    TY - JOUR

    T1 - Production of recombinant peanut allergen Ara h 2 using Lactococcus lactis

    AU - Glenting, J.

    AU - Poulsen, Lars K.

    AU - Kato, K.

    AU - Madsen, S.M.

    AU - Frøkiær, Hanne

    AU - Wendt, C.

    AU - Sorensen, H.W.

    PY - 2007

    Y1 - 2007

    N2 - Background: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/ or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high- level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/ L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N- terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.

    AB - Background: Natural allergen sources can supply large quantities of authentic allergen mixtures for use as immunotherapeutics. However, such extracts are complex, difficult to define, vary from batch to batch, which may lead to unpredictable efficacy and/ or unacceptable levels of side effects. The use of recombinant expression systems for allergen production can alleviate some of these issues. Several allergens have been tested in high- level expression systems and in most cases show immunereactivity comparable to their natural counterparts. The gram positive lactic acid bacterium Lactococcus lactis is an attractive microorganism for use in the production of protein therapeutics. L. lactis is considered food grade, free of endotoxins, and is able to secrete the heterologous product together with few other native proteins. Hypersensitivity to peanut represents a serious allergic problem. Some of the major allergens in peanut have been described. However, for therapeutic usage more information about the individual allergenic components is needed. In this paper we report recombinant production of the Ara h 2 peanut allergen using L. lactis. Results: A synthetic ara h 2 gene was cloned into an L. lactis expression plasmid containing the P170 promoter and the SP310mut2 signal sequence. Flask cultures grown overnight showed secretion of the 17 kDa Ara h 2 protein. A batch fermentation resulted in 40 mg/ L recombinant Ara h 2. Purification of Ara h 2 from the culture supernatant was done by hydrophobic exclusion and size separation. Mass spectrometry and N- terminal analysis showed a recombinant Ara h 2 of full length and correctly processed by the signal peptidase. The immunological activity of recombinant Ara h 2 was analysed by ELISA using antibodies specific for native Ara h 2. The recombinant Ara h 2 showed comparable immunereactivity to that of native Ara h 2. Conclusion: Recombinant production of Ara h 2 using L. lactis can offer high yields of secreted, full length and immunologically active allergen. The L. lactis expression system can support recombinant allergen material for immunotherapy and component resolved allergen diagnostics.

    U2 - 10.1186/1475-2859-6-28

    DO - 10.1186/1475-2859-6-28

    M3 - Journal article

    VL - 6

    JO - Microbial Cell Factories

    JF - Microbial Cell Factories

    SN - 1475-2859

    ER -