Integrated approach to produce a recombinant, his-tagged human α-galactosidase a in mammalian cells

Authors

  • José Luis Corchero,

    Corresponding author
    1. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
    2. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    3. Dept. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    • CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
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  • Rosa Mendoza,

    1. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
    2. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
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  • Julia Lorenzo,

    1. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
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  • Victor Rodríguez-Sureda,

    1. Biochemistry and Molecular Biology Research Center for Nanomedicine, Vall d'Hebron University Hospital, Barcelona, Spain
    2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain
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  • Carmen Domínguez,

    1. Biochemistry and Molecular Biology Research Center for Nanomedicine, Vall d'Hebron University Hospital, Barcelona, Spain
    2. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain
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  • Esther Vázquez,

    1. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    2. Dept. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    3. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
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  • Neus Ferrer-Miralles,

    1. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    2. Dept. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    3. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
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  • Antonio Villaverde

    1. Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    2. Dept. of Genetics and Microbiology, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
    3. CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), Barcelona, Spain
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  • Conflict of interest: The authors declare no conflict of interests.

Abstract

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30–40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

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