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Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects.

Abstract

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli and the high degree of similarity within this protein class, we are far from a generic platform technology. Indeed, feasible production of new Fab candidates remains challenging. In this study, we established a set-up that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated systems. Among the multitude of factors that influence Fab expression, we varied the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest. The resulting 32 production clones were characterized in carbon-limited micro-bioreactor cultivations with yields of 0 - 7.4 mg Fab/g cell dry mass. Antigen-binding region variations had the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performed better than the BL21(DE3) strain. Translocation mechanism variations mainly influenced leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the gene of interest integrated into the chromosome, reached Fab yields in the range of 80 to 300% of plasmid-based counterparts. Consequently, thegenome-integrated Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions. This article is protected by copyright. All rights reserved.

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  • Authors+Show Affiliations

    ,

    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

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    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

    ,

    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

    ,

    Boehringer Ingelheim RCV GmbH & Co KG, Dr. Boehringer-Gasse 5-11, A-1120, Vienna, Austria.

    ,

    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

    ,

    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

    Christian Doppler Laboratory for production of next-level biopharmaceuticals in E. coli, Department of Biotechnology, University of Natural Resources and Life Sciences, A-1190, Vienna, Austria.

    Source

    Biotechnology journal : 2019 Jun 24 pg e1800637

    Pub Type(s)

    Journal Article

    Language

    eng

    PubMed ID

    31231932

    Citation

    Fink, Mathias, et al. "Micro-bioreactor Cultivations of Fab Producing Escherichia Coli Reveal Genome-integrated Systems as Suitable for Prospective Studies On Direct Fab Expression Effects." Biotechnology Journal, 2019, pp. e1800637.
    Fink M, Vazulka S, Egger E, et al. Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects. Biotechnol J. 2019.
    Fink, M., Vazulka, S., Egger, E., Jarmer, J., Grabherr, R., Cserjan-Puschmann, M., & Striedner, G. (2019). Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects. Biotechnology Journal, pp. e1800637. doi:10.1002/biot.201800637.
    Fink M, et al. Micro-bioreactor Cultivations of Fab Producing Escherichia Coli Reveal Genome-integrated Systems as Suitable for Prospective Studies On Direct Fab Expression Effects. Biotechnol J. 2019 Jun 24;e1800637. PubMed PMID: 31231932.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Micro-bioreactor cultivations of Fab producing Escherichia coli reveal genome-integrated systems as suitable for prospective studies on direct Fab expression effects. AU - Fink,Mathias, AU - Vazulka,Sophie, AU - Egger,Esther, AU - Jarmer,Johanna, AU - Grabherr,Reingard, AU - Cserjan-Puschmann,Monika, AU - Striedner,Gerald, Y1 - 2019/06/24/ PY - 2019/6/25/entrez KW - DsbA KW - E. coli KW - Fab KW - OmpA KW - genome integration KW - microtiter fermentation SP - e1800637 EP - e1800637 JF - Biotechnology journal JO - Biotechnol J N2 - Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli and the high degree of similarity within this protein class, we are far from a generic platform technology. Indeed, feasible production of new Fab candidates remains challenging. In this study, we established a set-up that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated systems. Among the multitude of factors that influence Fab expression, we varied the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest. The resulting 32 production clones were characterized in carbon-limited micro-bioreactor cultivations with yields of 0 - 7.4 mg Fab/g cell dry mass. Antigen-binding region variations had the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performed better than the BL21(DE3) strain. Translocation mechanism variations mainly influenced leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the gene of interest integrated into the chromosome, reached Fab yields in the range of 80 to 300% of plasmid-based counterparts. Consequently, thegenome-integrated Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions. This article is protected by copyright. All rights reserved. SN - 1860-7314 UR - https://www.unboundmedicine.com/medline/citation/31231932/Micro-bioreactor_cultivations_of_Fab_producing_Escherichia_coli_reveal_genome-integrated_systems_as_suitable_for_prospective_studies_on_direct_Fab_expression_effects L2 - https://doi.org/10.1002/biot.201800637 DB - PRIME DP - Unbound Medicine ER -