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Microbioreactor Cultivations of Fab-Producing Escherichia coli Reveal Genome-Integrated Systems as Suitable for Prospective Studies on Direct Fab Expression Effects.
Biotechnol J 2019; :e1800637BJ

Abstract

Despite efforts to develop concepts for efficient antibody fragment (Fab) production in Escherichia coli (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon-limited microbioreactor cultivations with yields of 0-7.4 mg Fab per gram of cell dry mass. Antigen-binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80-300% of plasmid-based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions.

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, Muthgasse 18, 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, Muthgasse 18, 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, Muthgasse 18, 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, Muthgasse 18, 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, Muthgasse 18, 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, Muthgasse 18, A-1190, Vienna, Austria.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31231932

Citation

Fink, Mathias, et al. "Microbioreactor 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. Microbioreactor 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). Microbioreactor 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. Microbioreactor 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 - Microbioreactor 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 - 2018/12/20/received PY - 2019/06/11/revised PY - 2019/6/25/pubmed PY - 2019/6/25/medline PY - 2019/6/25/entrez KW - DsbA KW - Escherichia coli KW - OmpA KW - fragment antigen binding (Fab) KW - genome integration KW - microtiter fermentations 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 (E. coli) and the high degree of similarity within this protein class, a generic platform technology is still not available. Indeed, feasible production of new Fab candidates remains challenging. In this study, a setup that enables direct characterization of host cell response to Fab expression by utilizing genome-integrated (GI) systems is established. Among the multitude of factors that influence Fab expression, the variable domain, the translocation mechanism, the host strain, as well as the copy number of the gene of interest (GOI) are varied. The resulting 32 production clones are characterized in carbon-limited microbioreactor cultivations with yields of 0-7.4 mg Fab per gram of cell dry mass. Antigen-binding region variations have the greatest effect on Fab yield. In most cases, the E. coli HMS174(DE3) strain performs better than the BL21(DE3) strain. Translocation mechanism variations mainly influence leader peptide cleavage efficiency. Plasmid-free systems, with a single copy of the GOI integrated into the chromosome, reach Fab yields in the range of 80-300% of plasmid-based counterparts. Consequently, the GI Fab production clones could greatly facilitate direct analyses of systems response to different impact factors under varying production conditions. 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 -