title: Heterologous protein production using euchromatin-containing expression vectors in mammalian cells.
abstract: Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090, Austria.
authors.forename: Katalin
authors.initials: K
authors.lastname: Zboray
authors.affiliation: Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, 1190, Austria Polymun Scientific GmbH, Klosterneuburg, 3400, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sommeregger
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090, Austria.
authors.forename: Edith
authors.initials: E
authors.lastname: Bogner
authors.affiliation: Polymun Scientific GmbH, Klosterneuburg, 3400, Austria.
authors.forename: Andreas
authors.initials: A
authors.lastname: Gili
authors.affiliation: Polymun Scientific GmbH, Klosterneuburg, 3400, Austria.
authors.forename: Thomas
authors.initials: T
authors.lastname: Sterovsky
authors.affiliation: Polymun Scientific GmbH, Klosterneuburg, 3400, Austria.
authors.forename: Katharina
authors.initials: K
authors.lastname: Fauland
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090, Austria.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Grabner
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Stiedl
authors.affiliation: Institute of Pharmacology, Center of Physiology and Pharmacology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, 1090, Austria.
authors.forename: Herwig P
authors.identifier: http://orcid.org/0000-0001-6438-9068
authors.initials: HP
authors.lastname: Moll
authors.affiliation: The Antibody Lab, Vienna, 1210, Austria.
authors.forename: Anton
authors.initials: A
authors.lastname: Bauer
authors.affiliation: Vienna Institute of BioTechnology, Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, 1190, Austria renate.kunert@boku.ac.at.
authors.forename: Renate
authors.initials: R
authors.lastname: Kunert
authors.affiliation: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, 1090, Austria Institute of Pharmacology, Center of Physiology and Pharmacology, Comprehensive Cancer Center, Medical University of Vienna, Vienna, 1090, Austria emilio.casanova@lbicr.lbg.ac.at.
authors.forename: Emilio
authors.initials: E
authors.lastname: Casanova
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000235:genetics; D016466:CHO Cells; D022202:Chromosomes, Artificial, Bacterial; D006224:Cricetinae; D003412:Cricetulus; D022041:Euchromatin; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D015483:HIV Antibodies / Q000096:biosynthesis / Q000235:genetics; D015497:HIV-1 / Q000235:genetics / Q000276:immunology; D054298:Human Immunodeficiency Virus Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D057134:Antibodies, Neutralizing; D022041:Euchromatin; D006023:Glycoproteins; D015483:HIV Antibodies; D054298:Human Immunodeficiency Virus Proteins; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins