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Novel human kidney epithelial cell line in pharmaceutical biotechnology
SYMPOSIUM 19: EVOLUTIONARY STRATEGIES FOR CELL FACTORY DEVELOPMENT
procedures (passaging and cell banking) do not affect the ‘average’ genome structure and sequence to a great extent. The extraordinary chromosomal plasticity of this genome, however, seems to be the driving adaptive force when cells are put through a bottleneck. This feature underlies a novel application for which we provide proof of concept here: selection of 293 clones surviving stringent selective conditions (eg ricin toxin), followed by whole-genome analysis of copy number alterations, can effectively pinpoint the genomic region(s) that contain the gene(s) required for adaptation to those selective conditions. Furthermore, up to the level of sensitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In terms of tools, we optimized a workflow to detect human/vector genome breakpoints, and enabled visualization of the 293 genome data both through a user-friendly visualization web page, as well as through the Integrative Genome Browser (IGV) for rich data mining. http://dx.doi.org/10.1016/j.nbt.2014.05.1775
O19-4 Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16 Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ Graz University of Technology, Austria
The gram-negative -proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. Up to now some basic systems for targeted genetic manipulation of this bacterium were already established. However, the majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmids loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 hours. The utility of the new vector family is demonstrated by providing expression data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776
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www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O19-5 Novel human kidney epithelial cell line in pharmaceutical biotechnology Lukas Fliedl 1,∗ , Matthias Wieser 1 , Gabriele Manhart 1 , Matthias P. Gerstl 1 , Florian Kast 1 , Abdulhameed Khan 2 , Renate Kunert 2 , Johannes Grillari 2 , Regina Grillari-Voglauer 2 1
ACIB, Austria Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria
2
Mammalian cells are used as model systems, products themselves and as producers of recombinant proteins and vaccines. In these different applications a variety of different cell lines are used and although all have proven valuable for their specific application, there is still room for improvement in terms of posttranslational modifications. Especially novel human cell lines are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has maintained many differentiated characteristics of the normal nontransduced counterpart and tested its performance in the different fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well as specific non-human glycopatterns. Additionally, we used our cell line to produce influenza virus and proved high capabilities of our cell line in this application. Finally, we used our cell line to get insights into nephrotoxicity induced by cisplatin, which has important implications as a chemotherapeutic drug and hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. Therefore, the here established kidney epithelial cells combine applicability in various fields of pharmaceutical biotechnology, as they are capable of production as well as of pre-clinical testing of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1777
O19-6 De novo production of geranic acid with Pseudomonas putida Jens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markus Buchhaupt, Dirk Holtmann DECHEMA Research Institute, Germany
Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more
procedures (passaging and cell banking) do not affect the ‘average’ genome structure and sequence to a great extent. The extraordinary chromosomal plasticity of this genome, however, seems to be the driving adaptive force when cells are put through a bottleneck. This feature underlies a novel application for which we provide proof of concept here: selection of 293 clones surviving stringent selective conditions (eg ricin toxin), followed by whole-genome analysis of copy number alterations, can effectively pinpoint the genomic region(s) that contain the gene(s) required for adaptation to those selective conditions. Furthermore, up to the level of sensitivity afforded here (single copy plasmid insertions were easily detected), these cell lines have no inadvertent virus insertions. In terms of tools, we optimized a workflow to detect human/vector genome breakpoints, and enabled visualization of the 293 genome data both through a user-friendly visualization web page, as well as through the Integrative Genome Browser (IGV) for rich data mining. http://dx.doi.org/10.1016/j.nbt.2014.05.1775
O19-4 Versatile and stable vectors for efficient gene expression in Ralstonia eutropha H16 Steffen Gruber , Jeremias Hagen, Helmut Schwab, Petra Koefinger ∗ Graz University of Technology, Austria
The gram-negative -proteobacterium Ralstonia eutropha H16 is primarily known for polyhydroxybutyrate (PHB) production and its ability to grow chemolithoautotrophically by using CO2 and H2 as sole carbon and energy sources. Up to now some basic systems for targeted genetic manipulation of this bacterium were already established. However, the majority of metabolic engineering and heterologous expression studies conducted so far rely on a small number of suitable expression systems. Particularly the plasmid based expression systems already developed for the use in R. eutropha H16 suffer from high segregational instability and plasmids loss after a short time of fermentation. In order to develop efficient and highly stable plasmid expression vectors for the use in R. eutropha H16 a new plasmid design was created including the RP4 partitioning system, as well as various promoters and origins of replication. The application of minireplicons derived from broad-host-range plasmids RSF1010, pBBR1, RP4 and pSa for the construction of expression vectors and the use of numerous, versatile promoters extend the range of feasible expression levels considerably. Moreover, the implementation of the RP4 partition sequence in plasmid design increased plasmid stability significantly and enables fermentations with marginal plasmid loss of recombinant R. eutropha H16 for at least 96 hours. The utility of the new vector family is demonstrated by providing expression data with different model proteins. http://dx.doi.org/10.1016/j.nbt.2014.05.1776
S72
www.elsevier.com/locate/nbt
New Biotechnology · Volume 31S · July 2014
O19-5 Novel human kidney epithelial cell line in pharmaceutical biotechnology Lukas Fliedl 1,∗ , Matthias Wieser 1 , Gabriele Manhart 1 , Matthias P. Gerstl 1 , Florian Kast 1 , Abdulhameed Khan 2 , Renate Kunert 2 , Johannes Grillari 2 , Regina Grillari-Voglauer 2 1
ACIB, Austria Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria
2
Mammalian cells are used as model systems, products themselves and as producers of recombinant proteins and vaccines. In these different applications a variety of different cell lines are used and although all have proven valuable for their specific application, there is still room for improvement in terms of posttranslational modifications. Especially novel human cell lines are of ever increasing importance since they ideally represent the in vivo situation and might produce high quality biopharmaceuticals as similar to endogenous proteins as possible. Therefore we established a novel human continuously growing renal proximal tubular epithelial cell line (RPTEC) that has maintained many differentiated characteristics of the normal nontransduced counterpart and tested its performance in the different fields of pharmaceutical biotechnology. A complex model protein, erythropoietin, was stably produced in this cell line and the quality of the recombinant protein was compared to CHO derived product by analysis of isoforms as well as specific non-human glycopatterns. Additionally, we used our cell line to produce influenza virus and proved high capabilities of our cell line in this application. Finally, we used our cell line to get insights into nephrotoxicity induced by cisplatin, which has important implications as a chemotherapeutic drug and hypothesize that especially epithelial barrier formation and polarity of RPTECs need to be considered in toxicity models to validly predict the in vivo situation. Therefore, the here established kidney epithelial cells combine applicability in various fields of pharmaceutical biotechnology, as they are capable of production as well as of pre-clinical testing of biopharmaceuticals. http://dx.doi.org/10.1016/j.nbt.2014.05.1777
O19-6 De novo production of geranic acid with Pseudomonas putida Jens Schrader ∗ , Jia Mi, Daniela Becher, Patrice Lubuta, Markus Buchhaupt, Dirk Holtmann DECHEMA Research Institute, Germany
Production of plant terpenes by engineered microbes has become a prime example of applied synthetic biology with tremendous progress being made during the last decade. Whereas sesquiterpene titers reported have already reached g/L values in the bioreactor, efficient monoterpene production seems to be more