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Thesis Wagner 2019.P Identification of process-relevant kinases in human amniotic production cells Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulm Vorgelegt von Andreas Wagner aus Bad Köstritz 2019 Die vorliegende Arbeit wurde im Institut für Angewandte Biotechnologie der Hochschule Biberach unter der Anleitung von Herr Prof. Dr. Jürgen Hannemann durchgeführt. Amtierender Dekan: Prof. Dr. Peter Dürre Erstgutachter: Prof. Dr. Jürgen Hannemann Zweitgutachter: Prof. Dr. Bernd Eikmanns Wahlprüfer: Prof. Kerstin Otte Wahlprüfer: Prof. Dierk Niessing Tag der Promotion: 17. Juni 2019 Diese Arbeit wurde durch ein Promotionsstipendium nach dem Landesgraduiertenförderungsgesetz (LGFG) des Landes Baden- Württemberg finanziert und im Rahmen des kooperativen Promotionskollegs (KPK) „Pharmazeutische Biotechnologie“ der Universität Ulm und der Hochschule Biberach durchgeführt. Teile dieser Arbeit wurden bereits in den folgenden Artikel publiziert: Fischer, Simon, Andreas Wagner, Aron Kos, Armaz Aschrafi, René Handrick, Juergen Hannemann, and Kerstin Otte. 2013. “Breaking Limitations of Complex Culture Media: Functional Non-Viral MiRNA Delivery into Pharmaceutical Production Cell Lines.” Journal of Biotechnology 168: 589–600. “It’s a dangerous business, Frodo, going out of your door,” he used to say. “You step into the Road, and if you don’t keep your feet, there is no knowing where you might be swept to” Frodo Baggins, citing his uncle Bilbo Baggins The Lord of the Rings – The Fellowship of the Ring J.R.R. Tolkien Content I Content Content ..................................................................................................................................................... I Abbreviations ......................................................................................................................................... III Abstract .................................................................................................................................................. VI 1 Introduction ..................................................................................................................................... 1 1.1 Recombinant protein production in mammalian cell systems ............................................... 1 1.1.1 CEVEC’s amniocyte production cell technology .............................................................. 1 1.2 Protein expression optimization strategies ............................................................................. 2 1.2.1 Bioprocess development ................................................................................................. 2 1.2.2 Cell line development ...................................................................................................... 3 1.3 Phosphorylation as regulatory element and potential target for cell line development ..... 10 1.3.1 The human kinome and its relevance in biotechnological applications ....................... 10 1.3.2 Phosphorylation as an element for regulation of protein activity ................................ 12 1.4 Aim of this thesis ................................................................................................................... 14 2 Materials and Methods ................................................................................................................. 15 2.1 Cell culture ............................................................................................................................. 15 2.1.1 Cell lines, culture media and routine cell culture procedure ........................................ 15 2.1.2 Cryopreservation and thawing ...................................................................................... 15 2.1.3 Generation of CAP®-SEAP minipools and single cell cloning ......................................... 16 2.1.4 Establishment of stable cell pools ................................................................................. 16 2.1.5 Establishment of the siRNA screening procedure ......................................................... 17 2.1.6 High-throughput, high-content siRNA screening in recombinant CAP® cells ................ 21 2.1.7 Ectopic ERN1 overexpression ........................................................................................ 25 2.2 Analytical methods ................................................................................................................ 26 2.2.1 Trypan blue exclusion viability assay and cell count ..................................................... 26 2.2.2 Quantitative flow cytometry ......................................................................................... 27 2.2.3 Cell lysis and sample preparation for SDS-PAGE ........................................................... 32 2.2.4 BCA Assay ...................................................................................................................... 32 2.2.5 SDS-PAGE ....................................................................................................................... 32 2.2.6 Western Blot .................................................................................................................. 32 2.2.7 SEAP Reporter Assay ..................................................................................................... 33 2.2.8 Quantification of antibody concentration via HPLC ...................................................... 34 2.2.9 Statistic and functional analysis .................................................................................... 34 Content II 2.3 Molecular biology .................................................................................................................. 36 2.3.1 DNA plasmids ................................................................................................................ 36 2.3.2 Preparation of plasmid DNA .......................................................................................... 38 2.3.3 Total RNA extraction ..................................................................................................... 38 2.3.4 cDNA synthesis from total RNA samples ....................................................................... 38 2.3.5 Quantitative Real-Time PCR .......................................................................................... 39 3 Results ........................................................................................................................................... 40 3.1 Establishment of a high-throughput high-content RNAi screening platform with recombinant CAP®-SEAP cells .................................................................................................................................. 40 3.1.1 Establishment of a reliable siRNA transfection protocol for CAP® cells ........................ 40 3.1.2 Quality of the functional controls in the RNAi screening .............................................. 47 3.2 Identification of bioprocess-relevant kinases ....................................................................... 52 3.2.1 Overview of the results of the primary siRNA screening in CAP®-SEAP cells ................ 52 3.2.2 Functional analysis in silico............................................................................................ 59 3.2.3 Validation of selected targets in CAP®-IgG .................................................................... 68 3.3 Molecular analysis of putative cell engineering targets ........................................................ 72 3.3.1 STK24, DAPK3 and ERN1 are expressed in CAP® producer cells .................................... 72 3.3.2 Down-regulation of STK24 and DAPK3 to improve productivity ................................... 73 3.3.3 Cell line engineering – Ectopic expression of ERN1 ...................................................... 76 4 Discussion ...................................................................................................................................... 81 4.1 Suitability of the RNAi screening platform to identify process-relevant kinases .................. 81 4.2 Identification of bioprocess relevant kinases ........................................................................ 83 4.2.1 Proliferation and Viability .............................................................................................. 84 4.2.2 Recombinant protein productivity ................................................................................ 87 4.3 Cell line engineering with STK24, DAPK3 and ERN1 .............................................................. 94 4.4 Conclusion and Outlook ........................................................................................................ 98 5 References ..................................................................................................................................... 99 6 Appendix ...................................................................................................................................... 120 6.1 Supplementary material ...................................................................................................... 120 6.2 Content of Figures ............................................................................................................... 144 6.3 Content of Tables ................................................................................................................ 151 6.4 Online
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