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Proteomics-Based Analysis of Stress Responses During Recombinant Protein Production in Escherichia Coli''

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Proteomics-Based Analysis of Stress Responses During Recombinant Protein Production in Escherichia Coli'' PROTEOMICS-BASED ANALYSIS OF STRESS RESPONSES DURING RECOMBINANT PROTEIN PRODUCTION IN ESCHERICHIA COLI Inauguraldissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Greifswald vorgelegt von Katarzyna Magdalena Dolata geboren am 11.11.1989 in Poznań Greifswald, den 21.03.2019 1 Dekan: Prof. Dr. Werner Weitschies 1. Gutachter: Prof. Dr. Katharina Riedel 2. Gutachter: Prof. Dr. Wolfgang Liebl Tag der Promotion: 18.07.2019 2 Dedicated to my family and friends 3 4 Table of contents Table of contents Abstract .................................................................................................................................... 9 Zusammenfassung................................................................................................................. 10 Acknowledgements ............................................................................................................... 11 Original publications ............................................................................................................ 13 List of tables........................................................................................................................... 14 List of figures ......................................................................................................................... 15 Abbreviations ........................................................................................................................ 18 Introduction ................................................................................................................... 21 1.1 Overview of the recombinant therapeutic protein market ...................................... 21 1.2 E. coli as a favored bacterial host for recombinant protein production .................. 25 1.3 Strategies for the production of recombinant proteins in E. coli ............................ 28 1.3.1 Optimization of the protein expression vector .................................................. 30 1.3.2 Targeted expression of recombinant proteins ................................................... 32 1.3.3 Induction strategy.............................................................................................. 35 1.4 Cellular stress induced by recombinant protein production in E. coli .................... 36 1.5 Novel E. coli strains enhancing heterologous protein production .......................... 41 1.6 Application of proteomics to study protein production stress ................................ 43 Scope of the thesis ......................................................................................................... 46 Results ............................................................................................................................ 47 3.1 Cellular consequences of Tat pathway deletion in E. coli ...................................... 47 3.1.1 Loss of Tat function has a major impact on cell morphology .......................... 47 3.1.2 Proteome changes linked to the loss of Tat export ........................................... 49 5 Table of contents 3.2 Stress responses related to recombinant protein production and Tat-dependent secretion ................................................................................................................. 55 3.2.1 Production of scFv antibody in the oxidative cytoplasm .................................. 55 3.2.2 Co-expression of hGH with Tat system components ....................................... 65 3.3 Proteome changes under tunable recombinant protein expression and Sec- dependent secretion ................................................................................................ 78 3.3.1 Stress responses related to hGH expression and secretion via Sec system....... 80 3.3.2 Proteome changes associated with an excessive expression induction ............ 86 Discussion....................................................................................................................... 88 4.1 The Tat pathway has an important role in cell division and maintaining cell wall integrity in E. coli ................................................................................................... 88 4.2 Effect of foreign protein synthesis on growth kinetics ........................................... 90 4.3 Metabolic responses to protein overexpression ...................................................... 91 4.4 Recombinant protein overexpression suppresses periplasmic secretion of host's proteins ................................................................................................................... 95 4.5 Cell envelope stress and alterations of membrane integrity ................................... 98 4.6 Oxidative stress and redox regulation in the cytoplasm ......................................... 99 4.7 Proteostasis control by the protein production response....................................... 101 4.8 Cell adhesion mediated by misfolded protein production .................................... 103 Conclusions .................................................................................................................. 104 6 Materials and methods ............................................................................................... 105 6.1 Bacterial strains and plasmids ............................................................................... 106 6.2 Microbiological techniques ................................................................................... 107 6.2.1 Bacterial culture preservation ......................................................................... 107 6 Table of contents 6.2.2 Cell cultivation and recombinant protein expression...................................... 108 6.2.3 Transformation of E. coli ................................................................................ 110 6.3 Physiological assays.............................................................................................. 112 6.3.1 Monitoring of bacterial growth ....................................................................... 112 6.3.2 Biofilm formation ........................................................................................... 113 6.3.3 Colanic acid quantification ............................................................................. 113 6.3.4 Cell aggregation .............................................................................................. 114 6.4 Microscopy ........................................................................................................... 114 6.4.1 Transmission electron microscopy ................................................................. 114 6.4.2 Phase contrast microscopy .............................................................................. 115 6.5 Sample preparation for mass spectrometry ........................................................... 115 6.5.1 Subcellular fractionation and protein extraction ............................................. 115 6.5.2 Protein precipitation ........................................................................................ 117 6.5.3 Determination of protein concentration .......................................................... 117 6.5.4 Electrophoresis (SDS-PAGE) ......................................................................... 117 6.5.5 In-gel protein digestion ................................................................................... 118 6.5.6 In-solution protein digestion ........................................................................... 119 6.6 Analytical methods ............................................................................................... 119 6.6.1 Western Blot ................................................................................................... 119 6.6.2 LC-MS analysis .............................................................................................. 120 6.7 Bioinformatic analysis of proteomic data ............................................................. 123 6.7.1 Statistical analysis ........................................................................................... 123 6.7.2 Protein annotation ........................................................................................... 123 7 Table of contents 6.7.3 Graphical presentation .................................................................................... 124 6.7.4 Data deposition ............................................................................................... 124 References .................................................................................................................... 125 Appendix ...................................................................................................................... 139 8.1 CD-ROM contents ................................................................................................ 139 8.2 Protein and peptide sequences .............................................................................. 140 8.3 Equipment and consumables................................................................................. 142 8.4 Reagents and chemicals ........................................................................................ 145 8.5 Software ...............................................................................................................
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