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 ...... 149
8.5 Curriculum vitae ...... 150
8.6 List of publications and presentations...... 152
8.7 Eigenständigkeitserklärung ...... 153
8
Abstract
Abstract
Escherichia coli has been commonly used as a platform for recombinant protein production and accounts for approximately 30% of current biopharmaceuticals on the market. Nowadays, many recombinant proteins require post-translational modifications which E. coli normally cannot facilitate. Therefore, novel technological advancements are unceasingly being developed to improve the E. coli expression system. In this work, some of the most recently engineered platforms for the production of disulfide bond-containing proteins were used to study the E. coli proteome under heterologous protein production stress. The effects of protein secretion via the Sec and Tat translocation pathways were examined using a comparative LC- MS/MS analysis. The E. coli proteome responds to foreign protein production by activation of several overlapping stress responses with a high degree of interaction. In consequence, a number of important cellular processes such as cellular metabolism, protein transport, redox state of the cytoplasm and membrane structure are altered by the production stress. These changes lead to the reduction of cellular growth and recombinant product yields. Resolving the identified bottlenecks will increase the efficiency of recombinant protein expression processes in E. coli.
9
Zusammenfassung
Zusammenfassung
Escherichia coli stellt einen häufigen Produktionsstamm für die Herstellung rekombinanter Proteine dar und nimmt etwa 30% des derzeitigen Marktes für Biopharmazeutika ein. Heutzutage benötigen viele rekombinante Proteine post-translationale Modifikationen, welche in E. coli normalerweise nicht unterstützt werden. Deshalb werden stetig neue technologische Verfahren entwickelt, um E.-coli als Expressionssystem zu verbessern. In dieser Arbeit werden einige erst jüngst entwickelte E. coli Stämme für die Produktion von Proteinen mit Disulfidbindungen untersucht, um das E.-coli Proteom unter Stress durch heterologe Proteinproduktion näher zu charakterisieren. Die Effekte der Proteinsekretion via Sec- und Tat- Translokations-Weg wurden mittels eines vergleichenden LC-MS/MS Analyseverfahrens ermittelt. Das E.-coli Proteom reagiert auf eine Fremdproteinproduktion mit der Induktion von zahlreichen und sich überschneidenden Stressreaktionen. Als Konsequenz des Produktionsstresses werden viele wichtige zelluläre Prozesse wie der zentrale Metabolismus, der Proteintransport, der Redox-Zustand des Zytoplasmas sowie die Membranstruktur verändert. Diese Anpassung führt zu einer Reduktion des zellulären Wachstums und der Ausbeute an rekombinantem Protein. Eine Aufklärung des zellulären Engpasses während der rekombinanten Proteinexpression könnte letztendlich die Effizienz des Produktionsprozesses in E. coli erhöhen.
10
Acknowledgements
Acknowledgements