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Directed Sortase Evolution for Site-Specific Protein Engineering and Surface Functionalization

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Directed Sortase Evolution for Site-Specific Protein Engineering and Surface Functionalization Directed sortase evolution for site-specific protein engineering and surface functionalization Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Zhi Zou Master of Biochemistry and Molecular Biology aus Huanggang, Hubei, P.R. China Berichter: Univ.-Prof. Dr. rer. nat. Ulrich Schwaneberg Univ.-Prof. Dr. rer. nat. Andrij Pich Tag der mündlichen Prüfung: 26.02.2019 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek verfügbar. Table of Contents Table of Contents Acknowledgements ....................................................................................................................................... 6 Abbreviations and acronyms ......................................................................................................................... 7 Abstract .......................................................................................................................................................... 9 1. Chapter I: Introduction ............................................................................................................................ 11 1.1. Sortases: sources, classes, and functions ......................................................................................... 11 1.1.1 Class A sortases: sortase A ......................................................................................................................... 11 1.1.2. Class B sortase: sortase B ......................................................................................................................... 15 1.1.3. Class C, D, E, F sortases ............................................................................................................................. 17 1.1.4. Applications of sortase-mediated ligation in protein engineering and polymer functionalization ......... 18 1.2. Directed evolution ............................................................................................................................ 22 1.2.1 State of the Art .......................................................................................................................................... 22 1.2.2. High throughput screening methods ........................................................................................................ 24 1.2.3. Directed sortase evolution ....................................................................................................................... 26 1.3 Objectives .......................................................................................................................................... 28 2. Chapter II: A sortase-mediated high throughput screening platform for directed enzyme evolution ... 29 2.1. Declaration ........................................................................................................................................ 29 2.2. Sortase-mediated high throughput screening: challenges and applications ................................... 29 2.3. Results and discussion ...................................................................................................................... 30 2.3.1 Principle of SortEvove screening platform ................................................................................................ 30 2.3.2. Production of Sa-SrtA, GGG-eGFP-LCI and CueO-LPETGGGRR ................................................................. 32 2.3.3. Performance criteria of screening assays in directed sortase evolution (Application 1) and directed laccase evolution coupled with a semi-purification process (Application 2) ..................................................... 34 2.3.4. Validation of the SortEvolve screening system for directed Sa-SrtA evolution (Application 1) ............... 38 2.3.5. Validation of the SortEvolve screening system for directed laccase evolution coupled with a semi- purification process (Application 2) .................................................................................................................... 39 2.3.6. Characterization of identified Sa-SrtA variants in application 1 and identified CueO-LPETGGGRR variants in application 2...................................................................................................................................... 40 2.4. Conclusions ....................................................................................................................................... 44 2.5. Materials and Methods .................................................................................................................... 44 2.5.1. Cloning of plasmid constructs .................................................................................................................. 45 2 Table of Contents 2.5.2. Protein expression and purification ......................................................................................................... 47 2.5.3. Anchoring test of purified GGG-eGFP-LCI to PP-MTP ............................................................................... 49 2.5.4. Activity assay of purified CueO-LPETGGGRR ............................................................................................ 49 2.5.5. Screening protocol for Application 1: Directed Sa-SrtA evolution ........................................................... 49 2.5.6. Screening protocol for Application 2: Directed CueO laccase evolution .................................................. 50 2.5.7. Characterization of evolved Sa-SrtA variants ........................................................................................... 52 2.5.8. Characterization of evolved CueO-LPETGGGRR variants ......................................................................... 53 3. Chapter III: Directed sortase A evolution for efficient site-specific bioconjugations in organic co- solvents ........................................................................................................................................................ 54 3.1. Declaration ........................................................................................................................................ 54 3.2. Site-specific bioconjugation: challenges and scientific importance ................................................. 54 3.3. Results and discussions ..................................................................................................................... 55 3.3.1. Optimization of SortEvolve in DMSO co-solvent ...................................................................................... 55 3.3.2 KnowVolution of Sa-SrtA for DMSO co-solvent ......................................................................................... 57 3.3.3. Computational studies of Sa-SrtA in DMSO co-solvent ............................................................................ 71 3.3.4. Applications of sortase-mediated site-specific conjugation in organic co-solvents ................................ 78 3.4. Conclusion ......................................................................................................................................... 86 3.5. Materials and Methods .................................................................................................................... 87 3.5.1 Materials .................................................................................................................................................... 87 3.5.2. Optimization of SortEvolve screening assay in DMSO co-solvent ............................................................ 88 3.5.3 KnowVolution of Sa-SrtA towards organic solvents .................................................................................. 89 3.5.4. FRET assay of recombined Sa-SrtA variants ............................................................................................. 92 3.5.5 Characterization of Sa-SrtA WT and variants in absence/presence of 45% (v/v) DMSO .......................... 92 3.5.6 Activity profiles of Sa-SrtA in diverse organic co-solvents ........................................................................ 93 3.5.7 Sortase-mediated protein-protein ligation in DMSO or ethanol co-solvents ........................................... 93 3.5.8. In silico generation of sortase variants ..................................................................................................... 93 3.5.9. Molecular dynamics (MD) simulations ..................................................................................................... 94 3.5.10. Sostase-mediated ligation of hydrophobic antiviral peptide AVP 0683 and Abz-LPETGK-Dnp in DMSO or DMF co-solvent .............................................................................................................................................. 95 3.5.11. Sortase-mediated ligation of hydrophobic amines (tyramine or 4-(Trifluoromethyl)-benzylamine) and Abz-LPETGK-Dnp in DMSO co-solvent ................................................................................................................ 96 3.5.12. Sortase-mediated PEGylation of hydrophobic peptide Abz-LPETGK-Dnp in DMSO co-solvent ............. 96 3 Table of Contents 4. Chapter IV: A platform for covalent enzyme immobilization on surface of stimuli microgels via sortase- mediated ligation ........................................................................................................................................
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