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Protein Engineering of a Therapeutic Enzyme Arginine Deiminase

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Protein Engineering of a Therapeutic Enzyme Arginine Deiminase Protein engineering of a therapeutic enzyme arginine deiminase Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science (M. Sc.) Biochemical Engineering Feng Cheng aus Hangzhou, Zhejiang, P.R. China Berichter: Universitätsprofessor Dr. Ulrich Schwaneberg Universitätsprofessor Dr. Lothar Elling Tag der mündlichen Prüfung: 26.08.2015 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. ‘‘I am convinced that natural selection has been the most important, but not the exclusive, means of modification.’’ (Charles Darwin; ‘The origin of species’) Table of Contents Table of Contents Acknowledgements ....................................................................................................................... VII Abbreviations and acronyms ........................................................................................................ VIII Abstract ........................................................................................................................................... XI Graphical abstract ........................................................................................................................ XIV 1. Chapter I: Introduction ............................................................................................................. 1 1.1. Arginine deiminase (ADI) ................................................................................................... 1 1.1.1. ADIs in Nature ............................................................................................................ 1 1.1.2. ADIs’ structures .......................................................................................................... 2 1.1.3. The proposed catalytic reaction mechanism of ADI .................................................. 5 1.2. Anti-tumor application of ADI ........................................................................................... 7 1.2.1. Amino acid depriving enzyme for anti-tumor applications ....................................... 7 1.2.2. Arginine depriving enzyme for anti-tumor applications ............................................ 7 1.2.3. PEGylation of ADI ....................................................................................................... 9 1.3. Directed evolution ........................................................................................................... 12 1.3.1. Diversity generation ................................................................................................. 13 1.3.2. Screening systems .................................................................................................... 15 1.4. Objectives ........................................................................................................................ 17 2. Chapter II: Directed PpADI evolution for obtaining improved PpADI variants with high activity under physiological conditions using a sensitive screening system in MTP format ......... 18 2.1. Declaration ...................................................................................................................... 18 2.2. Introduction ..................................................................................................................... 18 2.3. Materials and Methods ................................................................................................... 19 Page I Table of Contents 2.3.1. Development of an ammonia detection based screening assay in 96-well plate format ……………………………………………………………………………………………………………..…………… 19 2.3.2. Generation of epPCR (error-prone PCR) library ....................................................... 21 2.3.3. Generation of a PpADI SeSaM (Sequence Saturation Mutagenesis) library ............ 22 2.3.4. Cultivation and expression of PpADI in 96-well MTP ............................................... 23 2.3.5. Screening procedures ............................................................................................... 24 2.3.6. Expression, purification, and quantification of PpADI WT, parent M6 and variants M19 and M21 ......................................................................................................................... 26 2.3.7. Determination of kinetic constants of PpADI WT, parent M6 and variants M19 and M21 …………………………………………………………………………………………………………………………… 26 2.3.8. Homology modeling ................................................................................................. 27 2.3.9. Cultivation conditions for melanoma cell line cultures ........................................... 27 2.3.10. Cell proliferation assay ......................................................................................... 27 2.4. Results .............................................................................................................................. 28 2.4.1. Development of ammonia detection system (ADS) for PpADI activity detection and library screening ..................................................................................................................... 28 2.4.2. Validation of the continuous ammonia detection system (ADS) for directed ADI evolution by screening an epPCR library and a SeSaM library .............................................. 31 2.4.3. Recombination of beneficial mutations by site-directed mutagenesis (SDM) ........ 34 2.4.4. Determination of kcat and S0.5 values of PpADI WT, parent M6, ‘best’ variants M19 and M21 ................................................................................................................................. 36 2.4.5. Anti-proliferation activity of PpADI WT, parent M6 and the selected variant M19 against two melanoma cell lines ............................................................................................ 38 2.5. Discussion ........................................................................................................................ 39 2.6. Conclusions ...................................................................................................................... 42 Page II Table of Contents 3. Chapter III: Directed evolution of ADI based on a competitive flow cytometry screening system ............................................................................................................................................ 43 3.1. Declaration ...................................................................................................................... 43 3.2. Introduction ..................................................................................................................... 43 3.3. Materials and Methods ................................................................................................... 44 3.3.1. Construction of arginine biosensor plasmid pArg-LiMEx ......................................... 44 3.3.2. Expression strains and media ................................................................................... 46 3.3.3. Characterization of pArg-LiMEx ............................................................................... 46 3.3.4. Optimization of LiMEx and identification of sorting parameters for flow cytometry ………………………………………………………………………………………………………………………….. 46 3.3.5. Protein expression and preparation of crude cell extracts in 96-well microtiter plate (MTP) ............................................................................................................................. 47 3.3.6. Citrulline-detection system in MTP format .............................................................. 47 3.3.7. Ammonia-detection system (ADS) in MTP format ................................................... 48 3.3.8. PpADI error-prone PCR (epPCR) library generation and cloning ............................. 48 3.3.9. Site-directed mutagenesis for recombination of selected amino acid positions .... 49 3.3.10. Expression and purification of PpADI WT, parent M6 and the ‘best’ variant M31 ………………………………………………………………………………………………………………………. 50 3.3.11. Kinetic characterization of the purified PpADI variants ....................................... 50 3.3.12. Cultivation conditions for melanoma cell line cultures ........................................ 50 3.3.13. Cell proliferation assay ......................................................................................... 50 3.4. Results .............................................................................................................................. 50 3.4.1. Construction of pArg-LiMEx vector (the biosensor) ................................................ 52 3.4.2. Characterization of pArg-LiMEx ............................................................................... 53 3.4.3. Development of pArg-LiMEx based ultrahigh-throughput screening system ......... 55 Page III Table of Contents 3.4.4. Sorting of model populations by Arg-LiMEx ultrahigh-throughput screening system …………………………………………………………………………………………………………………………… 57 3.4.5. PpADI error-prone PCR (epPCR) library generation and screening by Arg-LiMEx ... 58 3.4.6. Determination of kcat and S0.5 values of purified PpADI WT, parent M21, ‘best’ variant M31 ............................................................................................................................ 60 3.4.7. Anti-proliferation activity of PpADI WT,
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