Directed evolution of arginine deiminase (ADI) for anti-tumor application [Elektronische Ressource] / vorgelegt von Leilei Zhu

Lei Lei

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2010
• Nombre de lectures: 117
• Langue: Deutsch
• Poids de l'ouvrage: 36 Mo

Extrait

Directed Evolution of Arginine
Deiminase (ADI) for Anti-tumor
Application



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

Master of Biochemistry

Leilei Zhu

aus Weifang China


Berichter: Universitätsprofessor Dr. Ulrich Schwaneberg
ätsprofessor Dr. Lothar Elling



Tag der mündlichen Prüfung: 14.09.2010




Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.



Table of contents
Table of contents
Acknowledgement..................................................................................................I
Abstract................................................................................................................III
Abbreviations ....................................................................................................... V
List of figures....................................................................................................... VI
List of tables........................................................................................................ IX
Chapter 1 Arginine deiminase (ADI) .....................................................................1
1.1 Arginine deiminase (ADI).............................................................................1
1.2 ADI structure ...............................................................................................5
1.3 Catalytic reaction mechanism of ADI.........................................................10
1.4 Anti-tumor application of ADI.....................................................................11
1.5 Conclusion.................................................................................................16
Chapter 2 Directed evolution ..............................................................................17
2.1 Introduction to directed evolution...............................................................17
2.2 Directed enzyme evolution for pH tolerance of enzymes ..........................22
2.3 Directed enzyme evolution for medical application....................................24
2.3.1 Prodrug enzymes for tumor chemotherapy.........................................24
2.3.2 Oncolytic viruses for tumor chemotherapy..........................................25
2.3.3 Amino acid depriving enzyme for anti-tumor applications...................27
2.3.4 Affibody molecules for cancer prognosis ............................................28
2.3.5 Interferon for anti-tumor applicaiton ....................................................29
2.4 Conclusion.................................................................................................30
Chapter 3. Directed evolution of Arginine deiminase for increased activity at
physiological pH..................................................................................................31
3. 1 Abstract ....................................................................................................31
3.2 Introduction................................................................................................31
3.3 Materials and methods ..............................................................................33
3.3.1 Materials .............................................................................................33
3.3.2 Methods..............................................................................................34
i 3.3.2.1 Reagents used in assays .............................................................34
3.3.2.2 Cloning of ADI into pET42b(+) .....................................................34
3.3.2.3 Site directed mutagenesis of PpADI gene at position H404 .........35
3.3.2.4 Site sturation mutagenesis of PpADI gene at nine amino acid
position.....................................................................................................35
3.3.2.5 Construction of PpADI error-prone library ....................................36
3.3.2.6 Expression of PpADI in LB media with IPTG induction ................37
3.3.2.7 Expression of PpADI in auto-induction media ..............................37
3.3.2.8 Cultivation and expression of PpADI in 96-well plates .................37
3.3.2.9 Screening Procedure....................................................................38
3.3.2.10 Expression of ADI in shaking flask and purification....................39
3.3.2.11 Characterization of ADI wild type and mutants...........................40
3.3.2.12 Molecular Modeling ....................................................................41
3.3.2.13 Stability of PpADI in presence of serum .....................................41
3.3.2.14 Activity of PpADI (wild type, M1, M2) towards arginine derivative
dimethylarginine .......................................................................................42
3.4 Results ......................................................................................................42
3.4.1 PpADI cloning .....................................................................................42
3.4.2 PpADI expression in LB media with IPTG as inducer .........................44
3.4.3 PpADI expression in auto-induction media .........................................44
3.4.4 PpADI assay based on citrulline detection..........................................46
3.4.5 PpADI screening system for citrulline production in microtiter plates..47
3.4.6 Focused libraries generated by saturation mutagenesis.....................50
3.4.7 Validation of variant M1 (H404R) citrulline colorimetric assay in cuvette
format...........................................................................................................50
3.4.8 epPCR library generation and screening with citrulline detection assay
by screening error-prone mutant libraries ....................................................50
3.4.9 Characterization of PpADI variants.....................................................53
3.4.9.1 Determination of k and K of ADI wild type and the mutants ....53 cat m
3.4.9.2 Relative pH profile of wild type and mutant ..................................54
3.4.10 Stability of PpADI in the presence of human serum..........................55
ii G3.4.11 Activity of PpADI (wild type, M1, M2) towards arginine derivative (N ,
GN )-dimethylarginine....................................................................................56
3.5 Discussion.................................................................................................56
3.6 Conclusion.................................................................................................59
Chapter 4. PpADI reengineered for efficient operation under physiological
conditions............................................................................................................60
4.1 Abstract .....................................................................................................60
4.2 Introduction................................................................................................60
4.3 Materials and methods ..............................................................................62
4.3.1 Materials .............................................................................................62
4.3.2 Methods..............................................................................................62
4.3.2.1 Site saturation mutagenesis of PpADI M2 at position A128 .........62
4.3.2.2 Construction of PpADI error-prone library ....................................63
4.3.2.3 Site directed mutagenesis at position D38 of PpADI M4..............63
4.3.2.4 Site saturation mutagenesis at position D38 and E296 of PpADI
M3 ............................................................................................................64
4.3.2.5 Cultivation and expression of PpADI in 96-well plates .................64
4.3.2.6 Improved screening system in 96-well plate to identify PpADI
variants with high activities at low arginine concentration ........................64
4.3.2.7 Expression of PpADI in shaking flask and purification..................65
4.3.2.8 Characterization of PpADI variants ..............................................65
4.3.2.9 Native polyacrylamide gel electrophoresis of PpADI variants ......66
4.3.2.10 Homology modeling....................................................................67
4.4 Results ......................................................................................................67
4.4.1 Improved screening system in microtiter plates to identify PpADI
variants with high activities at low arginine concentration............................67
4.4.2 Genealogic tree of directed PpADI evolution ......................................68
4.4.3 EpPCR library construction and screening for variants with high
activities at low arginine concentration ........................................................69
4.4.4 Site saturation mutagenesis of PpADI at position D38 and E296 ...