Structural studies of tripeptidylpeptidase II [Elektronische Ressource] : expression and crystallization trials / Gönül Seyit

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Technische Universität München
Max-Planck-Institut für Biochemie
Abteilung für Molekulare Strukturbiologie
Structural Studies of Tripeptidylpeptidase II:
Expression and Crystallization Trials
Gönül Seyit
Vollständiger Abdruck der von der Fakultät für Chemie der Technischen
Universität München zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.) genehmigten Dissertation.
Vorsitzende: Univ.-Prof. Dr. Johannes Buchner
Prüfer der Dissertation: 1. Hon.-Prof. Dr. Wolfgang Baumeister
2. Univ.-Prof. Dr. Sevil Weinkauf
Die Dissertation wurde am 28.09.2006 bei der Technischen Universität München
eingereicht und durch die Fakultät für Chemie am 05.12.2006 angenommen.TABLE OF CONTENTS
1. SUMMARY ......................................................................................................... 4
2. INTRODUCTION ................................................................................................ 5
2.1 Cellular Proteolysis........................................................................................................................................... 5
2.2 Cellular roles of TPP II .................................................................................................................................... 5
2.3 Peptidase activity of TPP II ............................................................................................................................. 8
2.4 Structure of TPP II ........................................................................................................................................... 9
2.5 Size-activity relationship ................................................................................................................................ 12
2.6 Preparation of TPP II..................................................................................................................................... 13
2.7 The aims of this study..................................................................................................................................... 14
3. MATERIALS AND METHODS ......................................................................... 15
3.1 Growth and maintenance of Escherichia coli.............................................................................................. 15
3.1.1 E. coli host strains ..................................................................................................................................... 15
3.1.2 Storage of E. coli cultures......................................................................................................................... 15
3.1.3 Growing starter cultures of E. coli ........................................................................................................... 15
3.1.4 Preparation of competent E. coli cells...................................................................................................... 16
3.1.5 Transformation of competent E. coli cells ............................................................................................... 16
3.2 Nucleic acid techniques................................................................................................................................... 17
3.2.1 Isolation of plasmid DNA from E. coli.................................................................................................... 17
3.2.2 Restriction enzyme digestion of DNA ..................................................................................................... 17
3.2.3 Agarose gel electrophoresis...................................................................................................................... 17
3.2.4 Isolation of DNA from agarose gels......................................................................................................... 17
3.2.5 Quantitation of DNA................................................................................................................................. 18
3.2.6 Cloning of Drosophila melanogaster TPP II cDNA in E. coli ............................................................... 18
3.2.6.1 Clone dTPP II-NHis.......................................................................................................................... 18
3.2.6.2 Clone dTPP II-CHis.......................................................................................................................... 18
3.2.6.3 Clone dTPP II-wt .............................................................................................................................. 18
3.2.6.4 Clone dTPP II-Nmbp........................................................................................................................ 19
3.2.6.4.1 Elimination of the NdeI recognition site in the pMAL vector ................................................ 19
3.2.6.4.2 Exchanging the multiple cloning site of the pMAL vector..................................................... 20
3.2.6.4.3 Inserting the TPP II DNA into the pMAL vector.................................................................... 21
3.2.7 Introducing point mutations to the TPP II DNA...................................................................................... 22
3.2.7.1 Single residue exchange by site-directed mutagenesis.................................................................... 22
3.2.7.2 Double residue exchange by site-directed mutagenesis .................................................................. 22
3.2.7.3 Multiple residue exchange by site-directed mutagenesis ................................................................ 23
3.2.7.4 C-terminal truncations ...................................................................................................................... 24
3.2.8 Cloning of the C-terminal domain of TPP II ........................................................................................... 24
3.2.9 Cloning of Homo sapiens TPP II cDNA in E. coli.................................................................................. 26
3.3 Recombinant expression................................................................................................................................. 27
3.3.1 Over-expression of TPP II in E. coli ........................................................................................................ 27
3.3.2 Cell disruption methods............................................................................................................................ 27
3.3.2.1 Sonication.......................................................................................................................................... 27
3.3.2.2 Lysozyme treatment combined with sonication .............................................................................. 27
1TABLE OF CONTENTS
3.3.2.3 High-pressure homogenisation......................................................................................................... 28
3.4 Protein-chemical methods.............................................................................................................................. 29
3.4.1 Activity measurements.............................................................................................................................. 29
3.4.2 Determination of protein concentration ................................................................................................... 29
3.4.3 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) ............................................................... 30
3.4.4 Non-denaturing electrophoresis (Native PAGE) ..................................................................................... 30
3.4.5 Staining of proteins after electrophoresis................................................................................................. 31
3.4.6 Immunoblotting......................................................................................................................................... 31
3.4.7 Size exclusion chromatography................................................................................................................ 32
3.4.8 Purification of TPP II from E. coli (approach I)...................................................................................... 33
3.4.9 Purification of TPP II from (approach II)..................................................................................... 34
3.4.10 GdnHCl titration ..................................................................................................................................... 34
3.4.11 Purification of MBP-tagged TPP II by affinity chromatography.......................................................... 35
3.4.12 Purification of His -tagged TPP II by affinity chromatography ........................................................... 366
3.4.13 Disassembly of TPP II by dialysis ......................................................................................................... 36
3.4.14 Disassembly of TPP II by methylation .................................................................................................. 37
3.4.15 Disassembly of TPP II by β-octyl glucoside treatment......................................................................... 37
3.4.16 Large-scale purification of TPP II for crystallization............................................................................ 37
3.5 Bioph