TECHNISCHE UNIVERSITÄT MÜNCHEN Fakultät für Medizin Fachgebiet für Experimentelle Gynäkologie Recombinant production, purification and biochemical characterization of the three human kallikrein-related peptidases KLK4, KLK8 and KLK15 Josefine Theresia Maier Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Medizin genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr. E. J. Rummeny Prüfer der Dissertation: 1. apl. Prof. Dr. V. Magdolen 2. Univ.-Prof. Dr. M Schmitt Die Dissertation wurde am 22.06.2012 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 30.01.2013 angenommen. 1 I Abbreviations ..................................................................................................................... 3 II Introduction ........................................................................................................................ 6 II.1 Cancer in Germany ........................................................................................... 6 II.2 Gynecologic oncology ...................................................................................... 8 II.2.1 Ovarian cancer .................................................................................................. 8 II.3 Proteases ......................................................................................................... 12 II.3.1 Serine proteases .............................................................................................. 13 II.3.1.1 Trypsin-like serine proteases and zymogens .................................................. 14 II.3.2 Kallikrein-related peptidases (KLKs) ............................................................ 15 II.3.2.1 The beginning of the kallikrein era ................................................................ 16 II.3.2.2 Progress in the KLK research since the 1980s ............................................... 16 II.3.2.3 Genomic organization of KLKs ..................................................................... 17 II.3.2.4 Expression and regulatory mechanisms of KLKs .......................................... 18 II.3.2.5 Molecular features of KLKs .......................................................................... 19 II.3.2.6 KLKs form networks: Semen liquefaction .................................................... 19 II.3.2.7 Pathological functions of KLKs ..................................................................... 20 II.3.2.8 KLK4 ............................................................................................................. 22 II.3.2.9 KLK8 ............................................................................................................. 23 II.3.2.10 KLK15 ............................................................................................................ 23 III Aims of this project .......................................................................................................... 27 IV Material and methods ....................................................................................................... 28 IV.1 Materials and equipment................................ ................................................ 28 IV.1.1 Buffers and solutions ..................................................................................... 28 IV.1.2 Materials ........................................................................................................ 30 IV.2 General molecular biology methods .............................................................. 32 IV.2.1 Cloning and expression .................................................................................. 32 IV.2.1.1 DNA sequences and primers .......................................................................... 34 IV.2.2 Site-directed mutagenesis .............................................................................. 37 IV.3 Protein handling ............................................................................................. 42 IV.3.1 Protein analysis .............................................................................................. 42 IV.3.1.1 SDS polyacrylamide gel electrophoresis ....................................................... 42 IV.3.1.1.1 12% polyacrylamide gel ................................................................................. 42 IV.3.1.1.2 Electrophoresis ............................................................................................... 44 IV.3.1.1.3 Coomassie staining ......................................................................................... 45 IV.3.1.1.4 Silver staining ................................................................................................. 45 IV.3.1.2 Affinity chromatography ................................................................................ 45 IV.3.1.3 Protein structure and function ........................................................................ 47 IV.3.1.3.1 Protein refolding ............................................................................................. 47 IV.3.1.3.2 Drop-wise method .......................................................................................... 50 IV.3.1.4 Incubation with enterokinase ......................................................................... 51 IV.3.1.5 Enterokinase antibody .................................................................................... 52 IV.3.1.6 Benzamidine affinity column for trypsin-like KLKs ..................................... 53 IV.3.1.7 Protein blot from SDS-PAGE to PVDF-Membrane ...................................... 53 IV.3.1.8 Enzymatic kinetic studies ............................................................................... 54 IV.3.1.9 Effects of KLK4, 8 and 15 on pro-uPA ......................................................... 55 IV.3.1.10 Activity studies using fluorogenic substrates: Cleavage of PAR peptides by KLKs .............................................................. 55 V Experiments and results ..................................................................................................... 57 V.1 Cloning, expression and purification of KLK4, 8 and 15 ................................. 57 2 V.2 Purification of KLK proteases by Ni2+- NTA affinity ....................................... 57 V.3 Refolding ........................................................................................................... 58 V.4 Activation of recombinant KLK proteases by enterokinase .............................. 60 V.5 Site-directed mutagenesis: KLK15 S/A ............................................................ 62 V.5.1 Cloning: KLK15 S/A ......................................................................................... 63 V.5.2 Expression of KLK15 S/A in M15 E. coli cells ................................................ 63 V.5.3 Purification and refolding of KLK15 S/A ......................................................... 64 V.5.4 Incubation of KLK15 S/A using enterokinase ................................................... 65 V.5.5 Summary of enterokinase incubation ................................................................ 65 V.6 Removal of enterokinase using antibodies ........................................................ 65 V.7 Affinity column purification with benzamidine ................................................ 66 V.8 Enzymatic kinetic studies .................................................................................. 67 V.9 Cleavage of PAR peptides by KLKs ................................................................. 70 V.9.1 Proteases such as thrombin and trypsin cleave PARs ....................................... 70 V.9.2 Cleavage of KLKs upon PARs: Activity studies using fluorogenic substrates . 71 V.10 Effects of KLK4, 8 and 15 on pro-uPA ............................................................. 73 VI Discussion ........................................................................................................................ 77 VI.1 KLKs interplay with cancer-related targets ...................................................... 78 VI.2.1 Proteases and cancer-related substrates ............................................................ 80 VI.2.1.1 PARs .................................................................................................................. 80 VI.2.1.2 Involvement of PARs in colon cancer ............................................................... 80 VI.2.1.3 Plasminogen activation system mediates cellular events .................................. 81 VI.2.2 Recent work and research results: KLK15 ........................................................ 82 VI.3 Finding possible substrates of KLKs: extended substrate specificity ............... 84 VI.4 Future directions ................................................................................................ 85 VII Summary ......................................................................................................................... 87 VIII References and literature ................................................................................................. 90 IX Publications and Conferences attended ........................................................................... 95 X Curriculum vitae .............................................................................................................