Heterologous Expression of L-Amino Acid Oxidase from Calloselasma rhodostoma and Induction of Cell Death

ii Dedicated to my parents and teachers… iii i. Acknowledgements First of all, thanks to Prof. Sandro Ghisla, for providing me the opportunity to work in his lab. I am lucky indeed to have had such a friendly and lively person as my PhD supervisor. Prof. Peter Macheroux for his constructive criticisms and suggestions during this Ph.D. work and for critical reading of the manuscripts. Prof. Elisa May for her kindly advices and coordination in completing the publication work on LAAO induced cell death. Prof. Martin Scheffner for the lively discussions, suggestions and for providing the mammalian expression vectors and for the access to the cell culture facility. Herr Dr. Thomas Kapitza for patiently teaching me the techniques in baculovirus mediated expression system. Special thanks to Rajesh Kumar Singh and Michael Desilva for their help in cell culture experiments. Frau Susanne Feindler-Boeckh for her day-today assistance in the lab, without whose help this work would have been difficult to finish. Family members for their support and love. iv ii. Abbreviations α-anti-(used for antibodies) °C degree celcius aa(s) amino acid(s) Ab, mAb antibody, monoclonal antibody AIP apoptosis inducing protein AOX alcohol oxidase bp base pairs cDNA complementary DNA CIP calf intestinal phosphatase CV column volume Da, kDa dalton, kilodalton (molecular weight) DAAO D-amino acid oxidase DAPI 4´-6-Diamidino-2-phenylidole DMEM Dulbeccó s modified eagle medium DMSO Dimethylsulfoxide DNA deoxy ribonucleic acid e.g. for example EDTA ethylenediamine-tetra acetic acid ELISA enzyme-linked immunosorbent assay ER Endoplasmic Reticulum EtBr ethidium bromide FACS fluoresence activated cell sorting FAD flavin adenine dinucleotide FBS fetal bovine serum FCS fetal calf serum mA milli ampere MeOH methanol Min minute (s) Mut s , Mut + methanol utilization slow MWCO molecular weight cut off NAN N-acetyl neuraminic acid NP-40 nonidet P-40 O.D. optical density PBS phosphate buffered saline v PCR polymerase chain reaction POD Peroxidase RNA ribonucleic acid RPM/rpm rounds per minute RT room temperature S, sec Second SDS sodium dodocyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis Siglecs sialic acid binding Ig superfamily lectins Sp.activity specific activity TBE tris borate EDTA buffer TBS tris buffered saline TCA trichloroacetic acid UV-VIS ultraviolet-visible V volume V volt W weight x-gal