A Thesis submitted to The University of London for the degree of Doctor of Philosophy Expression Studies of Recombinant cAMP-specific Phosphodiesterase Type 4 Nadia Korniotis School of Pharmacy Department of Pharmaceutical and Biological Chemistry 29/ 39 Brunswick Square London January 2003 ProQuest Number: 10104247 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10104247 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 University of London Abstract Expression Studies of Recombinant cAMP-specific Phosphodiesterase Type 4 By Nadia Korniotis Cyclic nucleotides (cAMP and cGMP) are second messenger molecules that play a key role in many physiological processes, cAMP in particular regulates cell growth, cell differentiation, inflammation and glycogen metabolism. cAMP is degraded by phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4) which belong to a large family of PDEs consisting of 12 members. PDE4 isoenzymes which are highly specific for cAMP are most abundant in inflammatory cells making them good clinical targets for the therapy of asthma and other inflammatory disorders. The design of potential therapeutics will be helped by the structural analysis of PDE4. To date, there have been limited reports on expression systems capable of producing milligram quantities of full length PDE4. This has been due to the protein’s paucity and susceptibility to proteolysis. Although, the catalytic domain of PDE4 has been recently analysed by X-ray crystallography (Xu et a l , 2000), information regarding the regulation of the enzyme by its N-terminal regulatory domain and the binding of rolipram a PDE4 specific inhibitor is still poorly understood. Therefore, the main objective of this work was to establish a suitable expression system capable of expressing full length c AMP-specific phosphodiesterase 4 in quantities necessary to crystallise and analyze by X-ray crystallography. To achieve this objective Met^^RDl clone (Shakur et a l, 1993) supplied by Professor Miles Houslay was used. This clone consisted of rat brain cAMP-specific phosphodiesterase 4 gene which was cloned originally using Drosophila dunce gene as a probe hence the name RDI (rat dunce 1). The PDE4 gene consisted of both regulatory and catalytic domains but was missing the first 25 amino acids hence the name Met^^ which helped solubilisation and prevented membrane attachment. 11 PDE gene Met^^RDl was cloned and expressed in two systems, bacterial and mammalian. The bacterial system expressed Met^^RDl as a Glutathione S-transferase fusion protein in Escherichia coli. A substantial portion of phosphodiesterase expressed in bacteria was found to be inactive, insoluble and very highly degraded by proteolytic enzymes. Consequently, PDE4 expression in other systems was investigated. However, the insoluble protein produced was solubilised in urea and used to raise antibodies in rabbits as there were no commercial antibodies available against this protein. Phosphodiesterase was also expressed in mammalian cells using Semliki Forest Virus (SPY) system. SFV system parameters were optimised for BHK-21 cells using p- galactosidase gene due to the ease in which cells expressing this gene could be assayed and analysed in-situ compared to cells expressing the gene. Semliki Forest viral particles carrying Met^^RDl mRNA were produced by co-transfection of BHK-21 cells with pSFV-lMet ^^RDl mRNA and packaging-deficient helper RNA molecules at a ratio of 1:1. Met^^RDl was expressed by infecting BHK-21 cells with the recombinant virus at a multiplicity of infection of 0.5-1. This produced approximately 0.1-0.3 mg of Met^^RDl protein per 10^ cells (75-90 ml) with K^ of 7.9 ± 0.86 pM and IC 50 of 0.7 pM to rolipram (PDE4 selective inhibitor) consistent with previously published data forMet^^RDl (Shakur et a l, 1993). Preliminary purification strategy to purify Met^^RDl protein was developed using immunoaffinity column prepared from the antibody produced against the bacterial expressed Met^^RDl protein. Ill Acknowledgment The work described here would not be made possible without the help and support of many people. However, my principle debt goes to my husband who showed me support, help and encouragement throughout the project and without whom this work would not be possible or worth doing. I would like to thank my supervisor Dr Andy Wilderspin for giving me the chance to accomplish such an achievement and for providing me enough supervision and guidance to keep me on the right track. Many thanks go to the many people that I had the opportunity of meeting over the last four years at the School of Pharmacy. In particular I wish to thank Pedro Baptisa, Bela Chopra, Liz Khatri and Seema Sharma for making the School of Pharmacy a happy and enjoyable environment to work in. My special thanks go to Lynda Hawkins for her technical help and support, to Jurgen for his assistance with DNA sequencing, and to Colin James for his computing expertise. I must also thank Kieran Brickley and Paul Chazot for their help and technical support with antibody production. To my colleagues, Adi Moloudi and Ravinda Sidhu go my best thanks for their help and support in the lab. and to whom I wish all the best for the future. A word to my financial sponsors the BBSRC and to the Registrar Peggy Stone for her support and backing during the loss of my mother-in-law a year ago ... THANK YOU. I would like to thank my current employers at GlaxoSmithKline and my future employer Novartis Institute of Medical Sciences for their help, support and patience through the past six months and through the coming months. Finally I would like to thank my family for their encouragement and my cats Ebony and Ivory for keeping me sane throughout the duration of the writing period. IV List of Abbreviations Approximately a Alpha Akt Protein Kinase B APC Antigen presenting cells APS Ammonium persulphate ATP Adenosine triphosphate P Beta P-gal p-galactosidase BHK Baby hamster kidney bp Base pair BSA Bovine serum albumin Ca'+ Calcium ion CaM Calmodulin cAMP Cyclic adenosine 3', 5'-monophospate cDNA Complementary DNA cGMP Cyclic guanosine 3', 5'-monophosphate CHO Chinese hamster ovary Chr Chromosome cpm Counts per minute C-terminal Free carboxyl group of a protein CREB cAMP response element-binding protein CRE cAMP-response element °C Degrees centigrade Da Dalton DAB 3', 3-Diaminobenizine DAG Diacylglycerol DNA Deoxyribose nucleic acid dATP Deoxyadenosine triphosphate dCTP Deoxycytosine triphosphate DEAE Diethylaminoethyl- DEPC Diethyl pyrocarbonate dGTP Deoxyguanosine triphosphate dhfr dihydrofolate reductase DMF Dimethylformamide DMSO Dimethylsulphoxide DMP Dimethyl pimelimidate dNTP Deoxynucleotide triphosphate DTT Dithiothreitol E. coli Escherichia coli EDTA Ethylenediaminotetraacetic acid EtBr Ethidium bromide ERK Extracellular-signal-regulated kinase Fab Fragment antigen binding Fc Antibody fragment that is most readily crystallised fmole Femtomole, multiple of Y Gamma g Gram g Acceleration due to gravity GAF cGMP-specific phophodiesterases, cyanobacterial Anabaena adenylyl cyclases, and Escherichia coli transcriptional regulator fhl A GDP Guanosine 5'-diphosphate Gi Inhibiting G-proteins GMEM Glasgow Minimal Essential Media Gs Activating G-proteins GST Glutathione S-Transferase GTP Guanosine 5'-triphosphate H Helix Tritium h Hour HESS Hanks’ balanced salts pH]-cAMP Tritiated cyclic adenosine monophosphate HCl Hydrochloric acid vi HRP Horse Radish Peroxidase HSL Hormone-sensitive lipase IBMX 3-isobutyl-1 -methylxanthine IgG Immunoglobulin G EL-2 Interleukin 2 IP3 Inositol triphosphate EPTG Isopropyl-p-D-thiogalactopyranoside IRS-1 Insulin receptor substrate-1 kb or Kb Kilobase pairs kDa or KDa KiloDalton K. Michaelis-Menten constant < Less than L Litre Lac Z Reporter gene that encodes for p-gala LB Luria-Bertani LBA Luria-Bertani Agar M Molar, moles per litre mA Milliamps Divalent metal ion MCS Multiple cloning site Magnesium ion |LAg Microgram ml Millilitre ml Microlitre |jM Micromolar ^imole Micromole, multiple of 10"^ MOPS 3-(N-Marpholino) propane sulphonic mRNA Messenger RNA ng Nanogramme nm Nanomitre nmole Nanomole, multiple of 10'^ NO Nitric oxide NP-40 Nonidet P-40 vil N-terminal Free a-amino group of a protein OD Optical density O N orO /N Overnight Pi Inorganic phosphate PAGE Polyacrylamide gel electrophoresis PBS Phosphate buffered saline PDE Phosphodiesterase PEST Proline, Glutamic acid, Serine, and Threonine amino acid sequence pH -logioPT PKA Protein Kinase A pKa Dissociation constant, -logioKa PMSF Phenylmethylsulphonylfluoride PCR Polymerase chain reaction pmole Picomole, multiple of 10’*^ RDI Rat dunce 1 RNA Ribonucleic acid RNase A Ribonuclease A RNasin Ribonuclease inhibitor rNTP Ribosomal nucleotide triphosphate rpm Revolutions per minute SDS Sodium dodecyl sulphate SFV Semliki forest virus SH3 Src homology 3 Sj26 Schistosoma japonicum 26 SV Sindbis virus X Times TAE Tris, Acetic acid, EDTA buffer TEMED N,N,N’ ,N’ -tetramethyethylethylenediamine TLCK Tosyllysine chloromethyl