Synthesis of recombinant human relaxin H2 in bacteria and its effects on differentiation of carcinoembryonic stem cells by Roman Stanisław Poterski A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy In Biomedical Sciences Guelph, Ontario, Canada © Roman Stanisław Poterski, 2017 ABSTRACT SYMTHESIS OF RECOMBINANT HUMAN RELAXIN H2 IN BACTERIA AND ITS EFFECTS ON DIFFERENTIATION OF CARCINOEMBRYONIC STEM CELLS Roman Stanislaw Poterski Co-Advisors: University of Guelph, 2017 Doctor Alastair J.S. Summerlee Doctor Tarek Saleh Experiments described in this thesis were designed to develop a reliable method for the synthesis of the recombinant human hormone relaxin H2. Several methods were tested to produce highly pure, biologically active protein quickly, inexpensively and consistently from batch to batch. Relaxin H2 synthesized in bacteria was assessed by SDS-PAGE gel electrophoresis, Western Dot Blotting, Liquid Chromatography/Ultra High Definition Mass Spectrometry, and in vivo blood pressure response experiments using rats. The recombinant relaxin H2 described in this study was compared with commercially available relaxin H2 and was determined to be of equal quality, purity, and biological activity. In addition, to determine whether or not relaxin H2 induced differentiation of murine carcinoembryonic stem cells P19CL6 into cardiomyocytes was compared with a standard methodology using DMSO-induced differentiation. Relaxin H2 induced differentiation but the onset was delayed by four days compared with DMSO treatment. The endpoint of differentiation was determined as the start of spontaneous contractions within clusters of cells in culture. The results revealed that both commercially obtained and bacterially produced relaxin H2 equally caused a four-day delay in the initiation of contractions by cardiac myocytes compared with DMSO-treated controls. Moreover, cDNA microarray analysis of P19CL6 cells in three separate cultures, induced with DMSO (1%, as a control), and experimental treatments with serelaxin (16.7 nM), and bacterially-derived RLN H2 (16.7nM), respectively. There were no differences observed between cell treatments with the serelaxin- and bacterially-derived RLN H2 in the activation/deactivation patterns of 125 affected genes during the progression of the differentiation process. ACKNOWLEDGEMENTS I would like to thank all of the many people who have supported me during the course of my doctoral project. First and foremost, I thank Doctor Alastair J. S. Summerlee for his guidance and unreserved support. Despite being incredibly busy with his administrative, teaching, research and humanitarian endeavors, Alastair always found time to answer my questions and provided advice. There are not many professors of Alastair’s standing who would take on a part-time graduate student and would also support the candidate’s full-time workload. There were also absences from my graduate program due to the extenuating circumstances. Thank you, Alastair, for your continued support. I thank Dr. Tarek Saleh for agreeing to serve as my co-advisor and for his support. The members of my Advisory Committee, Drs. Coral Murrant and Brian Wilson were so important that without their support this project would not have been successful. Thank you. Members of the Biomedical Sciences Department of whom there are too many to name all, thank you. Dr. Jeff Thomason and the anatomy team, thank you for your support and encouragement. The DVM students with whom I have contact during their anatomy laboratory classes have been my unwavering supporters. I especially thank OVC Classes of 2013 and 2018 for cheering me on and electing me their Honorary Class President. iv My family, Barbara, Marta, Dave, Noah, and Seth were the true pillars and foundation on which I was able to build my doctoral aspiration. Thank you all with all my heart. v DECLARATION OF WORK PERFORMED I declare that the work described in this thesis has been completed by me with the following exceptions requiring the expertise and equipment not available locally: Chemical synthesis and sequencing of DNA’s were performed by University of Guelph Molecular Laboratory Services. Mass spectrometry analyses were done by at the Advanced Analysis Centre, Mass Spectrometry Laboratory, University of Guelph. Microarray hybridizations and partial statistical analyses were completed with the assistance of University Health Network Microarray Centre, Toronto. vi Table of Contents ACKNOWLEDGEMENTS............................................................................................................... iv DECLARATION OF WORK PERFORMED ................................................................................... vi LIST OF FIGURES .......................................................................................................................... x LIST OF TABLES ......................................................................................................................... xiii LIST OF ABBREVIATIONS .......................................................................................................... xv INTRODUCTION .............................................................................................................................. 1 OUTLINE OF THE THESIS ............................................................................................................. 5 CHAPTER 1............................................................................................................. 7 REVIEW OF THE LITERATURE ...................................................................................................................... 7 The discovery of relaxin................................................................................................................... 9 Tissue distribution of relaxin isoforms and their receptors ................................................. 10 BIOLOGICAL ACTIONS OF RLN ................................................................................................................ 14 Central effects of relaxin ................................................................................................................ 14 Relaxin, the heart, and circulation .............................................................................................. 16 Tissue remodeling ........................................................................................................................... 18 Relaxin and cancer .......................................................................................................................... 20 Relaxin and cell differentiation .................................................................................................... 21 GENERAL STRATEGIES FOR PRODUCTION OF RECOMBINANT PROTEINS ................................................ 23 Production by secretion................................................................................................................. 26 Production within the cell .............................................................................................................. 28 Fusion proteins ................................................................................................................................ 29 Protein tags ....................................................................................................................................... 30 Bacterial culturing systems .......................................................................................................... 30 Cell-free synthesis of proteins ..................................................................................................... 31 THE PRINCIPLES OF DESIGN AND CONSTRUCTION OF VECTORS FOR BACTERIAL EXPRESSION OF RECOMBINANT PROTEINS ......................................................................................................................... 35 The rationale of design and construction of eukaryotic expression vectors .................. 44 Expression of proteins in mammalian cells ............................................................................. 47 Expression of proteins in E. coli .................................................................................................. 47 HISTORICAL SYNOPSIS OF METHODS USED FOR PRODUCTION OF RELAXIN............................................ 48 RATIONALE .................................................................................................................................. 51 CHAPTER 2 ................................................................................................................................... 54 EXPRESSION OF HUMAN RLN H2 IN E. COLI USING A HEXA-HISTIDINE TAG AND MALTOSE-BINDING PROTEIN DUAL-AFFINITY FUSION SYSTEM ......................................... 54 INTRODUCTION .......................................................................................................................................... 54 RATIONALE ............................................................................................................................................... 61 MATERIALS AND METHODS ....................................................................................................................... 62 Methodology used to prepare bench-top RLN H2 ................................................................... 62 Plasmids and bacterial strains ....................................................................................................