Synthesis of Recombinant Human Relaxin H2 in Bacteria and Its Effects on Differentiation of Carcinoembryonic Stem Cells

Synthesis of recombinant human relaxin H2 in bacteria and its effects on differentiation of carcinoembryonic stem cells

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.

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.

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.

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 ...... 63 Preparation of DNA to produce the modified RLN H2 expression plasmid ...... 63 Transformation of bacteria ...... 67

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Culture media ...... 68 Cell growth conditions ...... 69 Harvesting of cells ...... 70 Confirmation of the biochemical integrity of bench-top RLN H2 ...... 72 Activity of bench-top relaxin H2 in vivo ...... 73 RESULTS ...... 76 Construction of novel plasmid ...... 76 Protein expression experiments using traditional media ...... 80 Alternative approach and media ...... 80 Detection of fusion protein ...... 81 Efficiency of the novel system for bacterial production of RLN H2 ...... 86 In vitro validation of bacterially expressed RLN H2 ...... 87 Mass Spectrometry Analysis ...... 89 In vivo assessment of recombinant RLN H2 ...... 99 DISCUSSION ...... 105 CHAPTER 3 ...... 115 EFFECTS OF RELAXIN H2 ON DIFFERENTIATION OF MURINE CARCINOEMBRYONIC STEM CELLS INTO CARDIOMYOCYTES ...... 115

INTRODUCTION ...... 115 RATIONALE ...... 119 MATERIALS AND METHODS ...... 120 P19CL6 cell culture ...... 120 Experimental protocol ...... 121 Morphological assessment of living cells ...... 122 RNA extraction and quantification ...... 122 Microarray Protocol ...... 123 RESULTS ...... 125 Morphological results ...... 125 Microarray analysis of gene expression profiles ...... 128 DISCUSSION ...... 136 GENERAL DISCUSSION ...... 141

THE CHALLENGES FOR PRODUCTION OF BENCH-TOP RLN...... 142 CHALLENGES OF PROTEIN PURIFICATION ...... 145 PRODUCTION SYSTEM DESIGN ...... 147 SUMMARY OF TECHNICAL CONSIDERATIONS ...... 147 A NOVEL DESIGN STRATEGY THAT WORKED ...... 148 BIO-ASSAY FOR RLN H2 ...... 150 THE CHALLENGE OF RLN RECEPTOR ...... 155 SCIENTIFICALLY AND REGULATORILY APPROVED METHODS USED FOR CHARACTERIZATION OF RECOMBINANT FORMS OF RLN H2...... 157 EFFECTS OF RLN H2 ON DIFFERENTIATION OF P19CL6 STEM CELLS ...... 165 MICROARRAY RESULTS SHOW THE GENES AFFECTED BY RLN H2 ...... 166 VALIDATION OF MICROARRAY RESULTS BY QUANTITIVE PCR ANALYSIS ...... 166 LIMITATIONS...... 171 OPPORTUNITIES FOR FUTURE RESEARCH ...... 172 OVERALL SUMMARY ...... 173

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LITERATURE CITED ...... 175 APPENDIX 1 ...... 199 SYNTHESIS OF RECOMBINANT HUMAN RELAXIN H2 IN E. COLI USING THE MALTOSE BINDING PROTEIN SYSTEM ...... 199

PURPOSE ...... 199 MATERIALS AND METHODS ...... 200 RESULTS ...... 207 DISCUSSION ...... 212 APPENDIX 2 ...... 215 APPENDIX 3 ...... 244

LIST OF FIGURES

Figure 1. Schematic cross-section through the cell wall and an approximate number of proteins present in each compartment of E. coli.

Figure 2. A schematic diagram of all required elements and features of a bacterial expression vector.

Figure 3. Amino acids and their corresponding nucleotide sequences of mature human RLN H2.

Figure 4. A diagram comparing designs of the RLN H2 expression cassettes.

Figure 5. Map of a plasmid vector pET 28a+.

Figure 6. An agarose gel picture showing linearized pMal-p2x plasmid vector.

Figure 7. An agarose gel photograph of pET28a+-6His-MBP-RLN-H2.

Figure 8. A plasmid DNA on 1% agarose gel.

Figure 9. Representative protein SDS-PAGE gel comparison of the two strains of

Rosetta cells.

Figure 10. Representative protein SDS-PAGE gel of MBP-RLN H2 fusion protein.

Figure 11. Representative protein SDS-PAGE gel of protein samples collected after digestion of MBP-RLN H2 fusion protein with FXa and carboxypeptidases.

Figure 12. Representative protein SDS-PAGE gel of desalted RLN H2.

Figure 13. Western Dot Blot analysis of bacterially expressed RLN H2.

Figure 14. Mass Spectrometry data of trypsin-digested, bacterially-expressed

RLN H2.

Figure 15. A representative trace of arterial blood pressure recording from an anaesthetized rat.

Figure 16. The effects of bacterially derived recombinant RLN H2 on systolic, diastolic and mean arterial blood pressures in anaesthetized rats.

Figure 17. Changes in heart rate in rats treated with bacterially produced RLN

H2.

Figure 18. Ten times magnification of a monolayer of differentiated P19CL6 cells stained with Rhodamine 123 dye.

Figure 19. One thousand times magnification of a contracting focal point of

P19CL6 cells treated with bench-top RLN H2 and mitochondrial-specific

Rhodamine 123 laser fluorescent dye.

Figure 20. The graph showing cumulative changes in expression levels of all 125 affected genes.

Figure 21. Positive 1 group of genes.

Figure 22. Positive 2 group of genes.

Figure 23. Positive to a negative group of genes.

Figure 24. Negative 1 group of genes.

Figure 25. Negative 2 group of genes.

Figure 26. Negative 3 group of genes.

Figure 27. Negative 4 group of genes.

Figure 28. Shown in this figure is the most recent proposed concept of RLN H2-

RXFP-1 interaction.

Figure 29. Proposed model of RXFP1/RXFP2 receptor binding and activation.

Figures in Appendix 1 and 3

Figure A1-1. A map of the pMal-p2x-ppRLN H2 vector.

Figure A1-2. A fragment of the polylinker sequence illustrating the Factor Xa cleavage site at the 5’ end of the RLN insert.

Figure A1-3. A typical bacterial culture growth characteristics.

Figure A1-4. A scanned image of a 1% agarose gel.

Figure A1-5. A scanned image of 1% agarose gel illustrating pMal-p2xppRLN plasmid DNA isolated from 3 different strains of bacteria.

Figure A1-6. A scanned photograph of a representative SDS-PAGE protein gel.

Figure A3-1. Plasmid DNA sequence graph.

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LIST OF TABLES

Table 1. The occurrence of RLN and related peptides in human, rat, and mouse.

Table 2. Relaxin family peptide receptors and their ligands.

Table 3. Organ and tissue distribution of receptors for RLN and RLN-related peptides.

Table 4. Recombinant protein expression systems.

Table 5. A comparison of advantages and disadvantages of cell-free systems for protein expression.

Table 6. Commonly used prokaryotic promoters and their inducers.

Table 7. Comparison of the primary components and features of prokaryotic and eukaryotic translational processes.

Table 8. Relaxin amino acids and codons rarely used in E. coli.

Table 9. Multi-cloning sites and expression regions of plasmid vector pET 28a+.

Table 10. Summary of results obtained from two strains of Rosetta E. coli expressing recombinant RLN H2 from plasmid vector pET28a+His-MBP-RLN H2.

Table 11. Quantification of the relative amounts of the fusion protein in the media.

Table 12. A summary of peptide masses obtained during mass spectrometry analysis of bacterially produced RLN H2.

xiii

Table 13. The effects of bacterially derived recombinant RLN H2 on systolic, diastolic and mean arterial blood pressures in anaesthetized rats.

Table 14. Changes in heart rate in rats treated with bacterially produced RLN H2.

Table 15. Compilation of significantly changed fold expression of 125 genes.

Tables in Appendix 1 and 2

Table A1-1. A typical bacterial culture growth profile and treatments with IPTG as measured by optical density OD600.

Table A2-1. Functional descriptions of the genes with up-regulated and differentially altered (positive at day 4 and negative at day 8 and 12) expression levels by RLN H2 treatment.

Table A2-2. Functional descriptions of the genes with down-regulated expression levels by RLN H2 treatment.

Table A2-3. Comparison of microarray gene expression levels down-regulated in cells treated with serelaxin and bacterially expressed RLN H2, respectively.

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LIST OF ABBREVIATIONS

0C degree Celsius

Da Dalton, molecular weight

E. coli Escherichia coli g gram h hour

IPTG isopropyl beta-D-1-thiogalactopyranoside kDa kilo Dalton, molecular weight l Litre

LB-L Luria-Bertani, Lennox broth

LB-M Luria-Bertani, Miller broth mm Hg millimeter of mercury, blood pressure

NaCl sodium chloride, salt

OD600 optical density measurement

ONE-TB Over-Night-Express Terrific Broth p plasmid vector

RLN H2 relaxin H2 rpm rotations per minute

SDS-PAGE sodium dodecyl sulfate poly-acrylamide gel

electrophoresis

xvi

INTRODUCTION

Phylogenetically, relaxin (RLN) is an old hormone (Yegorov and Good,

2012) that was discovered relatively early in the twentieth century. However, synthesizing the hormone and understanding its physiology has been challenging for a number of reasons that have emerged over the century since it was first discovered. Although all contemporary references suggest that RLN was first discovered in 1926 (Hisaw, 1926), the earliest reference to the possibility of the existence of the hormone dates back a further ten years. Frederick Lee Hisaw

(1916) reported changes in the pelvises of pocket gophers. He suggested that there was a flexible, and stretchable region palpable on the mid-ventral aspect of the pelvises that was only present in sexually mature female pocket gophers.

Later, he concluded that there was a softening and lengthening of the pelvic ligament in the late pregnant guinea pigs immediately prior to parturition. This elasticity of pelvic ligament allowed the safe passage of young. In 1926, Hisaw reported that an injection of serum from pregnant rabbits caused a loosening of the pelvic ligament in virgin guinea pigs (Hisaw, 1926). This relaxative substance was then extracted from pregnant pig corpora lutea and formally named “relaxin”

(RLN) (Fevold et al., 1930). Subsequent purification of RLN from the rat (Hudson et al., 1981), pig (Haley et al., 1982), and human (Hudson et al., 1983) led to the deduction of its DNA sequence, as well as the analysis of the hormone’s peptide structures that placed it within the insulin family of peptides (Shabanpoor et al.,

2010).

At present, the relaxin family consists of seven peptides (Table 1), which are structurally related to insulin (Bathgate et al., 2013; Halls et al., 2015). These small peptide hormones weighing approximately 6 kDa are produced in mammalian species and non-mammalian species of both sexes (Yegorov et al.,

2014). The relaxin family of peptides (RFP) are implicated in a number of physiological activities including: in male and female reproduction (Sherwood

2004; Ivell and Grutzner, 2009), as a neuropeptide in the brain (Bathgate et al,

2002), as a potent vasodilator and cardio-stimulant (Sarwar et al., 2016), and as an agent reducing tissue and organ fibrosis (Wilkinson et al., 2005; Unemori,

2017).

Relaxin peptides are synthesized as pre-pro-hormones (Bathgate et al.,

2006). The majority of work reported in the literature centres on one variant of the hormone designated human-2 or H2 relaxin (Table 1). Pre-pro-RLN H2 is comprised of 185 amino acids. Initially, the signal peptide (25 amino acids) is cleaved off and the pro-hormone undergoes further processing during which a C- peptide (107 amino acids), that connects the A- and B-chains, is removed to produce the mature form of the RLN hormone. The mature form of RLN H2 comprises an A chain containing 24 amino acids and a B chain having 29 amino acids. For the hormone to be biologically active, the A- and B peptides must be spatially arranged and folded correctly (Bathgate et al., 2006). This is achieved through two unique disulfide bonds between the B- and A-chains and one disulfide bond within the A-chain (Bullesbach et al., 2002). The assembled product is finally

2 deposited in secretory vesicles ready to be released in response to specific stimuli

(Bathgate et al., 2006).

Although RLN is a relatively simple protein, the heterodimeric structure, and the formation of the disulfide bonds create technical difficulties for artificial production of the hormone. There are reports in the literature of efforts to produce recombinant human RLN H2 in a number of different ways including: bacteria E. coli, (Canova-Davis et al., 1991), yeast (Yang et al., 1993; Cimini et al., 2017) and in mammalian cell systems (Soloff et al., 1992). Most of these reports suggest that

RLN H2 produced using these approaches is expensive, frequently difficult to purify and usually misfolded, rendering it unusable. Chemical methods for production of RLN H2 (Bullesbach and Schwabe, 1991) are available but are also expensive. In theory, bacterial E. coli production of recombinant proteins would be the preferred system for the production of RLN H2 due to the low cost of production, simpler purification and easier scale-up compared to other host systems. It is likely that the commercially available RLN H2 is produced in an E. coli expression system, but that process is proprietary. To date, there has been no simple, bench-top method of producing RLN H2 of reliable purity and activity reported.

Relaxin peptides bind to a variety of transmembrane receptors to induce various biological activities. In general, the receptors comprise seven- transmembrane components, which belong to the guanidine protein-coupled receptors (GPCRs, Bathgate et al., 2006). Complicating our understanding of the physiological actions of RLN peptides is the fact that some RLNs bind to more than

3 one specific receptor (Table 2), and there is species variability in those responses.

The lack of agonist-receptor specificity hampers investigations into the physiological properties, molecular and intracellular actions of the RLNs. These differences have also precluded the development of an internationally accepted bioassay for the RLNs.

OUTLINE OF THE THESIS

This thesis is focused on the development of a bench-top method for production of the recombinant relaxin H2 using bacteria E. coli. The literature review that follows, describes the discovery of relaxin, its well-known biological actions as well as novel activities including cell differentiation. Further, in the thesis, methods for production of recombinant proteins in general and for the expression of relaxin, in particular, are reviewed. Despite previous challenges,

(Appendix 1, Expression of pre-pro-relaxin H2 using maltose-binding protein fusion system.) there are three major objectives of the current thesis:

1. To determine whether or not it was possible to synthesize a bench-

top relaxin H2,

2. To validate the bench-top synthesized relaxin H2 by comparing it

with commercially available relaxin H2 in a series of experimental

tests,

3. To further validate the bench-top synthesis we will explore some

potentially novel actions of relaxin H2 on stem cell differentiation.

Preliminary data exists to suggest that, in addition to the myriad of other actions of relaxin, the hormone may be involved in differentiation of stem cells

(Bigazzi and Bigazzi, 2005). Therefore, a secondary purpose of the current research was to explore the possibility that bench-top (in comparison with

5 commercially available RLN H2) might be involved in the process of differentiation of murine carcinoembryonic stem cells.

CHAPTER 1

Review of the literature

The progress in basic biological research, biotechnology, and medical applications relies on the availability of proteins (Wolfe, 1993; Weaver, 1999).

Purification of native proteins from tissues is of a limited value: there are concerns about supply, costs, antigenicity and finally, the ethics of such an approach, particularly if human-specific proteins are required. It is vital to produce proteins in a laboratory and to be able to manipulate their structures and properties to enable scientific investigation. Recent advances in sequencing the genomes of human and of other species, combined with improvements in the recombinant DNA technologies in the past two decades, all contribute to expanding possibilities for the production of increasing quantities of synthetic and/or modified proteins.

The synthesis of proteins from non-natural sources can be categorized into biosynthetic and chemical processes or a combination of both. In general, each of these approaches has its advantages and disadvantages and the more complex the structure of the protein sought, the more complex and costly becomes the process for its production.

Biosynthetic production of proteins is carried out in a variety of systems and cell types. The most common cell types include bacteria, for example, Escherichia coli (E. coli; Swartz, 2001) and yeast, for example, Saccharomyces cerevisiae and

Pichia pastoris (Yang et al., 1993; Cimini et al., 2017). Alternatively, mammalian cells such as the Chinese hamster ovary cells (CHO, Lucas et al., 1996) or murine myeloma NSO cells (Werner et al., 1998), insect cells (Altman et al., 1999) and plant cells have been and continue to be used (Larrick and Thomas, 2001).

Production of proteins using transgenic animals is directed at their expression in animal products such as milk or eggs. However, due to commercial interests of such systems, there are no detailed data available on how these systems are designed.

Chemical methods for production of proteins were first introduced with the development of solid-phase synthesis technology (Canova-Davis et al., 1990).

This technique, combined with appropriate chemistry for protecting active groups, allows synthesis of peptides containing approximately 60 to 70 amino acid residues. The major advantage of this approach is the possibility to use substitutions (or analogs) of amino acids, which can be different from the naturally occurring amino acids. In addition, side chains, stereochemistry, or even the peptide’s backbone chemistry and bonds can be manipulated. It is also possible to control chemically post-translational modifications, which could include phosphorylation, glycosylation, and lipidation. However, this approach is often technically challenging and expensive. To overcome the chain length limits of the chemically obtained peptides (60-70 amino acids), methods for chemical and enzymatic ligation of smaller fragments into larger proteins, are currently being used (Goody et al., 2002). Finally, chemically produced peptides are initially obtained in a denatured form and it is not always possible to re-naturalize proteins

8 in one simple reaction. The yield of final product in a single synthesis and cost- effectiveness are both considerably lower, compared to the equivalent cellular expression.

Cell-free expression systems using purified components, or cell extracts for the synthesis of proteins in purpose designated bio synthesizers, are both useful for high-efficiency screening applications (Sawasaki et al., 2002). This technology

(Rapid Translation System) is guarded by a number of international patents, owned by Roche Biochemical International Company, who is the sole proprietor, manufacturer, and distributor of the equipment and supplies. Currently, there are only two institutions in Canada licensed to use the technology: University of

Manitoba and University of British Columbia.

The discovery of relaxin

In 1916 Frederick Lee Hisaw noticed significant differences in the pelvic girdle structure between adult males and females of the plains pocket gophers.

After his initial observations, Hisaw changed his animal research model to guinea pigs, which had been long known to undergo a loosening of the pelvic symphysis connective tissue prior to parturition. In 1926, Hisaw discovered that injecting serum from pregnant guinea pigs or rabbits into the estrogen-primed guinea pigs caused a relaxation of the pelvic ligament. In 1930, Hisaw and his two graduate students extracted an active substance from pig corpora lutea and initially named it releasin, which was later changed to relaxin (Fevold et al., 1930).

Tissue distribution of relaxin isoforms and their receptors

Since its discovery in 1926, RLN has been recognized as a reproductive hormone of pregnancy (see reviews by Sherwood, 1994, 2008). In the majority of mammals studied to date, the main sources of RLN are the ovaries, or placentae of pregnant females (Sherwood, 1994) but RLN is also synthesized in the male by the prostate gland (Yki-Jarvinen et al., 1983; Table 3). Samuel and colleagues

(2003) provided evidence to support the role of RLN in development and functioning of the male reproductive tract. There is also evidence to imply that RLN could be involved in fetal testicular descent, during late pregnancy in rats (Parry et al., 2001). Most recently, evidence has been reported to show that a unique RLN is produced in the brain (H3 in humans, M3 in mice [Bathgate et al., 2002], and R3 in rats [Burazin et al., 2002]). This provides evidence for the view that RLN may have a specialized role in the brain.

At present, we know that there are various forms of RLN and RLN-like peptides that are associated with different receptors. The three tables below summarize these forms of RLN by species, tissue distribution, and ligand differences.

Table 1. The occurrence of RLN and related peptides in human, rat, and mouse.

(Adapted from Bathgate et al., 2013).

Gene Protein Human Gene Mouse Gene Rat Gene ID

ID ID

RLN1 RLN H1 6013 Not present Not present

RLN2 RLN H2 6019 19773a 25616a

RLN3 RLN H3 117579 212108 266997

INSL3 INSL3 3640 16336 114215

INSL4 INSL4 3641 Not present Not present

INSL5 INSL5 10022 23919 Pseudogeneb

INSL6 INSL6 11172 27356 50546

Gene identification (ID) numbers are from Entrez Gene. a Genes for rat and mouse relaxin are named RLN1 in the databases but are the rodent equivalents of RLN2 in the human. b INSL5 is a pseudogene in the rat (Liu et al., 2005).

Table 2. Relaxin family peptide receptors and their ligands (Hsu et al., 2002;

Bathgate et al., 2013).

Receptor Receptor Ligands Activates Inhibits name name (Current) (Former) RXFP1 LGR7 RLN1, Adenylate cyclase, Not known RLN2, protein kinase A, RLN3 protein kinase C, phosphatidylinositol 3-kinase, extracellular signal- regulated kinase (Erk1/2) RXFP2 LGR8 RLN1, Adenylate cyclase Not known RLN2, INSL3 RXFP3 GPCR135, RLN3 Erk 1/2 signaling Adenylate SALPR cyclase RXFP4 GPCR142, RLN3, Not known Adenylate GPR100 INSL3 cyclase

Table 3. Organ and tissue distribution of receptors for RLN and RLN-related peptides (Bathgate et al., 2006).

RXFP1 RXFP2 RXFP3 RXFP4 Tissue human human Rat Mouse Human Rat Mouse Human Ovary mRNA Protein mRNA mRNA mRNA Oviduct mRNA mRNA Uterus mRNA, mRNA mRNA, mRNA mRNA protein protein Cervix, Vagina Protein mRNA protein Placenta mRNA mRNA mRNA Nipple Protein mRNA Protein Breast Protein Protein Protein Testis mRNA mRNA mRNA mRNA mRNA mRNA mRNA mRNA Prostate mRNA mRNA mRNA Gubernaculum mRNA mRNA Brain mRNA, mRNA mRNA mRNA mRNA mRNA mRNA mRNA protein Pituitary mRNA mRNA Kidney mRNA mRNA mRNA mRNA mRNA Heart mRNA, mRNA mRNA mRNA protein Lung mRNA mRNA Liver mRNA Intestine mRNA mRNA mRNA Colon mRNA mRNA Adrenal mRNA mRNA mRNA mRNA Thyroid mRNA mRNA mRNA Thymus mRNA mRNA Salivary gland mRNA mRNA Muscle mRNA mRNA mRNA Peripheral mRNA mRNA mRNA blood cells Monocyte cell mRNA, mRNA line THP-1 protein Bone marrow mRNA mRNA mRNA Skin mRNA mRNA

Biological actions of RLN

Central effects of relaxin

The pioneering work of Summerlee and colleagues (Summerlee et al.,

1984) revealed that intravenous injections of porcine RLN caused a suppression of reflex milk ejection (RME) in urethane-anesthetized, lactating rats in a dose- dependent manner. Furthermore, these researchers injected RLN into the cerebral ventricles of anesthetized, lactating rats and discovered that the pattern of RME was disrupted, without affecting the response of the mammary glands to oxytocin.

Summerlee and colleagues (1984) proposed that RLN could be inhibiting not only the release of oxytocin for the period of RME but also that RLN could be playing an important role in the overall central control of the oxytocin system (Summerlee et al., 1984). These findings were later supported and confirmed by showing that

RLN stimulated the release of both oxytocin and vasopressin from the posterior pituitary (Dayanithi et al., 1987) and other hypothalamic-pituitary hormones

(Geddes and Summerlee, 1995). It has been reported also that RLN may have a role in the timing of birth (Summerlee et al., 1998), hemodynamic control (Geddes et al., 1994) and maintenance of water balance during pregnancy (Summerlee et al., 1998, McKinley et al., 2001). Sunn et al. (2002) reported that circulating RLN acted directly on neurons of the subfornical organ to stimulate water drinking in rats. In 1995 Summerlee et al. suggested that RLN might be produced and could be acting locally within the brain in a manner that was separate from its production and actions in the periphery. Osheroff and Ho (1993) localized for the first time

RLN binding sites in various regions of the rat brain and showed that mRNA for

RLN was produced in sites different from regions where binding of the exogenous labeled RLN occurred, which supported the possibility of a central RLN system.

Relaxin mRNA was identified in tenia theca, pyriform cortex and lateral orbital cortex (Osheroff and Ho, 1993) whilst RLN binding sites in 29-day postnatal and adult rat brains identified using P32-labeled RLN, were reported in olfactory-related regions, deep layers of neocortex, numerous hypothalamic and thalamic nuclei and certain areas of the midbrain (Osheroff and Ho, 1993).

With the discovery of RLN H2 receptor LGR7 (leucine-rich-repeat- containing G-protein-coupled receptor 7; presently named RXFP1 [Table 2], above, Hsu et al., 2002; Bathgate et al., 2013), more detailed studies to localize precisely the distribution RXFP1 expression throughout rat and mouse brains appear to be attainable. As well, further genes for RLN expressed in brains of mice

(M3 RLN, Bathgate et al., 2002), and rats (R3 RLN, Burazin et al., 2002) have been reported.

In a recent report by Hai-Jian et al., (2016) it was shown that bilateral microinjections of RLN H2 into the hypothalamic paraventricular nuclei (PVN) in spontaneously hypertensive rats, induced an increased sympathetic activation, angiotensin vasopressin (AVP) release and pressor responses in comparison with normotensive Wistar-Kyoto rats. It was concluded that the exogenous RLN H2, in addition to the locally released RLN from RLN-positive neurons in the PVN, acted on RXFP1 receptors present in sympathetic neurons and AVP neurons, causing sympathetic activation and AVP release. In contrast, in in vitro studies of RLN H3

15 binding to its cognate receptor RXFP3 in PVN demonstrated an inhibition of oxytocin and vasopressin release (Kania et al., 2017).

Relaxin, the heart, and circulation

There is limited evidence in the literature to support the presence of RLN production centers in the heart. The cellular mechanisms and actions of RLN in the cardiovascular system were reviewed by Sarwar et al., (2016). Relaxin mRNA has been identified in the human (Dschietzig et al., 2001) and mouse (Bathgate et al., 2002) hearts using RT-PCR techniques. However, there are no reports to confirm these finding by extraction of RLN mRNA or native peptide from cardiac tissue samples (Samuel et al., 2003). Dschietzig et al. (2001) produced evidence for RLN synthesis in the human heart by Western blot analysis but could only confirm the presence of an 18 kDa form of proRLN in heart tissues. They concluded that the amounts of mature 6.5 kDa form of RLN might have been too small for detection. It has also been suggested that pro-RLN might be the bioactive form of RLN in the heart. Du et al. (2003) reported that RLN expression in the heart tissue was elevated in senescent, or in chronically ill animals (Bigazzi et al., 2001).

In addition, Du et al. (2003) reported that M1 RLN-deficient senile mice showed an abnormal build-up of collagen in the left ventricle. This finding was specific to male mice only and was not observed in any of the senescent females.

High-affinity binding sites for RLN in the cardiac atria, but not the ventricles, of rats and mRNA for the receptor have been reported as early as one day after birth (Osheroff et al., 1992, Tan et al., 1999). Relaxin receptor density may be determined by steroid environment although this may be gender specific: treatment of female rats with estrogen had no effect on RLN receptor density in the atria (Tan et al., 1999) whilst similar treatment in male rats decreased receptor density in the atria. It is interesting to note that RXFP1 receptors are specifically localized in the atria, but not in the ventricles of rats (Hsu et al., 2002) although to date, this has been supported only by RT-PCR methodology.

Hemodynamic effects and actions of RLN were first discovered in the middle of the last century (Miller et al., 1957) but did not receive much interest until the mid-nineteen-eighties. Initially, Miller et al. (1957) reported that ovarian extracts of porcine RLN caused a hypotensive episode in anesthetized dogs. In contrast,

Jones and Summerlee (1986) reported a substantial pressor effect and noticeable release of vasopressin in urethane-anesthetized, lactating rats treated intravenously with highly purified porcine RLN. A detailed study of the hypertensive action of RLN confirmed that in urethane-anaesthetized rats this pressor action is caused by the release of vasopressin from the pituitary (Parry et al., 1994), probably in response to activation of the forebrain angiotensin II system in the brain

(Parry and Summerlee, 1991).

In contrast to studies of Parry et al. (1991 and 1994) are the reports that

RLN has a strong vasodilatory action (hypotensive effects) on isolated mesenteric vessels (Bigazzi et al., 1986), coronary blood vessels (Bani et al., 1998) and in the

17 penile circulation (Bigazzi et al.; 1995, Bani et al., 1995). In a study by Fisher et al., (2002) RLN was shown to be a “potent vasodilator in its own right”. These authors used human donor gluteal biopsies and human lung tissues from patients undergoing pneumonectomies’ for lung cancer. In gluteal tissue in vitro preparations, Fisher et al., (2002) showed that RLN at pathophysiological concentrations was a strong vasodilator of small systemic resistance arteries and that this action was endothelium-dependent. Removal of the endothelium nearly completely obliterated RLN-induced responses, which would suggest that this vasodilator action could be nitric oxide-dependent (Di Bello et al., 1995). In the same study using the identical in vitro protocol for lung tissues, it was shown that

RLN had no vasodilator effect in pulmonary arteries. In a more recent study, using intact, blood-perfused hamsters having their cremaster muscles prepared by dissection in situ Willcox et al., (2013) showed that human RLN H2 induced a rapid and temporary vasodilation in the microcirculation of skeletal muscles in this species.

Tissue remodeling

Relaxin is well known for its role in the widening of the birth canal and softening of the cervix in preparation for parturition (Sherwood, 1994). These effects of RLN are caused, in part, by an increase in collagen turnover resulting from both, a decrease in collagen production and an increase in matrix metalloproteinases expression (Unemori and Amento, 1990) affecting the stability

18 of the extracellular matrix (ECM). It is possible that fibrosis caused by the accumulation of connective tissue resulting from the proliferation and adaptation of fibroblasts (components of the ECM) to overproduction of a surplus of collagen, could be due to a relative deficiency of RLN. This might result in an imbalance between factors promoting the production of matrix metalloproteinases (MMPs) or their tissue metalloproteinases inhibitors (TIMPs).

A number of human and animal diseases such as scleroderma, chronic renal failure, cirrhosis of the liver, pulmonary, and myocardial fibrosis (Kuwano et al., 2001) all share similar disequilibria between MMPs and TIMPs. The endpoint of such uncontrollable excessive accumulation of collagen is the loss of normal organ architecture, elasticity, and function of the affected tissues, leading to the whole body part fibrosis and terminal organ failure (Kissin and Korn, 2002).

Relaxin has been reported to induce a decrease of collagen accumulation in animal models of induced fibrosis: a mouse model of lung fibrosis (Unemori et al., 1996) and rat models of renal (Gerber et al., 2003) and hepatic (Williams et al.,

2001) fibrosis. Moreover, in 1999 Zhao et al. reported that mice lacking functional

RLN M1 gene have numerous developmental deficits. These abnormalities include impaired development of the pubic symphysis, reduced development of mammary gland and nipple during pregnancy and an increased buildup of collagen, especially in the nipples and vagina (Zhao et al., 2000). In male mice, deficient in

RLN M1 increased deposits of collagen were identified in the testis, epididymis, and prostate, all of which exhibited delayed tissue maturation and growth (Samuel and Amento, 2001). More recently, other connective tissue abnormalities have

19 been reported in RLN-deficient mice, in the heart (Du et al., 2003), skin (Amento et al., 2002), and lungs (Samuel et al., 2003).

Relaxin and cancer

It has been reported that primi- or multiparous women are less likely to develop cancer, particularly breast cancer, compared with nulliparous women (Ivell and Einspanier, 2002). It would be tempting to suggest that there may be a correlation between cancer and some, or all of the hormones of pregnancy. Bigazzi and colleagues (1992) reported that there was a bi-phasic effect of RLN H2 in

MCF-7 human breast cancer cell line: growth was stimulated at low concentrations of RLN and was inhibited at high levels of RLN. These effects were later attributed to differential stimulation of the nitric oxide pathway (Di Bello et al., 1995). Bani et al. (1999) followed these investigations by showing that RLN promoted differentiation of mammary tumor xenografts (derived from MCF-7 cell line) in athymic nude mice but RLN did not cause tumor enlargement. More accurately, the tumors were encapsulated in a thick layer of connective tissue. Tashima et al.

(1994) showed that RLN was synthesized within normal, benign and neoplastic breast tissues. These findings suggested that alterations in local concentrations of

RLN might influence cancer progression. Furthermore, high concentrations of RLN have been correlated with poor prognosis in disseminated breast cancer (Binder et al., 2001). Binder and colleagues (2002) suggested that the progression of tumors was associated with effects of RLN on MMPs and ECM remodeling.

Silvertown et al., (2007) investigated the role of relaxin H2 in prostate cancer progression in PC-3 and LNCaP cell lines in vitro. They reported that mutation of receptor binding domain produced an analog of relaxin H2 that exhibited antagonistic properties.

Brown et al., (2003) using second harmonic generation (SHG) luminescence and multiphoton laser scanning microscope, measured and compared the content and structure of collagens in a variety of tumors: mouse mammary adenocarcinoma MCa-IV, human colon adenocarcinoma LS174T, melanoma MU89 and soft tissue sarcoma HSTS26T. In RLN H2-treated mice, evidence suggests that the structure of ECM may have become loosened and tumor hindrance to chemotherapeutic agents reduced. The authors reasoned that the effects of RLN were due to the increase in activity of MMPs and that the “new” matrix of the tumors, altered after exposure to RLN, may have been created by both degradation of existing, and production of new collagen at the same time.

Relaxin and cell differentiation

There is contradictory evidence of the potential role of RLN in cell differentiation (Einspanier, 2001). For example, Pawlina and co-workers (1989) investigated the effects of RLN on the differentiation of murine embryonic fibroblasts 3T3-L1 in cell culture. In this study, recombinant RLN H2 delayed the normal course of cell division during the induction stage but did not have any effects on the expression of adipocyte phenotype, as demonstrated by lipid

21 deposits and insulin-dependent glucose transport. Relaxin-treated cells were from

2 to 3 times larger than the cells that were induced using a no-RLN induction protocol. These studies demonstrated that RLN affected proliferation during induction of differentiation of murine 3T3-L1 fibroblasts to pre-adipocytes without affecting differentiation directly. In later studies, (Pawlina et al., 1990) evidence was provided to show that human, recombinant RLN H2 had the ability to delay cell division in murine 3T3-L1 fibroblasts in vitro. In addition, it was shown that RLN

H2 was ten times more effective than porcine RLN in delaying the rate of cell division. In both these experiments, cell division delays were neutralized by addition of monoclonal antibodies specific for porcine relaxin and polyclonal antibodies specific for relaxin H2 to the culture medium. Morphological studies revealed that the RLN H2 treated murine fibroblasts progressed to differentiate into adipocytes at a slower rate and that it was a dose-dependent effect (Pawlina et al.,

1990). There are no other published data that supports the work reported by

Pawlina et al., (1989, 1990). Therefore, Einspanier, (2001) may have been correct in saying that there is “contradictory evidence” for the role of RLN in differentiation.

Despite the lack of solid evidence that relaxin has a role in cell differentiation, a patent application was filed by Bigazzi and Bigazzi (2005) who suggested that RLN could be involved in differentiation of stem cells. Therefore, in the second part of the thesis, we explored the possibility that both bench-top and commercially available RLN H2 might be involved in the process of murine carcinoembryonic stem cells differentiating into contracting cardiomyocytes.

General strategies for production of recombinant proteins

In theory, it should be relatively simple to produce a recombinant protein.

Fundamentally, DNA sequence encoding a protein of interest is cloned into an expression vector, downstream of a promoter. This vector is then introduced into a host cell, where the cell’s protein synthesis mechanisms take over control to produce the protein of interest. In practice, however, expression of proteins that are foreign to the host’s cells can be difficult because there can be many factors affecting the process. For instance, each protein folds in its own unique manner, a process that may predetermine the choice of expression host. Some proteins require post-translational modifications and the mechanisms of these modifications may or may not be present in the host cell. Also, some proteins may be toxic to the host cells and lead to their demise. In practice, there is no single solution for a successful production of all recombinant proteins. The choice of a host organism for protein expression predetermines which vector system can be used. There are many vectors available that range from simple, to highly specialized, containing DNA sequences needed for directing the recombinant protein into the desired cellular localization. Each potential expression system offers different advantages and disadvantages and these are summarized in Table

The following sections describe in greater detail the various options that were considered and explored experimentally, leading to an effective bench-top method for production of the recombinant RLN H2.

In the process of developing a technique and a method that was successful, a number of different approaches were attempted. Most of these were not successful. These failures are documented in Appendix 1 for completeness.

Table 4. Recombinant protein expression systems.

Protein Host or strain Starting Advantages Disadvantages expression used material system Bacterial E. coli DNA or Simple No post- synthetic genetics, easy translational nucleotide to manipulate, modifications, sequence inexpensive codon usage cloned into a conflicts, low vector yield Yeast Pichia pastoris, DNA or Some post- Improper Saccharomyces synthetic translational and/or cerevisiae nucleotide modifications, excessive sequence low-cost media glycosylation cloned into a vector Insect Sf-9, Sf-21, S- DNA or Post- Very long 2, Hi-5 synthetic translational production nucleotide similar to time, high costs sequence mammalian of media low cloned into a systems yield vector Mammalian CHO, HEK-293, DNA or Comprehensive Very long HeLa synthetic post-translation production time nucleotide modification is high costs of sequence possible, good media, protein cloned into a method for the yields low vector production of bioactive proteins Cell-free Bacterial or DNA, RNA or Some post- Low yields of mammalian cell synthetic translational proteins, extracts nucleotide modifications difficult to scale sequence possible, up cloned into a affordable vector Chemical Synthetic amino DNA Relatively easy Extremely synthesis acids sequence to manipulate expensive, the synthesis specialized process equipment and facilities must be available, very low efficiency

Production by secretion

Once the host cell has been activated into producing the protein of interest, it is important to have a methodology to isolate and purify the synthesized protein from the rest of the proteins normally present in the cytoplasm. The number of native proteins can be substantial. For example, on average E. coli cells contain more than 4000 proteins. However, the unique feature of E. coli is that the proteins are separated into two components: the majority of proteins (4290) are present in the cytoplasm and a minority (about 100 proteins) are restricted to a periplasmic compartment (Makrides, 1996). (See Figure 1).

Figure 1. Schematic cross-section through the cell wall and an approximate number of proteins present in each compartment of E. coli.

Culture medium ±10 proteins Lipopolysaccharide 70 Ä 70 Ä Outer membrane Peptidoglycan 210 Ä Periplasm ± 100 proteins 70 Ä Inner membrane

Cytoplasm ± 4290 proteins

Therefore, directing proteins for expression in the periplasmic space would be beneficial, because there would be considerably fewer proteins from which to purify the expressed protein of interest. In addition, the oxidizing environment of the periplasm and the enzymes foldases present in the periplasm could contribute to proper folding and formation of disulfide bonds within the expressed protein.

Ostenmeier and Georgiou (1994) showed that coexpression of periplasmic foldases with bovine pancreatic trypsin inhibitor enhanced the yield of correctly folded product.

To direct a protein for secretion into the periplasm, a fusion of signal sequence from normally secreted protein to N-terminal of the target protein needs to be assembled. Most frequently used signal sequences are derived from the E. coli “native” periplasmic proteins for instance: PhoA and MalE (Donovan et al.,

1996), or the outer membrane proteins: OmpA, LamB, and DsbA (Swartz, 2001).

The presence of a signal peptide per se, in a fusion protein to be translocated to the periplasm, however, does not always guarantee efficient processing. In addition, proteases that reside in the periplasm have the potential to destroy the newly formed protein (Baneyx and Georgiou, 1992). Protease III, OmpT, DegP, and Tsp are among the most frequently reported periplasmic proteases responsible for degradation of heterologous proteins (Baneyx and Georgiou,

1992). Escherichia coli strains deficient in the above-listed proteases (Protease III,

OmpT, DegP, and Tsp) have been reported to decrease degradation of various heterologous proteins secreted into the periplasm (Meerman and Georgiou, 1994).

Production within the cell

A recombinant protein can be accumulated in a soluble form in the cytoplasm, and/or the product could be precipitated and deposited in insoluble aggregates also known as inclusion bodies (Georgiou and Valax, 1996). There are distinct trade-offs for producing recombinant proteins in inclusion bodies: principally the yield is higher but the efforts to extract the inclusion bodies may be time-consuming and expensive (Georgiou and Valax, 1996). Several expression strategies are available to enhance production and extraction of recombinant proteins from inclusion bodies including using molecular chaperones; employing thioredoxin-deficient strains of E. coli to maintain a less reducing milieu; decreasing the rate of protein synthesis; lowering cultivation temperatures and using highly soluble fusion partners (Makrides, 1996).

The co-expression of chaperones contributes to increased production of mono- and multimeric proteins, but the success is highly protein specific (Georgiou and Valax, 1996). This could be due to the self-protective redox state of the bacterial cytoplasm protecting against the formation of disulfide bonds. Indeed, in

E. coli there are two paths contributing to the reduction of disulfide bonds: first, the thioredoxin system composed of thioredoxin reductase and thioredoxin, and second, the glutaredoxin system, which includes glutathione, glutathione reductase and three glutaredoxins (Prinz et al., 1997). It would be reasonable to propose that creating a less reducing environment in the cytoplasm would be beneficial for the formation of disulfide bonds. The additional difficulty would be

28 encountered, however, during purification of the target protein from the cytoplasm biomass of more than four thousand native bacterial proteins.

Fusion proteins

Fusion protein methods were developed to assist with retention of recombinant-fusion-protein complexes on matrices during purification (Banyx,

1999). Serendipitously, certain fusion partners improve the solubility of packaged proteins and prevent their deposition in inclusion bodies (Banyx, 1999). Most commonly used fusion systems include: thioredoxin (Trx); glutathione S- transferase (GST); and maltose-binding protein (MBP; Kapust and Waugh, 1999).

Banyx (1999) suggested that an explanation for the improved yield of fusion proteins would be that the fusion partner could be reaching its native conformation as it emerges from the ribosome, (or soon after its release). This would make the fusion complex resistant to degradation and would facilitate transport to its final destination within the cellular compartments.

Kapust and Waugh (1999) compared the above three fusion partners as solubility enhancers. They reported that MBP significantly improved solubility of the aggregate-prone proteins, compared with only marginal improvements for Trx and GST. They suggested that MBP could be interacting with its passenger protein in a way similar to intramolecular chaperones.

Protein tags

The last stage of recombinant protein production, the purification, is most difficult and cumbersome. To ease this challenge and, in addition to the above- described fusion proteins, a number of further affinity tags and affinity chromatography methods have been developed (Terpe, 2003). Ideally, a tag should be as small as possible and have a minimal effect on structure, activity, and properties of the recombinant protein. For instance, the 6-His tag is 0.84 kDa, the glutathione S-transferase tag is 26 kDa and maltose binding protein is 43 kDa. The

FLAG tag consists of 8 amino acids, but it is highly immunogenic, which means that the FLAG tag must be removed before a recombinant protein attached to it can be used to produce antibodies. In contrast, the His tag has extremely low immunogenicity and hardly ever interferes with protein structure or function. This makes His-tagged proteins the most suitable for many downstream applications without the need to sever off the tag (Terpe, 2003).

Bacterial culturing systems

Two types of culture systems are commonly used in a laboratory practice: batch or fed-batch (Sambrook and Russell, 2001). The bench-top batch culture containing all nutrients required to sustain the growth of cells from start to finish is the simplest way for the unrestricted growth of bacteria. However, unrestricted growth can lead to changes in the culture that might be detrimental to growth. For example, oxygen limitations and/or lowering pH values may change the speed and

30 efficiency of the growth activity. Nevertheless, this method is cheap and in some cases effective.

To achieve a higher density of cells and a larger amount of protein, fed- batch culturing system in a bioreactor is commonly used. This system provides technical means for flow of nutrients into, and outflow of by-products from the bioreactor in a controlled fashion and according to the rate of consumption and production yields, respectively. This approach is more expensive as it requires considerably larger investment for the equipment and supplies.

The final choice of a culturing system also depends on the expression mode i.e. is the protein to be expressed going to be secreted or not, is it going to be produced via an intracellular method or a combination of both methods.

Cell-free synthesis of proteins

Littlefield et al. (1955) reported that protein biosynthesis does not require the integrity of a cell and that the synthesis can be continued even after cell disruption. Soon thereafter, cell-free models, based on E. coli were reported by

Schachtschabel and Zilling (1959) and Lamborg and Zamecnick (1960). In both methods, however, ribosomes remained programmed with endogenous mRNA and simply continued reading the messages to which they had been already attached at the time of cell disruption.

The next stage in the development of cell-free translation systems was the introduction of exogenous RNA into bacterial-based extracts, which was first reported by Nirenberg and Matthaei (1961). This was followed by the development of an efficient cell-free system for translation of exogenous messages using the rabbit reticulocyte lysate that has been cleaned of its endogenous mRNAs by pretreatment with micrococcal calcium-dependent RNase (Pelham and Jackson,

1976).

Another method for expression of exogenous messages without cells was derived from wheat germ extract. Roberts and Paterson (1973) and Marcus et al.

(1974), showed that wheat germ cells have intrinsically low levels of endogenous mRNA and could be used for expression of exogenous messages without any pretreatment. In recent years, the selection of cell-free expression systems has expanded to a number of cell types (reviewed by Endo and Sawasaki, 2006). In particular HeLa cells (Mikami et al., 2008), Chinese hamster ovary (CHO) cells and many others (Sorkin, 2004) have been reported as effective.

In cases where no RNA is available, it is presently possible to use DNA

(instead of isolated RNA) in extracts that are free of endogenous mRNAs.

Lederman and Zubay, (1967) and Schweiger and Gold, (1969), reported on the successful use of DNA in bacterial based cell-free systems. A DNA in the form of a plasmid vector, as an isolated gene, or a synthetic DNA fragment, could be added to the translation reaction mix and the corresponding mRNA would be synthesized in situ by the endogenous RNA polymerase present in the bacterial extract, or in its supernatant fraction. Under these conditions, translation continues

32 while mRNA is still elongating, and the rates of transcription and translation are coordinated. This method is typical of bacterial extracts and is called coupled transcription-translation system.

Eukaryotic extracts are prepared from the cytoplasmic fraction, hence there is a lack of endogenous RNA polymerase activity (Mikami et al., 2008). This obstacle can be curtailed by the addition of an exogenous RNA polymerase. For instance, bacteriophage, T7, and SP6 RNA polymerases were both found to be the most convenient and efficient for use in cell-free systems. These enzymes were used in eukaryotic extracts to produce mRNA from DNA constructs with cognate promoters (Spirin, 1991; Craig et al., 1992). In this case, however, no real coupling between transcription and translation takes place, which is different from the above-described bacterial systems. In eukaryotic extracts the bacteriophage

RNA polymerases work much faster than the translation process, therefore mRNA is synthesized mainly in advance of protein synthesis. This non-coupled processing has been named combined transcription-translation. The advantages and disadvantages of cell-free systems for protein expression are compared in

Table 5.

Table 5. A comparison of advantages and disadvantages of cell-free systems for protein expression.

Extract source Advantages Disadvantages (system) E. coli High protein yield Many eukaryotic proteins Relatively tolerant to additives insoluble after expression Eukaryotic co- and post- translational modifications are not possible Codon usage is different from that of eukaryotes Rabbit reticulocyte Mammalian system Sensitive to additives Cap-independent translation Protein glycosylation not possible Co-expression of off-target proteins Wheat germ Translation of large proteins is Eukaryotic co- and post- possible translational modifications are No off-target endogenous not possible mammalian proteins Premature termination of High protein yield products Insect Translation of large proteins is Non-mammalian possible No endogenous mammalian proteins Some forms of protein glycosylation are possible Human and animal Human system Very sensitive to additives Co- and post-translational Much lower yield than that of modifications are possible E. coli Promises synthesis of functional proteins Potential to make virus-like particles (VLPs)

The principles of design and construction of vectors for bacterial expression of recombinant proteins

For the expression of a protein in bacteria E. coli, it is necessary to introduce the gene coding for it into the bacterial cell. However, not every gene can be expressed efficiently in bacteria due to several factors. As reported by Baneyx

(1999) these may include:

 Unique and structural features of the gene sequence,

 The translational efficiency and stability of mRNA,

 Degradation of the expressed protein by proteases of the host cell,

 Protein folding,

 Codon usage bias between the bacterial host and the foreign gene,

 And potential toxicity of the protein to the host.

The cDNA (i.e. not containing introns as does genomic DNA) coding for the heterologous protein is usually cloned into plasmid vectors by means of enzymatic, or PCR ligation. Plasmids are extra-chromosomal, self-replicating entities that may exist in cells in their supercoiled, nicked or relaxed forms. For a successful self- replication, relaxed plasmids are suited best. Plasmids are introduced into the bacterial hosts by means of chemical transformation or electroporation. The number of plasmids per cell varies from low, (1-60, pColE1, pMB1 derivatives) to high (up to hundreds, pUC - derivatives). In a typical bacterial culture, such multi- copy plasmids are distributed at random, as a result of cell divisions. Moreover,

35 with each next generation of cells, the number of plasmids decreases, and this occurs most frequently in the case of high copy number vectors. In addition, when plasmids carry genes that are toxic to the host’s cells, the growth rate is reduced, or the bacteria die off.

Historically, two kinds of promoters have been used: constitutive and regulatable. The constitutive promoter expression system is a simple way of overproducing a foreign protein in a bacterial cell because the protein is produced continuously. The continuously high level of expression is often too demanding on the cell’s energy sources, leading to a reduction, or complete inhibition of cell growth. The total yield of the target protein depends on both the amount of product per cell and the number of cells in a given volume of culture. The reduced cell growth associated with the constitutive expression frequently results in a lower quantity of the target protein comparing with regulated promoter system.

The regulatable promoter expression systems have the ability to switch on, adjust the level of, or turn off the expression of a foreign gene. For instance, most often used are environmental factors such as the temperature of growth environment, or concentration of a particular ingredient in the growth medium. Two bacterial promoters, phoA, and trp are induced after depletion of phosphates or tryptophan, respectively, from the growth medium. Another frequently used option is the lac promoter, which is induced by lactose, or a synthetic compound isopropyl-β-D-thiogalactoside (IPTG) presence in the growth medium. This ability to regulate the expression of a foreign gene allows for a separation of cell growth from the induction and/or product synthesis phases. When the desirably high

36 concentration of cells in the culture is achieved during the growth phase, then the foreign gene can be induced to produce the protein of interest. This approach leads to an increased total yield of the foreign protein when compared with the constitutive expression. Illustrated schematically in Figure 2, are all the required features and sequence elements of a bacterial expression vector (adopted from

Makrides, 1996).

Figure 2. A schematic diagram of all required elements and features of a bacterial expression vector:

P – Promoter (arrow indicates the direction of transcription)

R – Repressor, regulates promoter

RBS – Ribosome binding site

SD – Shine-Dalgarno sequence

SEQ – Coding sequence for recombinant protein

TT – Transcription terminator

Ampr – An ampicillin resistance gene

Ori – Origin of replication (determines the vector copy number in a cell)

RBS

R P SD SEQ TT Ampr Ori

Start

-35 -10 mRNA 5’ UAAGGAGG(N)8 AUG (91%) UAAU

16S rRNA 3’ AUUCCUCC GUG (8%) UGA UUG (1%) UAG TTG…(17)…TAAT Stop

Most bacterial mRNAs are polycistronic, hence contain information from multiple genes or cistrons. Each cistron has its own initiation codon and ribosome- binding site. The primary element of a vector is its promoter, which is usually positioned about 10 to 100 base pairs upstream from the ribosome-binding site

(RBS; Shine, Dalgarno et al., 1974) and is under the control of a regulatory gene present on the vector itself, or integrated as an internal part, into the host’s chromosome. The promoter of E. coli consists of two pairs of hexanucleotide sequences; the first starts at position –35 (upstream from the transcription initiation base) and is separated from the second hexanucleotide sequence at position –10.

Both are joined by a short “separator” sequence. Table 6 lists the many promoters commonly used for expression of genes in E. coli.

Table 6. Commonly used prokaryotic promoters and their inducers.

Promoter Inducer

Lac IPTG

Tac IPTG

T7 IPTG

Trc IPTG

Trp trp starvation

AraBAD L-arabinose

lac(ts) Thermal

PSPA Constitutive

A promoter should include the following features (Banyex, 1999):

 To be simple and cost-effective to induce

 To be easy for transfer to other strains of bacteria

 Should be strong enough to provide 10-30% or more of the total

proteins

 To possess a low or nil basal expression prior to induction.

Downstream of the promoter is positioned the RBS consisting of about 54 nucleotides. The sequence of RBS starts at position –35 (±2) and ends at position

+19 to +22 of the RNA coding sequence. The Shine-Dalgarno (SD, Shine et. al.,

1974) site (UAAGGAGG) binds to the 3’ end of the 16S rRNA during translation initiation. The optimal distance between the SD site and the start codon (of the gene to be expressed) ranges from 5 to 13 bases. The sequence in this region should avoid the risk for formation of secondary structures in the RNA transcript, hence, to increase the rate of success for proper translation initiation. Located downstream of the coding sequence (of the gene of interest) is the transcription terminator, which functions as a signal to stop transcription and, by forming a stem- loop structure, protects the mRNA from auto-nucleolytic destruction, therefore extending the half-life of the mRNA.

Next, in the backbone of a vector design should be placed a gene for antibiotic resistance to enable selection of the plasmid and its propagation.

Supplementation of the culture medium with antibiotics eliminates cells devoid of plasmids. The disadvantage of this approach is the decreased selection resulting

40 from degeneration of antibiotics, their loss of activity, or detoxification by periplasmic enzymes. The best example is an ampicillin, rapidly neutralized by β- lactamase secreted by bacteria into the growth medium, and which is also sensitive to changes in its culturing pH values. Paradoxically, resistance to, and supplementation with, the ampicillin are most commonly used in the majority of laboratories. Therefore, other than ampicillin antibiotics should be incorporated.

For instance: kanamycin, chloramphenicol, and tetracycline. From the regulatory or medical point of view, however, contamination of the final product, or biomass with excessive amounts of residual antibiotics should be avoided and ought to be incorporated into the experimental design also. To minimize or eliminate the negative effects of antibiotics on cells containing plasmids, other approaches have also been reported. One strategy is to design vectors that carry repressors, or genes causing the death of the cell upon the loss of the plasmid. Although valuable, this approach imposes restrictions on the composition of growth medium and causes the risk of metabolic exhaustion or even death of cells by additional processing of genes encoded by plasmids. Finally, as indicated earlier, the copy number of plasmids per cell is determined by the origin of replication, a sequence which completes the vector’s design.

A candidate promoter for use in E. coli should be strong enough to deliver at least 10-30% (or more) of the total cellular protein. The second requirement for such a promoter is to maintain the basal expression (prior to induction) at a minimal level. The reasons for the two above requirements are the following:

 In large-scale experiments involving liters of cultures, where cell density is

high, and even minimal promoter activity could cause premature

spontaneous induction, leading to depression of the promoter at the time of

the “programmed” induction.

 Proteins toxic to the host cells must be produced in tightly regulated

circumstances because even the smallest amount of the gene product

would be lethal to the host.

 If not completely repressed, expression systems can cause plasmid

instability and as a result, lowered or no production of the recombinant

protein is achieved.

 The requirement of the promoter is its ability to be reliably induced in a

simple, controllable and cost-effective fashion. Two closely related

promoters utilizing lactose, lac and lacUV5 were first, and continue to be

the most commonly used. Relatively weak and not used often in high-level,

large-scale production of recombinant proteins, both promoters require

mutant E. coli hosts deficient in lacY to allow for induction with lactose or its

synthetic analog, IPTG.

In recent years, synthetic promoters such as tac and trc have been reported to result in the production of about 15-30% of total cell proteins. The tac and trc are both constructed from –35 region of the trp (tryptophan) promoter and the –10 region of the lac promoter. The only difference between the two hybrid promoters is one base pair in the length of spacer fragment between the two hexamers in

42 each of them. For these two promoters as little amount as 50-100μM IPTG is often sufficient to achieve full induction. However, IPTG is too expensive for large-scale production and there are a number of issues with its toxicity, which frequently causes problems. To avoid this, temperature sensitive variants of lacI repressor protein can be used for thermal rather than chemical induction of recombinant protein production. However, there are also some concerns about the “leakiness” of the lac-derived promoters. This drawback can be minimized in low to medium copy number plasmids (e.g. pBR322) and sufficient repression of the promoter can be maintained by employing hosts that carry the lacIQ allele. In these strains, full activation of plasmid-encoded tac promoters can be achieved with as little as 3-10

μM IPTG.

A newer and innovative class of vectors named pET (Studier and Moffatt,

1986) became the leading systems for expression of proteins. In this design, the gene of interest is positioned downstream of the bacteriophage T7 late promoter on medium copy number plasmids. The disadvantage of pET strong promoter systems (T7/lacUV5) is that the target proteins are often not able to reach native conformation quickly enough and are instead deposited in inclusion bodies. This problem could be minimized by co-over-expressing such proteins with fusion proteins, or by using promoters that are activated by either up or downshift in temperature of the culture. For proper protein folding, lower temperature (30 vs.

370C) of culture is also sometimes helpful.

Another group of promoters is the nutritionally inducible class, for instance, phoA and trp. Both promoters are induced by limitation of available phosphate and

43 tryptophan, respectively, in the bacterial culture medium. Guzman, et al., (1995) reported on arabinose promoters (araBAD or pBAD), which use inexpensive, non- toxic sugar L-arabinose supplementation as an inducer. The arabinose promoter is believed to be weaker than tac promoter. The drawback here is that there is a large amount of heterogeneity in cell generations treated with sub-saturating concentrations of arabinose. Some cells are fully induced and other cells are not induced at all, what makes this promoter not quite a precisely controlling element.

An entirely different solution to the problem of plasmid instability is to insert the gene of interest into the chromosome of the E. coli, (Olson, et al., 1998). For instance, using bacteriophage λ as a way of entry. However, there appears to be little interest in this strategy because it was assumed that the dosage of the inserted gene would be too low. Indeed, the gene inserted into the chromosome of E. coli would need to be combined with some means of selection (e.g. antibiotic resistance) and little or no stability gain of the construct could be achieved.

Furthermore, purification of the expressed protein from all the proteins in the cytoplasmic biomass would be more complicated.

The rationale of design and construction of eukaryotic expression vectors

As described above, the most important feature of bacterial vectors is the

Shine-Dalgarno ribosome binding site. In eukaryotic, and specifically in mammalian expression systems, the mRNA also contains vital information that is essential for proper translation. This information, named the Kozak consensus

44 sequence (Kozak, 1986), is not equivalent to the prokaryotic RBS/translation initiation, but it is rather a translation initiation enhancer. The sequence is

ACCATGG, where the ATG (Met) is the optimal codon for initiation by eukaryotic ribosomes. Mutations within this 8 nucleotide sequence can dramatically change the expression levels. Subsequently, Kozak, who studied 699 vertebrate mRNAs, extended the consensus sequence for translation initiation to

GCCGCCACCATGG, where the A in the underlined ATG start codon is coordinate number 1 and the A (bold type) at position -3 could also be a G. Functional studies

(Kozak, 1986) on pre-pro-insulin and alpha-globin translation in cells additionally showed that a purine (typically A in position -3) is crucial for efficient initiation of translation, and in its absence, a G at position +4 is essential. Moreover, and perhaps most importantly, replacing purine with pyrimidine can reduce the expression levels by up to 95%.

Table 7 illustrates a comparison of the primary constituents of prokaryotic and eukaryotic translational processes.

Table 7. Comparison of the primary components and features of prokaryotic and eukaryotic translational processes.

Component Prokaryotic Eukaryotic Ribosomes The 30S and 50S subunits The 40S and 60S subunits Template or mRNA Further processing of mRNA After transcription, the mRNA transcript does not occur transcript is spliced to remove after transcription the non-coding regions (introns), and cap structure (M7 mRNA is polycistronic and methyl guanosine) and contains multiple initiation polyadenosine sequences are sites added to the 5’ and 3’ ends of the message, respectively. The cap structures and the poly A ends are both important for export of mRNA to the cytoplasm, proper initiation of translation and stability of mRNA. The mRNA is usually mono-cistronic. Features of The Shine-Delgarno Translation initiation occurs in translation sequence is present on the two ways: mRNA transcript and the complementary sequence is Cap-dependent translation: Cap present in the ribosomal structure and the cap-binding subunit. This facilitates proteins are responsible for binding and alignment of the proper ribosome binding to ribosome on the mRNA at mRNA and recognition of the the translation initiation site initiation codon. The first AUG (AUG). codon in the 5’ end of mRNA functions as the initiation The first amino acid of the enhancer codon. Kozak peptide being built is sequence may be present formylated methionine. around the initiation codon.

Cap-independent translation: Ribosome binding to mRNA occurs through “internal ribosome entry site” (IRES) on mRNA. Initiation factors Three initiation factors are More than three initiation known: IF1, IF2, and IF3 factors, which are regulated by phosphorylation. The initial step is the rate-limiting phase of eukaryotic translation. Elongation factors EF-Tu, EF-Ts, and EF-G EF1 (α, β, ɤ) and EF2

Expression of proteins in mammalian cells

A molecule of protein begins to be assembled in the endoplasmic reticulum.

As it is moved from one compartment to another, its structure is synthesized along the way, by continuous addition, or removal of ionic, or hydrophobic side chains.

Although pH level within the endoplasmic reticulum and the cytoplasm is neutral, compartmental pH value can vary greatly. For instance, insulin secretory granules have a lumenal pH of 4.5-5.5, where insulin can be stored in a soluble form, but the same insulin will have an only marginal solubility in the bacterial cytoplasm with pH of 7.8. Also associated with the endoplasmic reticulum is a large protein (489 residues, with homology to thioredoxin) called protein disulfide isomerase (PDI).

This protein is responsible for creating and isomerizing disulfide bonds within the expressed protein.

Expression of proteins in E. coli

A DNA sequence coding the protein to be synthesized must be first delivered and made readable to the E. coli host. This is accomplished first by means of constructing a plasmid vector carrying the DNA sequence for a protein of interest. This new plasmid is then introduced into the E. coli host by means of transformation. The majority of mammalian proteins expressed in bacteria will be recognized as foreign and will be degraded by the cellular proteolytic environment, or accumulated in an insoluble form as inclusion bodies. The prokaryotic

47 equivalent of the mammalian endoplasmic reticulum is the periplasmic space with its highly oxidizing milieu. There is no PDI present in bacteria, but the disulfide bonds can be formed with the aid of a group of enzymes called dsb foldases (dsb’s for di-sulfide-bonds). A number of dsb’s have been reported in the literature

(reviewed in Zhang et al., 2011). The best known is the dsbA, which is thought to be involved in the processing of small proteins. Ostenmeier and Georgiou (1994) reported on the proper folding of bovine pancreatic trypsin inhibitor (BPTI), A 6.5 kDaltons protein with three disulfide bonds.

Historical synopsis of methods used for production of relaxin

Relaxin is approximately 6 kDa peptide hormone belonging to the family of insulin and insulin-like peptides (Bedarker et al., 1977). Three genes for human

RLN (designated H1, H2, and H3) have been identified to date (Bathgate et al.,

2002) but not all three genes have been found in other species (Ivell and

Einspanier, 2002). These genes share a similar coding pattern that produces an initial peptide (pre-pro-RLN) including a signal fragment followed by three distinct sections known as the B-chain, the C-peptide and A-chain (Bathgate et al., 2002).

The pre-pro-peptide is processed by enzymatic cleavage of the signal portion to produce pro-RLN. After another enzymatic cleavage of the C-peptide, the mature form of RLN is produced. It is composed of two chains A and B, linked together by two disulfide bonds. There is one additional disulfide bond within the A chain for a total of three disulfide bonds in each molecule of mature RLN. The peptide

48 encoded by the H2 gene is the major stored and circulating form of RLN in the human. Relaxin H1 is expressed in the decidua, placenta, and prostate. Relaxin

H3 expression and actions are localized in the brain (Bathgate et al., 2002).

Furthermore, studies using RLN in a rat bioassays showed that the H2 protein has from 10 to 50% higher biological activity than H1 and H3 RLN forms (Bathgate et al., 2002).

Our knowledge of biological actions of RLN is still incomplete, mainly due to a chronic shortage of highly purified, animal-tissue-sourced or recombinant forms of the hormone for research purposes. In 1974, Sherwood and O’Byrne published a method for the purification of RLN from animal tissues. For human

RLN however, this method is not feasible simply because there are no human tissues readily available for extraction of the peptide. A number of experiments have been reported in the literature to describe recombinant DNA methods for production of RLN. One of these methods, first described by Stewart et al., in 1983, was to express porcine pro-RLN construct in E. coli. The results were disappointing because the protein was deposited in insoluble inclusion bodies in the cytoplasm of bacterial cells. As a consequence, complicated, expensive and inefficient methods were used for extraction of minuscule amounts of the prohormone from inclusion bodies. In later experiments using recombinant DNA techniques, the RLN

B- and A-chains were produced separately (Canova-Davis et al., 1991) and the following extraction from inclusion bodies, the peptides were combined together in a highly oxidizing environment for disulfide bond formation. In comparison with the

49 method of Stewart et al. (1983), there was no improvement in the yield of protein produced.

Bullesbach and Schwabe (1991) chemically synthesized the RLN B- and A- chain peptides and attempted to combine the chains in an oxidizing milieu for disulfide bond formation. Again, the amounts of protein obtained were very small and when production costs were added, it proved to be prohibitively expensive.

In 1991 Genentech Corporation (San Francisco, California, USA) received a patent for the production method of recombinant RLN H2. In 1993, the original license was sold to Connective Therapeutics Company (Palo Alto, California,

USA). Since then several other pharmaceutical companies have been involved including BAS Medical, Connetics, and Corthera, but in 2010 Novartis Corporation acquired control and now manages international license for RLN H2. This commercially available relaxin H2 has been renamed by the company as

“serelaxin”.

RATIONALE

Relaxin related research continues to be affected by the lack of a reliable and affordable source of the hormone. In the 1990’s, Genentech was first to patent and commercialize the human RLN H2. At that time, Genentech offered small quantities of the hormone to the research community at no charge. Since then, the license for RLN H2 has been transferred and since 2010 has been held by the

Novartis Corporation. Although Novartis continues to offer limited quantities of RLN

H2 to researchers, the gift of RLN H2 is accompanied by a Material Transfer

Agreement with a series of constraints and implications for intellectual property derived from research using serelaxin. There are other commercial sources of RLN

H2, but their purity is difficult to establish and to equate to Novartis serelaxin.

Not only are there seven members of the RLN peptide family but there are species differences between the variants. There is no naturally occurring RLN H2 that can be harvested from human donors. Therefore, recombinant DNA and chemical synthesis technologies are the only viable options to produce the relaxin

H2 hormone. Early reports on different technical approaches to synthesize relaxin were disappointing. Although the hormone is a relatively small and simple polypeptide, the folding and specificity of its tertiary structure are intricately sensitive to any change in the synthetic process. Even though production in E. coli systems offers potentially the cheapest, most reliable and possibly the approach that is most scalable, early results were unsatisfactory (Stewart et al., 1983). The

51 hormone appeared to be concealed in inclusion bodies in the bacterial cytoplasm and proved to be too costly for effective extraction.

Direct chain chemical synthesis was equally unproductive (Bullesbach and

Schwabe, 1991). These scientists synthesized the B- and A-chains separately by chemical means and combined them in an oxidizing milieu to create the disulfide bonds. The amounts of chemically synthesized relaxin H2 were very small and production costs were prohibitively high.

The current thesis focusses on whether or not it is possible to create a system for production of a recombinant, bench-top RLN H2 using a modified E. coli system. With a greater understanding of different approaches to directing a production of peptides to different cellular compartments, it is possible that recombinant technologies could be used to re-examine the synthesis of RLN H2 using E.coli systems. In fact, Cimini et al., (2017) have just reported a method for production of human pro-RLN H2 in the yeast Pichia pastoris. The rationale for their work was the need of a noncommercial source of supply of RLN H2.

Commercially available backbone plasmid vectors were used as a starting point to create various RLN H2 expression designs, which were tested in a number of strains of bacteria to develop an optimal system. Biological identity and activity of bacterially expressed RLN H2 were evaluated using SDS-PAGE gel electrophoresis, Western Dot Blotting, Mass Spectrometry and an animal model of blood pressure response.

Having demonstrated that it is possible to create a pure and reliable bench- top RLN H2, experiments were done to expand knowledge on the potential physiological actions of H2 RLN. Both the bench-top, recombinant RLN H2 and commercially available RLN H2 were studied to determine whether or not they affected the differentiation process of carcinoembryonic stem cells in a series of cDNA microarray experiments. This novel approach enabled measurement of a wide spectrum of gene expression levels in response to treatment with RLN H2 and comparison with established DMSO-induced differentiation of P19CL6 cells into contracting cardiomyocytes.

CHAPTER 2

EXPRESSION OF HUMAN RLN H2 IN E. COLI USING A HEXA-HISTIDINE TAG AND MALTOSE-BINDING PROTEIN DUAL-AFFINITY FUSION SYSTEM

Introduction

In humans, the relaxin (RLN) family consists of seven peptides: RLN1,

RLN2 (the same as RLN1 in other species), RLN3 and the insulin/RLN-like peptides INSL3, INSL4, INSL5 and INSL6 (Bathgate et al., 2013; Halls et al.,

2015). All peptides within the group evolved from an ancestral RLN3 gene (Hsu et al., 2003; Wilkinson et al., 2005), but set apart into various physiological functions.

Initially, RLN was recognized as a hormone of pregnancy (Hisaw, 1926; Ivell and

Grutzner, 2009), but at present it is well documented for its roles in the cardiovascular and renal systems (Sarwar et al., 2016) as well as for maintenance of tissue and organ collagen balance (Wilkinson et al., 2005; Unemori, 2017).

Relaxin 3 is primarily expressed and functions in the brain controlling appetite and anxiety (Bathgate et al., 2002). INSL3 has been shown to be responsible for testicular descent in men and for germ cell survival in both men and woman

(reviewed in van der Westhuizen et al., 2008).

Relaxin family peptides (RFP) are synthesized as pre-pro-proteins of similar lengths and sizes (Bathgate et al., 2006). Relaxin H2, the subject of the current research, consists initially of 185 amino acids. The signal peptide (25 amino acids) is the first element to be cleaved off, following which pro-RLN undergoes further processing in which the C-peptide, (107 amino acids), that connects the A- and B-

54 chains, is removed to create the mature form of the RLN. The completed form of

RLN H2 contains an A-chain of 24 amino acids and a B-chain having 29 amino acids.

For the RLN H2 to be biologically active, the peptide must be folded correctly by way of creating unique disulfide bonds between the B- and A-chains and one additional disulfide bond within the A-chain for a total of 3 disulfide bonds between all 6 cysteine residues (Bullesbach and Schwabe, 2000). The dynamics of chemically synthesized RLN H2 binding to its receptor suggested a novel

“gripping mode” of interaction between them (Bullesbach and Schwabe, 2000;

Chan et al., 2012). Two arginine residues positioned at B13 and B17 extend prongs from the helix, (like first digit and middle finger), respectively, to provide the electrostatic interaction. This is opposed by the hydrophobic constituent isoleucine at position B20, which is offset from the two arginine amino acids by about 40 degrees, resembling a thumb-like position. Bullesbach and Schwabe (2000) suggested that this “uniquely clear” receptor binding geometry of RLN would provide an excellent opportunity for the new design of RLN “peptidomimetics”.

At present, and perhaps in part inspired by Bullesbach and Schwabe

(2000), there appears to be a growing trend towards making RLN analogs constructed of shortened A- and B- chains. Hossain et al., (2011) chemically synthesized 24 analogs of RLN H2 with truncated termini of the peptide chains down to 38 from the standard 53 amino acids. It was shown that it is possible to significantly shorten both the N- and the C-termini of the B-chain and to retain the binding ability of RLN H2 to the RXFP1 receptor. The critical “active core” of the

55 chemically synthesized RLN H2 was made out of A4-24 and B7-24 of the native

RLN H2 sequence that makes it approximately one-third smaller in size and therefore considerably easier and significantly cheaper to make as a drug compared to the native form of RLN H2. However, the chemically synthesized RLN

H2 analogs were tested only in in vitro cyclic AMP assays and no other results were reported to date. In another development Chen et al., (2013) evaluated

365,677 chemical compounds deposited in the NIH library, and discovered only two agonists similar to RLN H2. Both these molecules were shown to bind to

RXFP1 or RXFP2 receptors in stably transfected HEK293T cells. The two chemical compounds showed similar potency to RLN H2 (3.7 μM and 3.8 μM) and efficacies of approximately 80%.

The two-chain structure and the formation of disulfide bonds create many technical difficulties for the non-natural production of the hormone. There are published reports of efforts to produce recombinant RLN H2 in different ways including bacteria E. coli, (Canova-Davis et al., 1991), yeast (Yang et al., 1993) and in mammalian systems (Soloff et al., 1992). However, most of these reports suggest that RLN produced using these approaches in not economical, often difficult to purify and usually misfolded making it unusable (review in Sorensen and

Mortensen, 2005). Chemical methods for production of RLN (Bullesbach and

Schwabe, 1991) are available but are expensive.

At the present time, there is no published method for a non-technically challenging, laboratory scale production of RLN H2 that would be of reliable purity and of acceptable activity. Hypothetically, production of RLN in a bacterial system

56 would be desirable due to the low costs of production, simpler purification, and easier scale-up compared to other host systems and chemical methods. It is likely that the commercially available RLN H2 is produced in an E. coli system but the process is proprietary, hence not accessible for comparison in the current study.

The commercial supply of RLN H2 is limited. In 2010, the Novartis

Corporation based in Basel, Switzerland acquired the World exclusive rights to the

RLN H2-making technology. It is possible that Novartis Corporation is now the primary supplier of recombinant RLN H2 and providing it for sale to research laboratories. Also, companies like Sigma-Aldrich, R&D Systems and Phoenix

Pharmaceuticals all list in their catalogs recombinant human RLN H2 produced in bacteria E. coli. The stability of the supplied product varies from merely 1-2 weeks to 3 months and at maximum up to a year when stored properly. There are also examples of companies like Abcam, who offers for sale recombinant RLN H2 that is approximately 18 kDa. The mature form of RLN H2 is approximately 6 kDa. This can be verified at the provided web link: http://www.abcam.com/recombinant- human-relaxin-2-protein-ab63227.html

The expression of foreign genes, particularly mammalian, in E. coli, is frequently inhibited by differences in amino acid codon usage. Some codons are preferably used in mammalian cells but the same codons are rarely used by E. coli. These infrequently used codons have been shown to reduce or completely diminish expression of mammalian recombinant proteins in bacteria (Novy et al.,

2001). Baca and Hol (1998) developed a method for overcoming codon bias by creating a synthetic plasmid and used it to overexpress Plasmodium falciparum

57 genes in E. coli. The hybrid vector is named RIG plasmid because it carries genes cloned from E. coli that encode three transfer RNA’s (tRNA): Arg (R), Ile (I), and

Gly (G). These genes direct the constitutive expression of tRNA’s that recognize the codons AGA/AGG (Arg, R), AUA (Ile, I) and GGA (Gly, G). Therefore, the increased levels of these three tRNA’s assisted E. coli in the more efficient translation of parasitic mRNA's (that are rich in these particular codons), leading to increased yields of recombinant parasite proteins.

To accomplish this codon usage advantage, a vector carrying the parasitic gene needs to be co-transformed into a strain of bacteria carrying the RIG plasmid.

In a further development of this approach, Novy et al., (2001) designed an analogous system for expression of mammalian proteins in bacteria. The plasmid is called pRARE and it codes for six amino acids containing codons seldom used in E. coli. At present, there are commercially available (Novagen Corporation) strains of bacteria pre-transformed with the pRARE plasmid. The Rosetta strains carry pRARE plasmid supplying six tRNAs for the following codons: AUA (Ile, I),

AGG (Arg, R), AGA (Arg, R), CUA (Leu, L), CCC (Pro, P) and GGA (Gly, G).

Plasmid pRARE confers resistance to chloramphenicol, which is an additional tool for selection of positive bacterial colonies in combination with the resistance of the plasmid carrying the recombinant protein sequence.

This novel design mimics naturally occurring RLN H2 B-chain comprising

33 amino acids. The last 4 amino acids are enzymatically cleaved off during processing to the mature form of RLN B-chain containing 29 amino acids. The cleaved off amino acids 29-33 are: K, R, S, and L were replaced with the Factor

Xa sequence of 4 amino acids: I, E, G and R. The molecular weight of this redesigned RLN B-chain was 29 Da less than the natural B-33 chain. In addition, this new 4 amino acid sequence acted as a mini C-peptide that is necessary for proper folding of RLN. The newly designed sequence was cloned into a pMal-p2x vector

(Appendix 1) and the plasmid was transformed into DH-5α recombination-deficient strain of E. coli. The RLN H2 sequence as used in the present series of experiments contains 15 amino acids encoded by codons that are not frequently used in bacteria. Table 8 below, shows the summary of RLN H2 amino acids having codon sequences rarely used in bacteria, which represents 28.3 % of its total amino acids.

Table 8. Relaxin amino acids and codons rarely used in E. coli.

Amino acid Codons Position in RLN sequence

Ile (I) AUA B8, B20, B22

2 Arg (R) AGG, AGA A18, A22, B13, B17

Leu (L) CUA A2, A6, A20, B10, B15

Pro (P) CCC Not present

Gly (G) GGA A14, B12, B24

The data presented in Appendix 1 on the efforts to express recombinant human RLN H2 from the pMal-p2x plasmid vector showed that there were a number of limitations and challenges encountered with that system. One of the major problems was that most MBP fusion proteins fail to bind efficiently to amylose resin during the purification process (Pryor and Leiting, 1997). There are also concerns that even when fusion proteins do bind, the amylose affinity chromatography does not produce samples pure enough to be admissible for further structural and/or functional studies of the expressed protein (Routzman and

Waugh, 2002).

In an effort to circumvent the difficulties associated with the amylose affinity chromatography, Routzman and Waugh (2002) experimented with a secondary, hexahistidine tag (6His-tag) cloned into various locations within the MBP reading frame. They reported that a 6His-tag can be added to the N-terminus of the MBP without hindering its ability to improve the solubility of its fusion partner.

Furthermore, in the same study, it was shown that 6His-MBP tagged proteins can be exported to the periplasm of E. coli making this approach feasible for recombinant proteins with disulfide bonds. Finally, Routzman and Waugh (2002) introduced a method for purification of 6His-MBP fusion proteins using an immobilized metal affinity chromatography (IMAC) on nickel ion-nitrilotriacetic acid

(Ni-NTA) resin, to which the 6His-tag binds with millimolar affinity. This method appears to be superior to the amylose chromatography used for purification of

MBP fusion proteins.

Rationale

The current chapter presents a unique approach for producing bench-top

RLN H2 and the steps to demonstrate that the synthesized hormone can be produced in a reliable and consistent manner, has sequence homology with naturally occurring RLN H2, shows biological activity (in vitro and in vivo) that is comparable with commercially available serelaxin and that can be stored and reconstituted successfully. The purpose of the experiments described in this chapter was to evaluate whether or not, combining 6His tag and MBP fusion proteins with bacterial hosts carrying the pRARE plasmid would be beneficial for expression of RLN H2.

Yang et al., (1993) reported the expression of human RLN in yeast

Saccharomyces cerevisiae using a single DNA sequence consisting of a B chain, a short interconnecting C chain of 6 amino acids and an A chain. A similar design for expression in bacteria was created for experiments described in this chapter.

Materials and methods

Methodology used to prepare bench-top RLN H2

The mature RLN H2 DNA sequence comprising of one string encoding 29 amino acids for the B chain and 24 amino acids for the A peptide was designed

(modeled after that reported by Hudson et al., 1984; Bullesbach and Schwabe,

1991 and Yang et al., 1993). In addition, one cleavage site for Factor Xa was inserted between the C-terminus of the MBP and N-terminus of the B-peptide, to separate RLN from MBP. A second Factor Xa site was placed between C-terminus of the B chain and N-terminus of the A chain to facilitate cleavage and proper folding of both peptides into mature molecules of RLN (Figure 3).

Figure 3. Amino acids and their corresponding nucleotide sequences of mature human RLN H2. Locations of Factor Xa sites incorporated into the present design are shown in red, B-chain is in blue and A-chain is in green.

I-E-G-R-D-S-W-M-E-E-V-I-K-L-C-G-R-E-L-V-R-A-Q-I-A-I-C-G-M-S-T-W-S-I-E-G-

R-Q-L-Y-S-A-L-A-N-K-C-C-H-V-G-C-T-R-R-S-L-A-R-F-C atc gag gga agg gac tca tgg atg gag gaa gtt att aaa tta tgc ggc cgc gaa tta gtt cgc gcg cag att gcc att tgc ggc atg agc acc tgg agc atc gag gga agg caa ctc tac agt gca ttg gct aat aaa tgt tgc cat gtt ggt tgt acc aaa aga tct ctt gct aga ttt tgc tga

Plasmids and bacterial strains

The pMal-p2x plasmid vector was purchased from New England Biolabs

Ltd., Mississauga, ON, Canada. A recombination-deficient strain DH-5α of E. coli was used for cloning, transformation, and DNA sequencing and was maintained in the laboratory collection. The plasmid vector pET-28a+ and Rosetta strains

(carrying the pRARE plasmid) of chemically competent bacteria were purchased from Novagen Inc. (Madison, WI, USA).

Preparation of DNA to produce the modified RLN H2 expression plasmid

Chemically synthesized oligonucleotides (Molecular Laboratory Services,

University of Guelph ON, Canada) were annealed using PCR methods, to create a DNA fragment coding for RLN H2 and consisting of B-29 and A-24 amino acids chains. This form of human RLN was shown to be the most abundant in the human and animal ovarian extracts and proved to be the most biologically active

(Bullesbach and Schwabe, 1991; Bathgate et al., 2002). This DNA fragment was cloned into a pMal-p2x plasmid to incorporate the MBP code. To facilitate separation of RLN from MBP after expression, a secondary fusion partner was employed: a 6-His tag positioned at the 5’ end of the MBP-RLN H2 coding sequence within the pET-28a+ plasmid vector. A cleavage site for Factor Xa was inserted between the 3’ end of the malE gene coding for MBP and the 5’ end of

RLN B-chain. To enable separation of both RLN chains and to facilitate their

63 folding, a cleavage site for Factor Xa was placed between the 3’ end of the B chain and the 5’ end of the A chain of RLN. Figure 4 below, illustrates schematically the details of this design. For cloning and DNA sequencing, DH-5α was used. For protein expression stages Rosetta strains of E. coli were used. The Rosetta strains carry multi-copy plasmid pRARE, which increases the number of tRNA genes that are not frequently used by native bacterial machinery (Brinkman et al., 1989). This minimizes the codon bias when expressing mammalian proteins in E. coli and allows for uninterrupted expression and incorporation of correct amino acids.

Figure 4. A diagram comparing designs of the RLN H2 expression cassettes.

Shown schematically are linearized expression vectors of MBP-RLN H2 fusion protein (A) and 6-His-MBP-RLN H2 modified fusion protein (B).

A: pMal p2x-RLN H2

malE Factor RLN B Factor RLN A Xa Xa

B: pET 28a+6-His-MBP-RlnH2

ATG-6His malE Factor RLN B Factor RLN A Xa Xa

The assembled plasmid was transformed into recombination deficient strain

DH-5α of E. coli for propagation. The resultant plasmid DNA was extracted, linearized and evaluated on an agarose gel. After plasmid DNA sequencing confirmed that the MBP-FXa-RLN-B-FXa-RLN-A DNA fragment was present and of the expected size, the constructed fragment was excised with restriction enzymes and inserted into the pET28a+ plasmid. Table 9 shows the multi-cloning and restriction enzymes of plasmid vector pET 28a+. In addition, shown in Figure 5 is a map of the same plasmid vector.

Table 9. Multi-cloning sites and expression regions of plasmid vector pET 28a+.

Type/region Name Description Start End position position Trx T 7 term T 7 termination 26 72 termination sequence sequence Multi-cloning MCS MCS: XhoI, NotI, 158 203 site (MCS) EagI, HindIII, SalI, SacI, EcoRi, BamHI, NheI, NdeI, NcoI Tag T 7 tag T 7 tag cds 207 239 Tag His tag His tag cds (6xHis) 270 287 Promoter T 7 T 7 promoter 370 386 Repressor LacI LacI coding 773 1852 protein gene sequence (cds) Bacterial Ori Bacterial origin of 3286 0 origin replication Selectable kanR Kanamycin 3995 4807 marker resistance gene ssDNA origin f1 ori f1 origin 4903 5358

Figure 5. Map of a plasmid vector pET 28a+ (adapted from Novagen Inc.).

This step was required to incorporate the second tag, the 6-His sequence.

The recombination-deficient strain DH-5α of E. coli was used to verify that the cloning was successful and that the fragment was in the proper reading frame by linearization with restriction enzyme (Not 1) digestion. Plasmid propagated in a

DH-5α strain of bacteria was used to collect sufficient quantities of pure DNA and was sequenced (Laboratory Services, University of Guelph).

The sequence verified plasmid pET28a+ carrying the correct 6-His-MBP-

FXa-RLN-B-FXa-RLN-A fragment was used to transform the following three strains of E. coli:

1. Rosetta – used to monitor the stability of the plasmid,

2. Rosetta DE3 and,

3. Rosetta DE3pLysS for protein expression.

Genotypes of the Rosetta strains:

Rosetta: F- ompT hsdSB (rB- mB-) gal dcm pRARE (CamR)

Rosetta DE3: F- ompT hsdSB (rB- mB-) gal dcm (DE3) pRARE (CamR)

Rosetta DE3 LysS: F- ompT hsdSB (rB- mB-) gal dcm (DE3) pLysS pRARE

(CamR).

Transformation of bacteria

Transformations of competent bacteria with plasmid DNA’s were performed according to the rubidium chloride protocol (RbCL, Sambrook and Russell, 2001).

The rubidium chloride chemical transformation method was used because this technique proved to be reliable (Roychoudhury et al., 2009). To transform, all three strains of bacteria, obtained from Novagen via Fisher Scientific (Nepean, Ontario,

Canada), twenty microliters of competent bacteria suspension were combined with

128 nanograms (measured using a Nano-Drop instrument) of the plasmid DNA for each of the three strains in transformation reactions. Details on the plating and propagation of the transformation reactions are shown in the Appendix section.

The genotypes of the three Rosetta strains of bacteria are shown above under a separate heading.

For a selection of positive clones in all transformed cells of the Rosetta strains, chloramphenicol was added to agar plates containing carbenicillin (a synthetic substitute for ampicillin; pMal-p2x-RLN H2) and kanamycin (pET-6His-

MBP-RLN H2).

Culture media

All ingredients of the various media were purchased from Fisher Scientific

(Nepean, ON, Canada) and were prepared in the laboratory. Two standard microbiological media (Atlas, 1997) were tested to determine the protein expression levels: Luria-Bertani-Miller (LB-M, 10 g NaCl/L) and Luria-Bertani-

Lennox (LB-L, 5 g NaCl/L). Because both media require isopropyl β-D-1- thiogalactopyranoside (IPTG) supplementation prior to induction of protein expression, and to avoid the negative effects of IPTG on cell growth, an additional medium called “The Over-Night Express TB Medium” (ONE-TB) was used for the reason that no IPTG addition is necessary to initiate protein expression. ONE-TB medium is a blend of the following solutions:

1. Carbon sources optimized for tight control of growth to high densities

prior to induction with lactose and continued growth.

2. A concentrated buffer that maintains constant pH during metabolic acid

production and provides an additional source of nitrogen required by

bacteria to support protein synthesis.

3. Provides a critical supply of magnesium for achieving maximum cell

density.

4. The ONE-TB medium contains no NaCl.

Cell growth conditions

Cell cultures in LB-M and LB-L media (114 ml volume each) were grown at

370 C for 16 hours on a shaker oscillating at 250 rpm. The induction of expression with 1 mM IPTG (Sigma-Aldrich, Canada) followed and the cell growth was continued for 3 additional hours. There was no need to use IPTG for experiments using Over-Night Express TB medium. These cultures (114 ml volume) were grown at 320 C for a total time of 16 hours on a shaker set up to 250 rpm. The optical density of cultures at 600 nm was monitored and measured using a Turner spectrophotometer.

For both LB-L and LB-M broth cultures not induced, media samples (1 ml) were collected at 16 hours and induced media samples (1 ml) were obtained after

3 hours post-induction. For experiments using ONE-TB medium, not induced

69 samples were taken at 10 hours, and self-induced (by the media ingredients) samples at 16 hours of growth. In addition, samples of induced media were collected and after removal of cells, were analyzed for the presence of fusion protein in the spent medium. All other samples were processed according to the cold osmotic shock procedure and periplasmic fractions were harvested for analytical SDS-PAGE protein gel electrophoresis (Schagger and von Yagow,

1987).

Harvesting of cells

As the system reported in the current chapter was designed to direct expression of the recombinant protein in the periplasmic space, the methods described by Neu and Heppel (1965) and Nossal and Heppel (1966) were used to harvest the expressed fusion protein. Briefly, in this procedure, E. coli cells were harvested from saturated cultures at a density of about 5x108 cells per milliliter by centrifugation at 13,000 rpm at 4º C. The bacterial cell pellets (one gram, wet weight) were washed twice with 40 ml of cold cell wash buffer containing 0.01 M

Tris-HCL (pH 7.1) and 0.03 M NaCl. Next, the washed cell pellets (one gram, wet weight) were suspended in 40 ml of a 20 % sucrose and 0.033 M Tris (pH 7.1) buffer supplemented with sodium EDTA (0.5 mM, to eliminate proteases). For the osmotic shock, for one gram of wet cells, 40 ml of 40 % sucrose, 0.033 M Tris-HCl

(pH 7.1) supplemented with 0.1 M sodium EDTA (pH 7.1) was used. The mixture was stirred on a rotary shaker for 10 minutes at 180 rpm and following this step,

70 centrifuged for 10 minutes at 13,000 rpm at 4º C and the supernatant was removed and discarded. The remaining cell pellet, containing periplasmic proteins, was rapidly dispersed in 80 ml/gram of ice-cold 5 mM magnesium chloride (MgCl2) buffer and deionized, sterile and ice-cold water (to verify solubility of the fusion protein).

To separate the 6His-tag MPB-RLN fusion protein from the rest of periplasmic proteins, purification was carried out on His-Bind cartridges

(Novagen). After cleavage with Factor Xa, another chromatographic step on columns packed with Ni-NTA resin was required to retain 6-HisMBP and to collect the recombinant RLN in the flow-through fractions. To remove the 4 remaining FXa amino acids attached to the C-terminal of the B-chain, digestion with carboxypeptidases C, B and A was performed according to the protocol supplied with the enzymes (a gift from Worthington Biochemical Corporation, Lakewood,

NJ, USA). The solutions of RLN were next desalted on Bio-Rad columns with a cut-off the size of 6 kDa and were used for all analytical and experimental purposes.

Endotoxin contamination of end-point solutions is a possibility with this bench-top method of preparation, therefore, the solutions of RLN produced using this method were subjected to an endotoxin contamination assay (Toxi-Sensor Kit,

Fisher Sci. Nepean, ON, Canada). All of the tested batches were negative for the presence of endotoxins.

For long-term storage, bench-top RLN H2 was precipitated by using acetone (Sambrook and Russell, 2001) as follows: An excess volume of acetone

71 was cooled down to -200 C in advance of the planned procedure. The sample bench-top RLN H2 solution was placed into an acetone-resistant tube. Four times the sample volume of pre-cooled acetone was added to the tube and vortexed briefly. The acetone-RLN suspension was incubated at -200 C for a minimum of 60 minutes. Next, the solution was centrifuged for 10 minutes at a maximum speed of

13,000 rpm. The acetone-containing supernatant was collected and discarded into an appropriate collection vessel. The tube containing bench-top relaxin was left uncapped on the bench top for a maximum of 30 minutes to prevent its over-drying.

For immediate use or short-term storage, the RLN pellet was dissolved in a sodium acetate (0.2 mM) buffer adjusted to pH=5.5.

Confirmation of the biochemical integrity of bench-top RLN H2

Mass Spectrometry

Three samples from different batches of bench-top RLN H2, 10 µg each, were submitted to the University of Guelph Mass Spectrometry Laboratory and analyzed using an AGILENT LC-UHD Q-Tof apparatus. Two samples were acetone-precipitated and stored at -200 C for two and three years, respectively.

The third sample was dissolved in a sodium acetate (0.2 mM) buffer adjusted to pH = 5.5.

Western Dot Blot Analysis

Using Western dot blot analysis, samples of bacterially derived RLN H2 were tested and compared with purified porcine RLN (prepared in the Department of Biomedical Sciences, Ontario Veterinary College, [OVC], University of Guelph), serelaxin, and human insulin. The duplicate tests were conducted in a double-blind fashion so that the person performing the procedure did not know the origin of samples and the person submitting samples was not informed about coding used for Western blot until revealing of the code during the reading of the results. The dot blot analysis was chosen due to the limited availability of RLN H2 antibodies.

Traditional Western blot requires tenfold amounts of antibodies.

Activity of bench-top relaxin H2 in vivo

Animals

The experimental protocol was reviewed and approved by University of

Guelph Animal Care Committee in accordance with guidelines of the Canadian

Council on Animal Care. Retired female breeder Sprague-Dawley strain of rats

(Charles River, Canada, St. Constant, QC, Canada) weighing between 320 and

350 grams were used. The animals were housed at the University of Guelph

Central Animal Facility and maintained under controlled conditions with 12 hours

73 of light and 12 hours darkness, room temperature 19° C with food and water available ad libitum.

On experimental days, the animals were transported to the operating room and anaesthetized with a double injection of 0.2 mL ketamine hydrochloride (100 mg/mL, i.p. Ketalean, Biomeda-MTC, Animal Health, Cambridge, ON, Canada) and 0.2 mL diazepam (5 mg/mL, i.p. Sabex, Boucherville, QC, Canada). A supplementary injection of 0.2 mL Rompun (1 mg/mL, i.m., Xylazine, Bayer Inc.,

Animal Health, Etobicoke, ON, Canada) was given intramuscularly (i.m.) into one of the hind legs. For direct recordings of blood pressure the right common carotid artery was cannulated (polyethylene tubing O.D. 0.96 mm, I.D. 0.58 mm,

Intramedic, Clay Adams, Becton Dickinson and Company, Sparks, MD, USA). The left hind leg was chosen for femoral vein cannulation (polyethylene tubing O.D.

0.61 mm, I.D. 0.28 mm, Intramedic) to allow for administration of drugs. To prevent blood from clotting, the cannulae were pre-washed and filled with physiological saline (physiological saline 0.9 %, Biomeda-MTC, Animal Health, Cambridge, ON,

Canada) containing 10 % heparin (Heparin sodium, 1000 USP/mL, Hepalean,

Organon, Toronto, ON, Canada). The carotid cannula was then connected to a blood pressure transducer (Spectra Med transducer model P23XL, Grass

Instrument Co., Quincy, MA, USA) linked to a calibrated blood pressure recorder

(Polygraph Grass-7, Grass Instruments). The animals were placed on- and covered with cotton wool pads to maintain their body temperature. Approximately

30 minutes after recording started and baseline blood pressures were established, animals were treated with 0.1 mL of saline injection through the leg vein cannula.

After another 30 minutes of stable blood pressure recording, 5 μg of recombinant bacterially-derived RLN H2 dissolved in 0.1 mL of saline was slowly administered via femoral cannula. Blood pressure was continuously recorded for one hour. At the completion of experiments, the animals were euthanized with an overdose of a barbiturate (Somnotol i.v., Bayer Inc., Animal Health, Etobicoke, ON, Canada) via the femoral vein cannula.

Results

Construction of novel plasmid

Plasmid DNA samples were collected, digested with restriction enzymes and evaluated by gel electrophoresis. Shown below are the results of all steps taken to assemble and evaluate the novel plasmid vector 6-His-MBP-RLN H2.

Plasmid DNA’s were extracted, restriction enzyme digested and evaluated by running agarose gel electrophoreses. Figures 6, 7, and 8, respectively, illustrate the positive results. Figures 9 to 12 illustrate the procedures done to extract and purify the fusion protein and the final product, RLN H2, assessed by running protein gel electrophoreses. The figure legends provide detailed information related to each gel image, respectively.

Figure 6. An agarose gel picture showing linearized pMal-p2x plasmid vector.

Lanes 1 and 8 show the 1kb molecular marker. Lanes 2 and 3 show linearized empty pMal-p2x plasmid of 6723 base pairs. Chemically synthesized DNA fragment of RLN H2 with FXa sites incorporated of 222 base pairs, lanes 4 and 7.

1 2 3 4 5 6 7 8

6723 bp

222 bp

Figure 7. An agarose gel photograph of pET28a+-6His-MBP-RLN-H2.

A 1% agarose gel image illustrating: lanes 1 and 8 DNA 1kb markers, supercoiled plasmid pET28a+-6His-MBP-RLN-H2 is seen in lanes 2, 5 and 6. Lane 3 shows the vector of expected size 6701 base pairs linearized with Not I enzyme uniquely present within the sequence of RLN H2.

1 2 3 4 5 6 7 8

6701 bp

Figure 8. A plasmid DNA on 1% agarose gel.

Lane 1 is the 1kb DNA marker. Lane 2 is an empty pET28a+ plasmid. Lane 3 is the same empty plasmid linearized with Sac I enzyme. Lane 4 shows pET28a+ with

MBP-RLN H2 fragment cloned into it. Lane 5 is the same plasmid linearized with

Not I enzyme.

1 2 3 4 5

6701 5369 bp bp

Protein expression experiments using traditional media

The three strains of E. coli were used for different purposes: the first

(Rosetta) was not used for protein expression. The strain was designed to allow for monitoring of the stability of the plasmid. The second and third strains (Rosetta

DE3 and Rosetta DE3pLysS) were both tested in protein expression pilot experiments using 10 mL of Luria-Bertani-Lennox (LB-L) and Luria-Bertani-Miller

(LB-M) media. After numerous attempts in larger scale up to 500 mL cultures, no quantifiable amounts of the fusion protein could be detected. Continuation of the experiments using LB media could no longer be justified and was therefore discontinued.

Alternative approach and media

An alternative approach was adopted. The LB media were replaced by

Over-Night Express TB (ONE-TB) culture medium (Studier, 2005). Rosetta strains were grown in the ONE-TB medium and after 6His-MBP-RLN H2 fusion protein was detected from 10 mL small-scale pilot experiments, cultures were expanded to grow in 100, 500 and 1000 mL volumes.

Detection of fusion protein

Samples of cultures were collected before and after induction of fusion protein expression. The cold osmotic shock protocol (Neu and Heppel, 1965, and

Nossal and Heppel, 1966) was used to harvest the periplasmic proteins. From this pool of proteins, the 6-His-MBP-RLN H2 fusion protein was separated on His-

IMAC columns. The isolated fusion protein was next digested with proteinase

Factor Xa to yield RLN H2 with no MBP or 6-His tags. Throughout the purification stages, samples were held for analytical purposes on SDS-PAGE gels according to a method by Schagger and von Jagow (1987) for separation of proteins between

1 and 30 kDa (Figures 9-11). Shown in Table 10 are the average yields of expressed periplasmic proteins harvested from cold osmotic shock fluid. From cultures of Rosetta DE 3 cells, media were collected at the end of experiments.

The amounts of the fusion protein are summarized in Table 11. Due to the high salt content in the IMAC elution buffer, purified samples of bench-top RLN H2 required desalting on columns with a 6 kDa cut-off size (Figure 12).

Figure 9. Representative protein SDS-PAGE gel comparison of the two strains of

Rosetta cells.

Proteins collected from Rosetta DE3 strain: Lane 1 is a protein marker, lanes 2 – not induced, 3 – induced, 4 – periplasmic, 5 – same as 4, 10-fold concentrated.

Rosetta DE3 pLysS strain: lane 6 – periplasmic fractions, lanes 10 – not induced,

9 – induced, 8 – diluted lane 9, 7 – 10-fold concentrated periplasmic extract. On the left, the upper arrow indicates MBP-RLN fusion protein of approximately

50.453 kDa and the lower arrow indicates the 42.5 kDa native MBP protein. The difference in molecular weights is noticeable.

1 2 3 4 5 6 7 8 9 10

50.45 kDa

42.5 kDa

Figure 10. Representative protein SDS-PAGE gel of MBP-RLN H2 fusion protein.

Protein samples obtained from Rosetta DE3 pLysS carrying 6His-MBP-RLN H2 fusion protein. Lane numbers: 1 and 6 – indicate not induced cell pellet; 2 and 7 – induced cell pellet; 3 and 8 – periplasmic fractions; 4 and 9 – His-IMAC purified periplasmic fractions and 5 and 10 show 10-fold concentrated periplasmic fractions.

1 2 3 4 5 6 7 8 9 10

Figure 11. Representative protein SDS-PAGE gel (not-reduced) of protein samples collected after digestion of MBP-RLN H2 fusion protein with FXa and carboxypeptidases. Lane 1 shows His-MBP fraction; lanes 2 and 3 show column wash fractions after separation of RLN; shown in lane 4 is RLN H2 (10 times concentrated). Lane 5 is a protein molecular marker with the weight of proteins

(kDa) indicated by the arrows on the right. The RLN H2 sample in lane 4 of approximately 6 kDa is seen just lower than the 6.5 kDa of protein marker seen in lane 5.

1 2 3 4 5

44.0

26.6

17.0 14.2

6.5

Figure 12. Representative protein SDS-PAGE gel of desalted RLN H2.

Lanes 1-4 show concentrated RLN H2 fractions eluted from His-trap column after digestion of fusion protein with FXa and retention of the 6His-MBP fragment on the column. Lanes 5-8 illustrate the same samples after purification on a desalting column with a cutoff size of 6 kDa. It is noticeable that despite the desalting, some impurities still remained in the eluted bench-top RLN H2 samples.

1 2 3 4 5 6 7 8

Efficiency of the novel system for bacterial production of RLN H2

Table 10. Summary of results obtained from two strains of Rosetta E. coli expressing recombinant RLN H2 from plasmid vector pET28a+His-MBP-RLN H2.

All results shown were obtained from cultures grown in Over-Night Express TB medium without IPTG supplementation. Optical densities were equilibrated by an addition of plain medium to culture samples.

Wet Wet Wet Not Induced weight of weight of weight of RLN H2 RLN Strain of induced OD600 all cells periplasm MBP- wet H2dry E. coli OD600 at harvest proteins RLN H2 weight weight mg in mg in mg in 1L mg in 1L mg in 1L 1 mL 1 mL 0.68 1.35 32.9 0.750 37.50 4.523 1.356

0.68 1.35 31.3 0.713 35.60 4.293 1.287 Rosetta 0.68 1.35 44.3 1.010 50.50 6.090 1.827 DE3 pLysS 0.68 1.35 40.9 0.932 46.62 5.623 1.686 Mean 37.35 0.851 42.55 5.13 1.53 ±SE ±3.12 ±0.07 ±3.57 ±0.43 ±0.13 0.68 1.35 30.1 0.686 34.31 4.137 1.241

0.68 1.35 29.5 0.672 33.63 4.055 1.216 Rosetta 0.68 1.35 26.4 0.601 30.09 3.629 1.088 (DE3) 0.68 1.35 26.6 0.606 30.30 3.654 1.096 Mean 28.15 0.64 32.08 3.86 1.16 ±SE ±0.96 ±0.02 ±1.09 ±0.13 ±0.03

Table 11. Quantification of the relative amounts of the fusion protein in the media.

Media samples collected at harvest time from four one liter cultures growing

Rosetta DE3 contained MBP-RLN H2 fusion protein that leaked out of the periplasmic space. Percentages given in column 1 are in relation to periplasmic fractions of MBP-RLN H2 listed in column 6, Table 10, previous page.

Percentage of MBP-RLN H2 RLN H2 RLN H2 periplasmic wet weight wet weight dry weight MBP-RLN H2 (mg) (mg) (mg)

19.15 6.57 0.79 0.237 15.41 5.13 0.62 0.1.87 22.07 6.64 0.80 0.240 17.87 5.41 0.65 0.195 18.62±1.280 5.93±0.389 0.71±0.045 0.214±0.013

In vitro validation of bacterially expressed RLN H2

The identity of the produced recombinant RLN H2 was confirmed by comparing it with known RLN standards using Western Dot Blot analysis. Figure

13 illustrates the results validating the recombinant protein as RLN H2. Listed in the legend to the figure are the RLN standards used for comparisons.

Figure 13. Western Dot Blot analysis of bacterially expressed RLN H2 compared with commercially available serelaxin, porcine RLN, and insulin (as a negative control). The detailed legend below, explains the figure.

Porcine RLN is shown in cells: 1b and 1c (1μg), 1d and 1e (100ng), 2a, 2b, 4a and 4b (10ng).

Serelaxin (Connetics) was spotted and detected in fields: 2c and 2d (1μg), 2e and 2f (100ng), 2g and 3a (10ng).

Bacterially expressed RLN H2 was spotted and detected in squares: 3b and 3c (1μg), 3f and 3g (100ng), 4a and 4b (75ng) 4c and 4d - 50ng, 4e, and 4f – (25ng).

Human recombinant insulin was spotted on fields: 1f, 1g (1μg), 3d and 3e (1μg) and was not detected.

First cell 1a and last cell 4g were both spotted with plain buffer in which all RLN samples were dissolved.

a b c d e f g

Mass Spectrometry Analysis

Three samples of bench-top RLN H2 collected after desalting were analyzed by the staff of the University of Guelph Mass Spectrometry Laboratory using an AGILENT LC-UHD Q-Tof apparatus. This AGILENT equipment is a combined liquid chromatography (LC) and ultra-high definition (UHD) mass spectrometry device capable of removing any impurities from the sample before analyzing the trypsin-digested protein fragments. Two lyophilized samples of bacterially-derived RLN H2 produced 3 and 2 years prior to analysis and stored at

-20ºC were degraded and no results were obtained. The third sample resolved in sodium acetate buffer and stored at -20ºC for one year was identified as RLN H2 matching the Gene Bank X00948, (RLN H2). Shown in Figure 14 are the results of mass spectrometry analysis showing presence and abundance of trypsin- digested peptide fragments of the submitted RLN H2 sample. The presence of basic amino acids at or near a cut peptide fragment N- or C-terminus results in producing N-terminal (a, b, a, and d) or C-terminal (x, y, z, v, and w) fragment ions as shown on the graphs, respectively. The identified peptide masses along with amino acid sequences confirmed that the analyzed sample was RLN H2, and are listed in Table 12.

Figure 14. Mass Spectrometry data of trypsin-digested, bacterially-expressed RLN H2.

The following eight graphs illustrate the mass spectra peaks (peptide products of a tryptic digestion). Absolute intensity (a.i.) is shown on the Y axes. Mass peaks were assigned using the XTOF software. Peptide sequences, detected masses and cut positions are listed in Table 12 below.

B Chain matches

Peptide fragment number 1, B 18-29, detected mass 1323.595 Da

Peptide fragment number 2, B 1-10, detected mass 1151.516 Da

Peptide fragment number 3, B 18-29, detected mass 1339.590 Da

Peptide fragment number 4, B 1-10, detected mass 1135.522 Da

Peptide fragment number 5, B 18-29, detected mass 1324.579 Da

Peptide fragment number 6, B 18-25, detected mass 862.404 Da

Peptide fragment number 7, B 3-9, detected mass 933.463 Da

A chain match

Peptide fragment number 8, A 10-17, detected mass 1020.394 Da

Table 12. A summary of peptide masses obtained during mass spectrometry analysis (as illustrated in graphs Figure 14), of bacterially produced RLN H2.

“Detected Mass” column lists the identified peptide masses (shown in Figure 14) after tryptic digestion of bacterially expressed RLN H2 acquired from the AGILENT LC-UHD Q-Tof device using the Expasy database. Theoretical pI: 8.66 / Mw (average mass): 5968.05 Da/ Mw (monoisotopic mass): 5963.88 Da.

Detected Peptide Sequence Cut at Peptide fragment Mass(Da) Positions number 1323.595 AQIAICGMSTWS B 18-29 1 1151.516 DSWMEEVIK B 1-10 2 1339.590 AQIAICGMSTWS B 18-29 3 1135.522 DSWMEEVIKL B 1-10 4 1324.579 AQIAICGMSTWS B 18-29 5 862.404 AQIAICGM B 18-25 6 933.463 WMEEVIK B 3-9 7 1020.394 CCHVGCTK A 10-17 8

In vivo assessment of recombinant RLN H2

In order to evaluate the biological activity of purified bench-top recombinant

RLN H2 a group of 8 retired breeder female rats was used in blood pressure experiments according to the protocol developed by Parry et al., (1990). The recombinant RLN H2 expressed in bacteria caused significant increases in both systolic and diastolic pressures, which lasted for more than 20 minutes. This is consistent with similar responses to equal doses of RLN reported by Parry et al.,

(1990). A sample blood pressure recording from a single animal is illustrated in

Figure 15. The compilation of blood pressure responses from the whole group of animals is presented in Table 13 and shown in Figure 16.

Heart rate was also monitored and the increases are comparable with that reported by Parry et al., (1990) and are summarized in Table 14 and illustrated in

Figure 17.

120 mm Hg

80 blood pressure increase PBS RLN H2 20 minutes

5 minutes

Figure 15. A representative trace of arterial blood pressure recording from an anaesthetized rat. The animal received an intravenous control injection of 0.01 ml of PBS and after 20 minutes of stable blood pressure, 5 μg of bacterially produced RLN H2 in 0.01 ml of PBS was administered via the leg vein. A significant increase in blood pressure was sustained for more than 60 minutes after which time the experiment was terminated.

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Table 13. The effects of bacterially derived recombinant RLN H2 on systolic, diastolic and mean arterial blood pressures (MAP; mm Hg) in anaesthetized rats

(n=8) after intravenous injection (5 µg) as recorded at set times (min). Data are expressed as mean ± SEM changes in blood pressure. All data were compared by two-way analysis of variance (ANOVA) with mean values of systolic, diastolic and

MAP pressures recorded after treatment with saline at four time points. Significant

(p˂0.05) elevations in arterial pressures are indicated by blue shading.

Blood Time (min)

Treatment pressure 0.0 5.0 15.0 30.0

Systolic 102.0±0.57 124.34±5.78 117.0±4.16 117.0±4.48

RLN H2 Diastolic 82.0±0.58 99.34±4.05 92.67±2.40 92.34±2.73

MAP 88.67±0.57 107.67±4.81 100.78±2.89 100.56±3.05

Systolic 102.0±1.0 102.0±1.0 101.9±0.9 101.8±0.89

Saline Diastolic 83.0±0.9 82.9±0.9 82.8±0.8 82.8±0.9

control MAP 89.27±0.95 89.2±0.97 89.1±0.89 89.07±0.96

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Figure 16. The effects of bacterially derived recombinant RLN H2 on systolic, diastolic and mean arterial blood pressures (mm Hg) in anaesthetized rats (n=8) after intravenous injection (5 µg) as recorded at set times (min). All data points shown in this graph are from Table 13, above.

Changes in blood pressures 140

120

100

40 Pressure (mmHg) Pressure 20

0 0 5 15 30 Time (min)

systolic (RLN) diastolic (RLN) MAP (RLN) systolic (SAL) diastolic (SAL) MAP (SAL)

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Table 14. Changes in heart rate in rats treated with bacterially produced RLN H2.

The effects of bacterially derived recombinant RLN H2 on heart rate in rats (n=8) after intravenous injection (5 µg) as recorded at four time (min) points after treatment with saline. Data are expressed as mean ± SEM changes in heart rate.

All data were compared by two-way analysis of variance (ANOVA) with mean values of heart rates recorded after treatment with saline at four time points.

Significant (p˂0.05) elevations in heart rates are indicated by blue shading.

Number of heart beats per minute Time after treatment (min) Treatment 0 5 15 30 Saline 223.70±3.42 223.15±6.76 227.07±6.32 224.95±5.89 RLN H2 5μg 227.25±2.69 247.65±6.96 249.07±6.83 247.95±6.77 Mean Percentage Change (MPC) 1.56 10.97 9.68 10.22

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Figure 17. Changes in heart rate in rats treated with bacterially produced RLN H2. The effects of bacterially derived recombinant RLN H2 on heart rate in rats (n=8) after intravenous injection (5 µg) as recorded at four time (min) points after treatment with saline. All data points shown in this graph are from Table 14, above.

Changes in heart rate 260

250

240

230

220 Heart Heart beats/minute 210

200 0 5 15 30 Time (min)

Saline RLN H2

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Discussion

The experiments described in this chapter summarize the synthesis and validation of a laboratory-synthesized RLN H2 protein using a bacterial system.

The synthesis was confirmed using Western Dot blots compared to several RLN

H2 standards obtained from multiple commercial sources. In addition, the mass of our synthesized RLN H2 (the A and B chains) was confirmed by MS-LC-UHD-Q-

Tof which determined the presence of all peaks. Finally, the biological activity of the RLN H2 protein was confirmed by intravenous injection into female rats which produced a comparable hypertensive and tachycardic response to that previously observed using RLN H2 from a commercial source (Parry et al., 1990). Taken together, these results suggest that our synthesized peptide is indeed RLN H2, and its biological activity suggests that the A and B chains are properly folded.

Numerous attempts to produce recombinant RLN H2 in a bacterial system were investigated in the current chapter. Initial efforts using the newly designed pET 28a+ plasmid vector with dual-tag: 6-His-MBP-RLN H2 fusion protein and

Rosetta strains of E. coli were carried out using conventional microbiological media: LB-M and LB-L. These media, however, still required supplementation with

IPTG to induce protein expression. The amounts of fusion protein obtained using these media supplemented with IPTG were too small to quantify and to justify the continuation of this line of experiments.

There could be an explanation for this experimental outcome. Cultures were grown for up to 16 hours, prior to induction with IPTG, followed by further cultivation

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for at least 3 hours in each attempt. Luria-Bertani broths can theoretically support

E. coli cell growth to an OD600 of around 7. However, the stable growth of cells in these broths ends surprisingly early after 3 to 4 hours of growth, reaching an OD600 of 0.3. This stage is followed by an extended period of time (12 to 16 hours), when the rate of growth and an average yield of cell mass both decrease progressively

(Sezonov et al., 2007). The reason for this, according to Sezonov et al. (2007) is that LB broths contain no fermentable sugars that could easily be utilized by E. coli. Luria-Bertani broths contain two organic ingredients: tryptone and yeast extract. Tryptone is an assortment of peptides created after the digestion of casein by the protease trypsin. Yeast extract is obtained by extruding the cell contents following discarding of the cellular walls. Bacteria E. coli readily process oligopeptides supplied by tryptone and yeast extract by making free amino acids accessible for their metabolic needs. These amino acids, while being the principal source of carbon for E. coli, contribute to the alkalization of the medium because the end product of amino acid catabolism is ammonium. The optimal pH of LB broths should be maintained at 7.0±0.2 but, when there is an excess of ammonium, pH quickly rises above 9.0, which causes cell growth to arrest due to an elevated pH rather than the deficiency in the supply of carbon. Attempts were made to control the pH of the medium by supplementing LB broths with glucose. Glucose as a source of carbon, however, also has certain negative effects:

1. It contributes to the promoter tightening, hence the level of protein

expression is minimal or no protein is expressed at all,

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2. Glucose as well contributes to lowering of medium pH below the optimal

7.0±0.2 what makes the environment too acidic for the growth of E. coli.

It is possible that large industrial fermenters could be equipped with means of continuous monitoring of pH, ways of supplementation with a carbon source and removing excess ammonium from the culture medium. In an average academic laboratory using a shaker flask culturing system, the precise control of culture environment might not always be attainable.

In recent years, Novagen Company promoted a new medium called “Over-

Night Express Auto-Induction Medium”, which is based on the formula developed by F. William Studier at the Brookhaven National Laboratory. This complex medium allows regulated expression of proteins in E. coli without the need of monitoring density of culture or addition of IPTG to the medium. The method utilizes media components that are metabolized differentially to promote growth reaching high cell densities and automatically inducing expression of recombinant proteins from the lac promoters. These unique, self-controlled induction media provide more reliable means for production of proteins from the pET series and other IPTG-inducible plasmids and the Rosetta strains of bacteria.

Expression of RLN H2 from a dual tag 6His-MBP constructed within the backbone of the pET28a+ plasmid vector and the Rosetta strains of E. coli maintained in Over-Night Express TB Auto-Induction medium was achieved. The

RLN H2 fusion protein was not intended for cytoplasmic expression. As indicated in Chapter 1, that approach would lead to the creation of insoluble inclusion bodies.

Instead of this, efforts were focused on the periplasmic destination, where the

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environment for the creation of disulfide bonds required by RLN is more favorable.

Pryor and Leiting (1997) reported that MBP does not contain any cysteines and therefore its protein stability is independent of reducing agents present in the cytoplasm.

During the purification stages, fusion protein expressed in Rosetta DE3 strain was detected not only in the fractions collected from the periplasmic space but in the auto-induced culture samples as well. The most likely reason for this could be that this host strain contains T7 RNA polymerase gene (a λDE3 lysogen) for expression of target proteins. In λDE3 lysogens, the T7 RNA polymerase is under control of the lacUV5 promoter. The inherent leakiness of the lacUV5 promoter allows for some degree of un-induced transcription due to the lack of additional control. Therefore, this strain of Rosetta is suitable for expression of genes coding for proteins that are not harmless to the host (Studier, 1991). Since in this series of experiments the cells were not harmed, it would be reasonable to propose that MBP-RLN product was not toxic. This is in contrast with statements made in Appendix 1, where one of the conclusions was that RLN was toxic to the

E. coli strains used in the experiments described therein. For more stringent control over the λDE3 an improved host, the Rosetta DE3 pLysS proved to be more advantageous. The pLysS plasmid encodes T7 lysozyme, a natural inhibitor of T7

RNA polymerase, which virtually eliminates its ability to transcribe target genes in not induced cells. The transfer (t) RNA genes are controlled by their own promoters, whilst the RARE and LysS genes are present on the same plasmid

(Studier, 1991). This strain proved to be most effective in the present work.

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In addition, Mergulhao et al., (2004) reported that during cell division leakage of the periplasmic contents can take place. The accumulation of recombinant protein in the periplasmic space may also cause osmotic pressure to build up, which could force the expressed protein to be pushed across the outer membrane (Hasenwinkle et al., 1997). Periplasmic secretion per se may also initiate cell lysis, and as a result, the periplasmic contents could be released prematurely (Lee and Bernstein, 2001). Oganesyan (2009) determined that approximately 40% of the expressed human interferon (exact-hIFNα2) a fusion protein directed for expression in the periplasm, was released by the osmotic shock and about 20% was secreted into the culture medium prior to the osmotic shock procedure.

Talmadge and Gilbert (1982) using pulse-chase methods reported on the biosynthesis of pre-pro- and proinsulin in bacteria. They observed that from the same plasmid DNA a variety of hybrid pre-pro-insulin could be concurrently made.

Molecules with intact bacterial signal sequences were directed into the periplasm and molecules with damaged signals were not sent to the periplasm and remained in the cytoplasm. These molecules were degraded within 2 minutes. In contrast, molecules that reached the periplasmic space remained stable for at least 10 times longer. Wickner et al., (1978) have suggested that in bacteria, the secretion mechanism is different from that of eukaryotes because the unprocessed pre- protein can be detected in vivo in bacteria. In eukaryotes, it has been reported that the pre-protein is processed before synthesis is complete. Talmadge and Gilbert

(1982) concluded that a full-length pre-pro-insulin precursor appears only

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transiently just before the signal sequence is removed by the bacterial signal peptidase. They observed that the synthesis of a full-length pre-pro-insulin precursor occurred at 30 seconds of the chase at 340C. These authors also suggested that the presence of unprocessed pre-proteins in bacteria could be indicative of slower processing comparing to eukaryotes. A possible explanation for this delay was that the entire pre-protein could be left in the periplasm and kept there anchored to one of the membranes until signal peptidase is able to cleave off the signal peptide and allow for the protein to be released.

There is another aspect worth noting here, which pertains to the sodium chloride content in a culture medium and the possibility that the expressed recombinant protein might be susceptible to desalting (or precipitation) into the medium. Indeed, the molarity at which RLN H2 can be eluted from purification columns can be as low as 14 mM, which translates into 0.876 g of NaCl per liter of solute (Sherwood, 1990). Luria-Bertani broths contain 5 and/or 10 g of NaCl per liter which equals to 85.47 and 170.94 mM, respectively. Therefore, it would be logical to assume that the high molar concentrations of NaCl in the media may have contributed to the low yield in periplasmic fusion protein obtained from cultures grown initially in LB broths in the discussed series of experiments.

Therefore, the change to using the salt-free ONE-TB media was the correct one in this series of experiments. A similar conclusion can be made regarding the purification system where 6-His binding enabled harvesting quantifiable amounts of both MBP-RLN-H2 fusion protein and after further enzymatic digestion, purification of RLN H2. As demonstrated in experiments presented here,

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bacterially produced RLN H2 was biologically active in comparison with commercially available serelaxin. Based on the Mass Spectrometry results, tryptic digestion of bacterially derived RLN H2 proved to contain peptides of correct sizes and sequences. There were, however, several minor unmatched peptides.

Canova-Davis et al., (1991) detected minor, unmatched peaks in their MS analyses of recombinant RLN H2. The explanation for this occurrence in that work was due to trans-amino peptidases, which are responsible for non-specific cleavage of RLN and insulin-like growth factor used in their experiments. It is, therefore, possible that similar non-specific trypsin digests happened here. Stults et al., (1990) suggested that deducing the amino acid sequence of RLN H2 from the known DNA sequence was sufficient enough when Mass Spectrometry data were reviewed and factored in.

Under normal, physiological conditions in non-anaesthetized animals, an increase in systemic blood pressure is typically followed by a compensatory decrease in blood pressure and heart rate, mediated by the immediate baroreceptor response (Brown, 1908). This was not the case in the present experiments using ketamine/xylazine anaesthetized rats. The pressor response to

RLN H2 lasted for more than 30 minutes. In addition, there was a sustained increase in heart rate. The lack of ability of the baroreceptor reflex to counteract hypertension induced by RLN may also explain the prolonged effect of RLN on vasopressin release and is likely to contribute to the appearance of the vasoconstrictor effect of vasopressin in RLN-treated rats.

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The pressor effects of RLN H2 after intravenous injection in anaesthetized rats are mediated primarily by the release of vasopressin (Parry et al., 1990).

Relaxin most likely acts centrally to stimulate the release of vasopressin by activating central angiotensin II-containing pathways, probably via the circumventricular organs (Parry et al., 1994). Relaxin appears to be transported into the cerebrospinal fluid to exert its effects on RLN H2 receptors (RXFP1) in brain regions devoid of a blood-brain barrier (Coldren et al., 2015). The prolonged heart rate increases in rats, following intravenous injection of bacterially-derived

RLN H2, may have been associated with the ketamine-xylazine anesthesia.

Sanford and Colby (1980) reported that this drug combination produces a prolonged anesthesia in rabbits within 10 minutes after injection. The anesthetics caused an initial drop in blood pressure by about 30%. At the same time, heart rate and the respiratory rate dropped 19% and 77%, respectively, also within 10 minutes post injection. Blood pressure returned to nearly normal level after 6 hours post initial anesthesia but no information was provided on the heart and respiratory rates.

Parry et al., (1998) compared the blood pressure and heart rate effects of

RLN in two strains of rats: normotensive Long-Evans and Brattleboro rats unable to synthesize bioactive vasopressin. After treatment with exogenous RLN, in Long-

Evans rats, there was an increase in blood pressure but there was no pressor response in Brattleboro rats. There were also differences in tachycardiac responses to RLN: in Long-Evans rats, there was a quick and sustained increase in heart rate that lasted for at least 15 minutes; in contrast, the beginning of RLN-

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induced tachycardia was delayed in Brattleboro rats and was attenuated compared with Long-Evans rats. The tachycardia response to RLN H2 could be three fold: there are RLN-binding sites in heart muscle and in vitro experiments confirmed that RLN treatment of cardiac tissue causes an increase in the rate of contractions

(Kakouris et al., 1992); the second possibility could be the indirect effect of RLN causing the release of vasopressin, which causes a pressor response and tachycardia (Geddes et al., 1995); the third possible mechanism could be by means of RLN-induced activation of the brainstem cardiovascular centres. It is likely that RLN could act through the circumventricular organs to activate the forebrain angiotensin II system (Geddes et al., 1994).

Relaxin has a role in cardio physiology (reviewed in Chapter 1.). Novartis

Corporation is currently sponsoring a multi-center stage 3 clinical trial on the use of relaxin H2 (RLX030, REASAN2™ or SERELAXIN™) in acute heart failure

(AHF). Initial application by Novartis to the American Federal Drug Administration

(FDA) was to obtain the status of a “breakthrough” rank treatment for RLN, which would allow for a speedy approval. Only 1161 patients were included in that study.

This application, however, was not successful as reported by both the Novartis and the FDA in May of 2014. Nevertheless, FDA suggested that Novartis expand on the “intriguing” initial data and advised the company to conduct a more extensive investigation. Accordingly, Novartis has currently 6300 patients enrolled in a more extensive, global program that follows a traditional drug approval process.

Recently, Novartis announced that in December 2017 the company will release the results of the current study.

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Based on the results presented in this Chapter, I would like to propose that the protein produced in this series of experiments is indeed a bacterially-produced, recombinant form of RLN H2. After the work for this chapter was already completed, Cimini et al., (2017) reported a method for the production and purification of human native and histidine-tagged forms of pro-RLN H2 using methylotrophic yeast Pichia pastoris. The authors reported that the product was biologically active and the amounts of harvested recombinant pro-RLN H2 were economically justified. The current method using bacterial system is easier to scale up without a need for specialized fermenters, typically required for the yeast cultivation. There is also no risk that RLN H2 produced in E. coli would become post-translationally glycosylated. The post-translational addition of sugar motifs to

RLN H2 could possibly complicate the purification process and could also potentially affect biological activity, function, antigenicity, and clearance from circulation.

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CHAPTER 3

EFFECTS OF RELAXIN H2 ON DIFFERENTIATION OF MURINE CARCINOEMBRYONIC STEM CELLS INTO CARDIOMYOCYTES

Introduction

It is well recognized that during embryonic development the heart is the first organ to become fully functional. The building blocks of a heart are muscles called cardiomyocytes, which upon maturation begin to contract rhythmically and continue to beat throughout the life of an organism. Based on the work using transgenic animals and various model cell lines, researchers have been able to establish that there are a variety of genes involved in cardiac myocyte differentiation (Robbins et al, 1990). Most notably, these genes include transcription factors such as GATA binding protein 4 (GATA4), myocyte enhancer factor 2C (Mef2c), a Nk2 transcription factor related, locus 5 (Nkx2.5), MEF2c, dHAND and T-box families (Yamaoka et al., 2017). In transgenic mice, inactivation of most of these genes produces profound abnormalities in cardiac morphogenesis. For example, there were numerous myogenic and morphogenic defects in the heart tubes of murine embryos lacking the homeobox gene Nkx2.5

(Lyons et al. 1995). The right ventricle was not developed in mice deficient in

MEF2c and dHAND genes (Lin et al., 1997). GATA4 deficiency resulted in failure to fuse into a heart tube of the bilaterally symmetric cardiogenic areas (Kuo et al,

1997; Molkentin et al, 1997). Each of the cited example genes is important for heart formation, but none of them is absolutely critical for the cardiac myocyte

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differentiation. Although there were noticeable differences in gene expression patterns, cardiac myocytes still developed. It would be reasonable to suggest then, that even though there was a loss of certain function in the knockout animals, there could be novel genes that might play important roles in cardiac myocyte differentiation.

There are limited numbers of model cell culture systems in which to study cardiac myocyte differentiation. Primary cultures of heart muscle cells and transformed cardiac myocyte cell lines are of limited use simply because both have already undergone terminal differentiation. Embryonic stem cells (Robbins et al,

1990) and bone marrow stromal cells (Makino et al, 1999) can be induced to differentiate into cardiac myocytes in vitro. However, both kinds of cells require feeder cells to grow on. The resulting populations of cells include undifferentiated cells, cardiac myocytes and other kinds of cells (Yamaoka et al., 2017).

Consequently, this is not an optimal system. Another option is to use P19 cells that are a euploid, multipotent murine cell line derived from an embryonal carcinoma as described by McBurney et al (1982). Aggregates of these cells differentiate into neurons and glial cells after treatment with retinoic acid. When the P19 cell aggregates are treated with dimethyl sulfoxide (DMSO) cardiac and skeletal myocyte populations arise. Similar to the embryonic stem cells, differentiation of

P19 cells into cardiac myocytes is inefficient and formation of cellular aggregates is required. It has been also reported that the aggregation itself could induce endoderm as a co-stimulant (Smith et al, 1987).

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In 1996 a cell line named P19CL6 (derived from P19 cells) was developed following the long-term culture of clones that were selected for their ability to contract after treatment with DMSO without the need for aggregation (Habara-

Ohkubo, 1996). P19CL6 cells treated with DMSO differentiate almost exclusively into cardiac myocytes spontaneously beating after 10 days in culture. More importantly, there was no evidence of skeletal myogenesis because transcripts encoding MyoD have not been detected (Habara-Ohkubo, 1996). Kitsis et al,

(2002) carried out a comprehensive evaluation of P19CL6 cells treated with DMSO and reported that a minimum duration of stimulation was 4 days. In addition, it was shown in the same report that the differentiated P19CL6 were indeed cardiac myocytes as assessed by the expression of cell type-specific proteins and electrophysiological characteristics.

Since there is limited published information about the effects of RLN on differentiation, the metabolic state of a cell undergoing differentiation, cell cycle, cellular development, and differentiation were assessed using the mitochondria- specific dye Rhodamine 123. The metabolic state of the cell, cell cycle, cellular development, and differentiation are most frequently measured by assessing the activity of mitochondria (Lincoln et al., 1980). In addition, changes in mitochondrial function were reported as contributing factor in human myocardial infarctions and cardiomyopathies (Murphy et al., 2016). The laser fluorescent dye, rhodamine 123, has been reported as an excellent compound for localization of mitochondria in living cells (Lincoln et al., 1980). Rhodamine 123 is highly specific to mitochondria because it does not stain the plasma membrane, nuclear envelope, lysosomes,

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endoplasmic reticulum or the Golgi complex. At concentrations below 10 µg/mL, there is no dye-associated toxicity and the growth rate of cells treated with

Rhodamine 123 does not differ from that of cells grown without the dye.

Furthermore, at an excitation wavelength of 485 nm, rhodamine 123 emits a bright green fluorescence at a wavelength of 529 nm. Rhodamine also remains in mitochondria for several days (Summerhayes et al., 1982).

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Rationale

Development of the mammalian heart is a complex process. Morphological stages are well established but the pathways regulating differentiation of cardiac myocytes from precursor cells are less well understood. Murine embryonic carcinoma stem cells P19CL6 differentiate into rhythmically contracting cardiac myocytes after treatment with DMSO in vitro. Although not a perfect copy of spontaneous development and differentiation, this approach has been used as a model system to identify potential components in the differentiation pathways

(Yamaoka et al., 2017). Since there is limited published information about the effects of RLN on differentiation, experiments were performed to assess whether or not P19CL6 cells would be a suitable model to study the effects of RLN H2 on the differentiation of these cells. Therefore, the current study was undertaken to investigate the effects of serelaxin and compared to laboratory produced RLN H2 in the differentiation of P19CL6 cells into cardiomyocytes. Further, complementary

DNA (cDNA) microarray analyses were done to systematically evaluate if RLN H2 had any effects at the genomic level on the differentiation of P19CL6 cells. A total of 22,400 known murine genes and expressed sequence tags (ESTs) were investigated.

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Materials and methods

P19CL6 cell culture

P19CL6 cells were obtained from Dr. Richard N. Kitsis (Albert Einstein

College of Medicine, Bronx, New York, NY, USA). The cells were maintained in a culture growth medium consisting of α-modified minimum essential medium

(αMEM), supplemented with 10% fetal bovine serum (FBS), 100U/mL penicillin and 100µg/mL streptomycin (all supplies were purchased from Invitrogen,

Burlington, ON, Canada). For the induction of differentiation, 1% DMSO (Sigma-

Aldrich, Oakville, ON, Canada) was added to the growth medium, subsequently referred to as differentiation medium for the control groups. Bacterially derived

RLN H2 was compared with recombinant human serelaxin, which was a gift to Dr.

A. Summerlee from Dr. Elaine Unemori, (formerly of Connetics Inc., Paolo Alto,

CA, USA). Tissue culture grade flasks and/or dishes and all other consumable plastic ware were purchased from Sarstedt (Montreal, PQ, Canada). Cell cultivation was carried out in a humidified incubator set at 37°C. The incubator atmosphere was maintained with a saturated mix of 90% air and 10% CO2.

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Experimental protocol

P19CL6 cells were grown in T–25 flasks containing 5 mL of growth medium until 75-80% confluence was achieved. At this point, the cultures were passaged as follows: used medium was discarded and the cell monolayer was washed twice with PBS. After removal of the second PBS wash, 2 mL of 0.05% trypsin solution in PBS was added just to cover the cell for a brief period of time and then the solution was discarded. The flasks were then placed in the incubator for 5 to 10 minutes. To neutralize trypsin, 2 mL of fresh growth medium was added to the flask and the cells were dissociated by pipetting in- and out several times. Using a standard hemocytometer cell count was performed. New flasks containing 5 mL of growth medium were seeded with 3.5×105 cells from the previous passage and grown again until 75–80 % confluence was reached. The cells were used for differentiation experiments after the third passage to exclude the risk of carrying over any DMSO used for cryopreservation of stock cultures.

Two sets of cultures were set up to carry out the differentiation protocol as follows:

1. Control groups: in T–25 flasks containing 5 mL of differentiation medium for

the DMSO treatment and

2. Relaxin treatment groups: in T–25 flasks filled with 5 mL of growth medium

supplemented with RLN (16.7 nM H2 RLN, bacterially-produced or

serelaxin obtained from Connetics Corporation, respectively).

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The media supplemented with treatments (DMSO control, and experimental serelaxin and bench-top RLN H2, respectively) were changed every two days throughout the experiments. At pre-set time points: day 4, day 8 and day 12, RNA extractions were carried out.

Morphological assessment of living cells

On day 8 the cell medium was supplemented with Rhodamine 123 (Sigma-

Aldrich) at a concentration of 1 µg/mL. Detailed information is provided in the introductory section.

RNA extraction and quantification

Pre-assigned cultures from both treatment groups were selected at days two, six and ten (counting from day two, at which time the cells reached nearly

100% confluence; it should be understood that the total times from start to harvest should count as days 4, 8 and 12, respectively) for RNA extraction using GE

Illustra™ RNA-spin Mini Kit (GE Health, Canada) according to the manufacturer’s protocol. The quality, quantity, and purity of all RNA samples were assessed using a Bioanalyzer apparatus (Agilent Technologies, Genomics Facility at the

Advanced Analysis Centre; University of Guelph). All RNA samples were stored in a freezer at –80°C until next stage of the experiment.

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Microarray Protocol

Complementary DNA (cDNA) microarray analyses are expedient and broad-ranging techniques to investigate the levels of expression for thousands of genes at the same time. There are no reported investigations on the effects of RLN on the differentiation of stem cells, and no RLN-related microarrays data are available in the literature to date. Therefore, the purpose of the current work was to explore the possible effects of RLN on the differentiation of stem cells and to determine whether or not the expression of genes were affected by the RLN treatment.

The microarray slides pre-printed with 22.4 k ESTs (expressed sequence tags) were purchased from the University Health Network Microarray Centre (UHN,

Toronto, ON, Canada). The RNA samples were labeled according to the University

Health Network Microarray Centre’s (UHN, Toronto, ON, Canada) standard indirect (amino allyl) labeling protocol, adapted from Agilent Technologies (Santa

Clara, CA, USA). Full-length protocols for preparation of microarray slides and standard procedures are presently available directly from the Agilent’s website at http://www.agilent.com/cs/library/usermanuals/Public/G4140-

90050_GeneExpression_TwoColor_6.9.pdf

Ten micrograms of total RNA were labeled with Cyanine dyes (Cy5 and

Cy3, Amersham Biosciences) and hybridized to Mouse 22.4 k arrays. The procedure was performed in a hybridization box placed in an incubator set to 37°C using DIG Easy Hyb hybridization solution (Sigma-Aldrich). Finished slides were

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scanned on the Agilent scanner G2565BA and quantified using Array Vision v 8.0

(Imaging Research Inc., USA). After normalization, the data were first filtered to eliminate spots that were flagged by Gene Traffic™, (a microarray data management program, Iobion Informatics, USA). The software flags spots for certain reasons but the majority of the seen flags are most likely due to the signal being near background levels. All genes that were not flagged were then analyzed by SAM (Significance Analysis of Microarrays, version 2.20, Stanford University).

SAM is a statistical program for identifying significantly changed genes in a set of microarray hybridizations. For more information on how the SAM program works, the reader is referred to the website at http://www-stat.stanford.edu/~tibs/SAM/.

The data were subjected to one–class unpaired SAM analysis. Next, the data were filtered to obtain a list of genes that did not contain any flagged spots and included only genes with absolute values of log2 ratio ≥1 in at least 2 observations. The list of genes that were found significantly changed by SAM program was uploaded into GeneTrafic™. This list was analyzed by a cluster view, using both k-means and hierarchical clustering methods.

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Results

Morphological results

The first objective was to compare whether or not relaxin would affect the time period required for the cells to start contracting. Young et al., (2004) reported that treating the P19CL6 cells with dimethyl sulfoxide (DMSO) for 6 days caused spontaneous contractions beginning at day 8. The control group of cells on the same treatment in present experiments also begun spontaneously contracting at day 8. Treatment of experimental groups with bench-top RLN H2 or serelaxin alone delayed initiation of contraction by 4 days, which is day 12 of culturing. Live video recordings of contracting cells were made as visual evidence of contractile ability.

Rhodamine 123 is highly specific to mitochondria because it does not stain the plasma membrane, nuclear envelope, lysosomes, endoplasmic reticulum or the Golgi complex. At concentrations below 10 µg/mL, there is no dye-associated toxicity and the growth rate of cells treated with Rhodamine 123 does not differ from that of cells grown without the dye. Furthermore, at an excitation wavelength of 485 nm, rhodamine 123 emits a bright green fluorescence at a wavelength of

529 nm. It also remains in mitochondria for several days (Summerhayes et al.,

1982). Following the above protocol and reported by Paquin et al., (2002) for labeling of living cells, the cultures of contracting cells were labeled with

Rhodamine 123. Pictures were taken at low, and high magnifications, Figure 18 and Figure 19, respectively.

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Figure 18. Ten times magnification of a monolayer of differentiated P19CL6 cells stained with Rhodamine 123 dye. The red arrows point to a contracting complex of cells illustrated at high magnification in Figure 19, below.

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Figure 19. One thousand times magnification of a contracting focal point of

P19CL6 cells treated with bench-top RLN H2 and mitochondrial-specific

Rhodamine 123 laser fluorescent dye. The intensely green accumulation of staining of cells forms a circular pattern around a focal ring indicated by three red arrows. This image and conclusion that the green stained layers of the cells were mitochondria-rich cardiomyocytes are comparable with similar results that were reported by Paquin et al., (2002).

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Microarray analysis of gene expression profiles

The murine microarray slides used in the current experiments each contained 22,400 DNA probes coding for known genes and expressed sequence tags (EST). Highly purified RNA samples were labeled with cyanine fluorophores

(Cy3 – green and Cy5 – red) and hybridized to the DNA probes. The luminescence of DNA-RNA complexes was then measured and analyzed. The endpoint of these experiments were differences between gene expression levels of the matching

DMSO-treated control versus RLN experimental treatments. For instance: experimental day 4 set against control day 4, etc. In this comparison, groups of genes with changed expression profiles were identified and these are illustrated in

Figure 20:

1. Having increased levels of expression, above the X-axis,

2. Increased at day 4 (above X-axis) and then decreased levels at day 8

and 12 of expression (underneath the X-axis) and,

3. Decreased levels of expression (beneath the X-axis).

This was the standard procedure for each day 4, day 8 and day 12 experimental treatments with serelaxin (Connetics RLN H2) compared with controls. Three sets of 2 matching microarray results were generated.

Compiled in Table 15 are 125 gene expression profiles divided into specific groups. Due to the large number (3x125) of data points shown in Figure 20, the groups were further subdivided into smaller, easier to read graphs were created and these are listed below:

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 “Positive 1 group of genes” – this subgroup represents 14 genes that had

increased expression level in response to RLN H2 compared to DMSO

control (Figure 21),

 “Positive 2 group of genes” – additional subgroup of 13 genes that had

increased expression levels in response to RLN H2 compared to DMSO

control (Figure 22),

 “Positive to negative group of genes” – this subgroup of 18 genes

represents genes that showed increased expression levels at day 4 and,

then reversed to negative expression levels in response to RLN H2

compared to DMSO control (Figure 23),

 “Negative 1 group of genes” – this subgroup represents 20 genes that

had decreased expression levels in response to RLN H2 compared to

DMSO control (Figure 24),

 “Negative 2 group of genes” - additional subgroup of 21 genes that had

decreased expression levels in response to RLN H2 compared to DMSO

control (Figure 25),

 “Negative 3 group of genes” - additional subgroup of 19 genes that had

decreased expression levels in response to RLN H2 compared to DMSO

control (Figure 26),

 “Negative 4 group of genes” - additional subgroup of 20 genes that had

decreased expression levels in response to RLN H2 compared to DMSO

control (Figure 27).

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Two additional series of microarray experiments with the same treatment days were done using bacterially produced RLN H2 creating six sets of 2 corresponding microarray outcomes. Listed in Appendix 2 are functional descriptions of the genes with altered expression levels. The information was obtained from the Mouse Genomic Informatics, http://www.informatics.jax.org/ curated by the Jackson Laboratory.

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Figure 20. The graph showing cumulative changes in expression levels of all 125 affected genes. There is a large number of data points, therefore the results were divided into smaller groups according to their change folds and are presented on the following pages.

Changes in gene expression levels 5

Mm.318250 4 unknown 3 unknown Mm.1231 2 unknown unknown 1 unknown

0 unknown DAY4 VS CON1 DAY8 VS CON2 DAY12 VS CON3 Mm.239117 -1 unknown Mm.181959 -2 Mm.426616

Fold change in level of in changelevel expression Fold Mm.332522 -3 unknown -4 Mm.200362 Mm.209071 -5

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Table 15. Compilation of significantly changed fold expression of 125 genes.

Variation of expression Number of Known Unknown genes genes genes Positive group 1 14 12 2 Positive group 2 13 7 6 Quantity of positive 27 19 8 Positive to negative 18 6 12 Negative group 1 20 15 5 Negative group 2 21 15 6 Negative group 3 19 13 6 Negative group 4 20 17 3 Quantity of negative 80 60 20 Total number of changed 125 85 40 genes

Figure 21. Positive 1 group of genes. This subgroup represents 14 genes that had increased expression level in response to RLN H2 compared to DMSO control.

Positive 1 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Changeof expression 0 Mm Mm Mm Mm Mm unk Mm Mm Mm Mm Mm Mm unk Mm .282 .432 .379 .399 .326 now .133 .634 .396 .227 .530 .245 now .360 706 028 939 372 799 n 704 3 243 23 5 522 n 075 Day 4 VS CON1 0.94 1.37 1.31 1.24 1.39 1.26 1.52 0.97 0.97 1.07 1.12 0.73 1 1.31 Day 8 VS CON2 1.21 1.56 1.6 1.06 1.54 1.45 1.69 1.2 1.22 1.02 1.31 1.01 1.46 1.37 Day 12 VS CON3 1.21 1.08 1.17 0.63 1.1 1.03 1.19 1.14 1.01 0.89 1.23 1.05 0.92 1.13

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Figure 22. Positive 2 group of genes. An additional subgroup of 13 genes that had increased expression levels in response to RLN H2 compared to DMSO control.

Positive 2 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 Changeof expression 0 unk Mm unk unk Mm unk unk unk Mm Mm Mm Mm Mm now .170 now now .399 now now now .272 .221 .307 .338 .66 n 31 n n 829 n n n 045 269 45 32 Day 4 VS CON1 1.05 0.97 1.1 0.95 1.02 1.36 1.32 1.22 1.28 1.01 1.01 0.73 1.24 Day 8 VS CON2 1.11 1.18 1.16 1.25 1.05 1.49 1.52 1.45 1.34 1.09 1.15 1.11 1.47 Day 12 VS CON3 1.01 1.19 0.96 1.06 0.88 0.84 1.23 1.15 1.06 0.74 0.93 1.11 1.01

Figure 23. Positive to a negative group of genes. This subgroup of 18 genes represents genes that showed increased expression levels at day 4 and, then reversed to negative expression levels in response to RLN H2 compared to DMSO control.

Positive to Negative 5 4 3 2 1 0 -1 -2 -3 -4 Changeof expression Mm Mm Mm Mm Mm unk unk Mm unk unk unk unk unk unk unk unk unk unk .13 .12 .33 .42 .15 no no .57 no no no no no no no no no no 005 645 564 330 865 wn wn 415 wn wn wn wn wn wn wn wn wn wn 4 0 1 2 0 Day 4 VS CON1 4.04 3.95 3.79 3.04 4.13 1.15 3.26 3.33 1.56 3.47 1.64 3.9 3.25 3.94 3.33 4.04 3.03 2.4 Day 8 VS CON2 -1.8 -1.6 -1.4 -1.1 -1.6 -0.7 -1.6 -1.2 -0.4 -1.5 -0.7 -1.4 -1.1 -1.5 -1.1 -1 -1.3 -0.9 Day 12 VS CON3 -3 -2.9 -2.5 -1.7 -2.8 -1.1 -2.1 -1.7 -1.3 -2.1 -1.1 -2.1 -1.9 -2.7 -1.3 -2.9 -1.7 -1.4

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Figure 24. Negative 1 group of genes. This subgroup represents 20 genes that had decreased expression levels in response to RLN H2 compared to DMSO control.

Negative 1 0 -0.5 -1 -1.5 -2 -2.5 -3 -3.5 -4

Changeof expression un M M un un M M un M M M M M M M un M M M M kn m. m. kn kn m. m. kn m. m. m. m. m. m. m. kn m. m. m. m. ow 301 213 ow ow 180 236 ow 123 396 332 153 405 123 410 ow 756 200 247 980 n 53 025 n n 032 067 n 7 053 720 272 46 1 445 n 2 362 480 Day 4 VS CON1 -2 -1 -1 -4 -1 -3 -2 -1 -1 -3 -2 -1 -1 -1 -2 -3 -1 -1 -1 -2 Day 8 VS CON2 -3 -1 -1 -4 -1 -2 -2 -1 -2 -3 -1 -1 -1 -1 -2 -2 -1 -1 -1 -2 Day 12 VS CON3 -3 -0 -1 -3 -1 -3 -1 -0 -1 -3 -0 -0 -1 -1 -1 -2 -1 -2 -0 -2

Figure 25. Negative 2 group of genes. An additional subgroup of 21 genes that had decreased expression levels in response to RLN H2 compared to DMSO control.

Negative 2 0 -0.5 -1 -1.5 -2 -2.5 -3 -3.5 M M M M M M M M M M M M M un M un un un un un M m. m. m. m. m. m. m. m. m. m. m. m. m.

Changeof expression kn m. kn kn kn kn kn m. 34 32 40 38 33 42 40 23 33 31 33 27 34 ow 38 ow ow ow ow ow 11 09 83 74 24 01 66 57 91 25 82 70 31 87 n 62 n n n n n 93 11 60 15 1 16 16 61 17 22 50 74 95 1 Day 4 VS CON1 -1 -1 -1 -1 -1 -1 -1 -1 -3 -2 -1 -1 -1 -0 -1 -1 -1 -2 -1 -1 -1 Day 8 VS CON2 -1 -1 -1 -1 -1 -1 -1 -1 -3 -2 -1 -1 -1 -1 -1 -1 -1 -2 -2 -1 -1 Day 12 VS CON3 -0 -1 -1 -1 -1 -0 -0 -0 -3 -2 -0 -0 -1 -2 -0 -0 -1 -1 -1 -1 -0

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Figure 26. Negative 3 group of genes. An additional subgroup of 19 genes that had decreased expression levels in response to RLN H2 compared to DMSO control.

Negative 3 0 -0.5 -1 -1.5 -2 -2.5 -3 -3.5 -4 Changeof expression M M M M M M M M M M M unk unk M unk unk M unk unk m.1 m.2 m.2 m.2 m.2 m.4 m.2 m.1 m.2 m.3 m.2 no no m.4 no no m.4 no no 827 504 777 903 495 004 796 825 914 918 800 wn wn 266 wn wn 159 wn wn 85 38 92 20 55 20 90 74 42 49 38 Day 4 VS CON1 -1.9 -1 -1.3 -1.4 -1.1 -3.3 -1 -1.4 -1.2 -1.6 -1.1 -1.3 -1.1 -0.5 -1.2 -1 -1.1 -3.3 -1.2 Day 8 VS CON2 -2 -1 -1.3 -1.4 -1.3 -3.3 -1.1 -1.4 -1.2 -1.8 -1.2 -0.9 -0.8 -1.2 -1.2 -1.1 -1.4 -3.6 -1.4 Day 12 VS CON3 -1.4 -1 -1.7 -1.4 -0.7 -3.2 -0.6 -1.5 -0.4 -1.7 -0.7 -1.1 -1.2 -1 -0.3 -1 -0.5 -3 -1.4

Figure 27. Negative 4 group of genes. An additional subgroup of 20 genes that had decreased expression levels in response to RLN H2 compared to DMSO control.

Negative 4 0 -0.5 -1 -1.5 -2 -2.5 -3 -3.5 -4 -4.5 M M M M M M M M M M M M M M M M M unk unk unk Changeof expression m. m. m. m. m. m. m. m. m. m. m. m. m. m. m. m. m. no no no 209 244 267 280 204 296 391 181 872 394 265 415 567 384 281 158 317 wn wn wn 071 825 998 225 4 150 808 959 46 798 40 865 69 634 744 143 947 Day 4 VS CON1 -1.1 -1 -1 -2.8 -1.2 -0.8 -0.8 -4.2 -1.2 -1.5 -1.1 -1.7 -1 -1.2 -1.6 -1.3 -1 -1.4 -1.9 -1.5 Day 8 VS CON2 -1 -1 -1.1 -2.9 -1.5 -1 -1.2 -4 -1.4 -1.3 -1.2 -1.6 -1.1 -1 -1.7 -0.7 -1.1 -1.4 -1.4 -1.5 Day 12 VS CON3 -0.4 -0.8 -0.8 -2.6 -1.7 -1.1 -1.2 -3.8 -1.2 -0.1 -1.2 -1.4 -0.8 -1.2 -1.7 -1.4 -1 -0.2 -1.2 -1.5

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Discussion

Standard methodology to examine the differentiation of stem cells into cardiomyocytes is to use DMSO-induced differentiation of P19CL6 carcinoembryonic stem cells (Yamaoka et al., 2017). The current study compared

DMSO-induced with RLN H2-induced differentiation of P19CL6 cells into cardiomyocytes.

When P19CL6 cells are allowed to grow in culture with no inducers, they do not differentiate (Kitsis et al., 2002, Yamaoka et al., 2017). In the present study, untreated P19CL6 cells remained viable for up to 20 days but no spontaneous contractions were detected. Induction of P19CL6 cells with 1% DMSO causes differentiation into mitochondria-rich cardiomyocytes, detectable with a laser fluorescent dye Rhodamine 123, and spontaneous contractions of cell layers by day 8 in culture (Kitsis et al., 2002, Yamaoka et al., 2017). Using the same indicators of the endpoint, RLN H2 (16.7nM) also induced differentiation of P19CL6 cells into cardiomyocytes but the process was delayed by 4 days compared with standard DMSO treatment. Excitation-contraction coupling in cardiomyocytes is controlled by a calcium-dependent process in which calcium levels are increased intracellularly (Han et al., 1993) and this results in contraction of actin and myosin bands. The delay in contraction of cardiomyocytes following RLN H2 treatment could be due to the delayed expression of several structural collagens as well as decreased expression of myosin light and heavy chains required for myocyte functioning. This could be separate or in addition to a decrease in the expression

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of genes coding for calcium ions (Ca+2) transport proteins which could also delay the development of the impulse generating and conductive properties of the differentiating cells.

Both commercially obtained standard, serelaxin (Connetics Corp.) and the bacterially-derived protein at a concentration of 16.7 nM equally affected the differentiation of P19CL6 carcinoembryonic stem cells in separate series of in vitro experiments. Relaxin delayed by four days the initiation of spontaneous contractions of cardiomyocytes, compared with DMSO control treatment. This morpho-functional effect was assessed by using Rhodamine 123 laser fluorescent dye to determine that the observed contractions of differentiated cell layers were caused by the mitochondria-rich cardiac myocytes.

Using microarray analysis to detect changes in gene expression profiles, it was possible to show consistent responses by P19CL6 cells in culture to a challenge from RLN H2. Eighty genes were downregulated, 27 genes were upregulated, and 18 genes were both, initially switched on and after day 4 switched off during the remaining period of testing. The groups of genes that were affected support the proposal that RLN may affect cellular differentiation and these are described below. The fact that a large number of genes affected by RLN treatment were downregulated may appear to be counterintuitive. However, cardio-genesis per se is known to be negatively regulated by a variety of signals. For instance, during early development of a mammalian embryo, cardiac GATA and Wnt subfamilies of signaling factors are normally delayed, and experimentally switching them on prematurely caused heart defects (reviewed in Zaffran and Frasch, 2002).

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In addition, there are reports to suggest that the cardiac phenotype is not dominant

(Evans et al., 1994). For these reasons and because RLN H2 also delayed cardiac differentiation, it is possible to suggest that RLN may also be involved in the regulation of signaling in early cell differentiation.

Moreover, four procollagen coding genes were downregulated: type 1 - alpha 2, type 3 – alpha 1, type 4 – alpha 3, and type 5 – alpha 1, all of which are responsible for maintenance of stable extracellular matrix. This finding was not unexpected because of the role RLN plays in the maintenance of collagen balanced milieu (Samuel et al., 2016). As well as collagens, genes involved in cell growth were downregulated such as insulin-like growth factor 2 and insulin-like growth factor binding protein 5, both of which are involved in regulation of cell growth, proliferation, and organ morphogenesis.

Among the downregulated genes were also: myosin light 6, myelin, actin alpha 2 and actin beta that are a part of the heart contractile elements. Shaw et al., (2009) reported that treatment of murine cardiac myofilaments in vitro with RLN

H2, induced changes in protein kinase C (PKC) affecting activation of myofilaments. Both of these findings were consistent with the cardio-stimulatory effect. In addition, Shaw et al., (2009) proposed that RLN contributed to the inotropic response of actomyosin. (An actomyosin is complex of the proteins actin and myosin that are constituents of muscle tissue interacting with ATP to cause muscle contraction). In support of the findings reported by Shaw et al., (2009) is that an upregulated gene Gnb2l1, classified as a receptor for activated C kinase

1, was identified in the present study. This gene is one of the G-protein

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components that are activated in response to treatment with RLN, which in turn also contributes to the contractile response in the differentiated cardiomyocytes.

Also contributing to the contractile actions could be a downregulated Cyclin D1 identified here. There were also two negatively affected genes: Tropomyosin 1 α that participates in the control of the calcium-dependent muscle contraction mechanisms and Tropomyosin 2 β that affects regulation of vertebrate striated muscle contraction. It would be worth mentioning that there was identified one downregulated gene named G-protein coupled receptor 177.

The next two groups of RLN affected genes were 27 upregulated genes and

18 that were upregulated up to day 4 and then were downregulated for the remainder of the treatment time. After analysis of these two groups of genes, it was noticed that the vast majority of these were coding for ribosomal proteins suggesting that they were involved in the processing of proteins. Detailed descriptions of these genes are provided in tables in Appendix 2, Tables A2-3, A2-

4, and A2-5. A search of the Mouse Genome Database provided descriptions for the known genes as they pertain to the mouse genome (without particular references). When searches of PubMed for specific genes from these two groups were combined with RLN, no positive results returned. This was disappointing at first, but later it became apparent that this negative outcome opens new opportunities for RLN research. Descriptions of large-scale alterations in gene expression profiles are not intended for the provision of detailed mechanical and/or functional information. Microarrays are by design programmed to provide a rationale and basic data for future hypothesis-driven studies (Peng et al., 2002).

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The limitations of microarrays are that there are no statistically acceptable methods for making a reliable comparison between representative experiments

(Jaluria et al., 2007). Most common evaluation simply relies on making a comparison between control and experimental data at a given point in time.

Nevertheless, the data shows that the results obtained from microarray experiments using serelaxin were in effect similar to both of the repeats using RLN produced locally in the laboratory. Miller et al., (2010) created a particular algorithm for comparison of specific data, but it is clear that algorithm for this kind of analysis must be customized for each set of experiments. It would be reasonable to assume that modifications or improvements to the current experimental design and availability of customized analytical tools would provide more detailed information.

The work described in this thesis on microarray experiments using stem cells as a model to study the effects of RLN on cell differentiation is a novelty and it should be explored further.

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GENERAL DISCUSSION

The rationale for the work reported in the present thesis was two-fold:

1. To demonstrate that it was possible to make recombinant relaxin that is

sufficiently stable, pure, easy to produce and affordable for use in scientific

experiments, and

2. To examine whether or not human relaxin affects differentiation of stem

cells in vitro.

The driving force behind this work was to create an affordable, considered reliable, and readily available source of RLN H2 at a time when commercially available RLN H2 is already expensive to purchase and, due to expected developments in the clinical use of RLN, likely to become more expensive. After a great deal of trial and error, it was possible to show that recombinant human RLN can be produced using specifically selected E.coli bacterial system and purpose- modified plasmids. The polypeptide was directed for expression in the periplasmic space where there are relatively few other peptides expressed, so it was easier to extract and purify the recombinant RLN. More notably, the periplasmic space in bacteria contains the essential cellular machinery for inciting the formation of disulfide bonds, which are an essential component of satisfactory folding for the

RLN H2 polypeptide.

The second objective of the current work was to explore the impact of RLN on the differentiation of carcinoembryonic stem cells and to investigate some of the likely cellular mechanisms of its actions. Bigazzi and Bigazzi (2005) submitted

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a patent application, which they were granted, for the idea that RLN might affect differentiation of primitive germ (stem) cells. The intriguing fact was that there were no data in the patent application to suggest that RLN actually affected differentiation and there has been no data published since to either support or refute this hypothesis.

The challenges for production of bench-top RLN

Cell-based production of research or therapeutic-quality recombinant proteins requires an appropriate host organism. It also needs to have efficient processing capabilities to manufacture not only the protein of interest but also to process post-translational modifications that occur normally in the production of the native protein. This is particularly important for the expression of multipart proteins with folded configurations and a number of disulfide bonds (Graslund et al., 2008). A number of challenges were encountered in attempts to produce bench-top RLN H2. These included:

Production of RLN is notoriously difficult mainly due to its complex structure comprising two peptides (chains A and B) linked together by two disulfide bonds and an additional disulfide bond within the A chain (reviewed in Eigenbrot et al.,

1991). In a living mammalian organism, the RLN mRNA is translated as a single chain precursor called pre-pro-RLN consisting of a signal peptide and three domains: B, C, and A (Hudson et al., 1984). The signal peptide is cleaved off during insertion into the endoplasmic reticulum to generate pro-RLN. The interconnecting

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C domain is next removed by endopeptidases present within the endoplasmic reticulum, to generate the mature form of RLN. Finally, RLN and free C-peptide are packaged separately in the Golgi into secretory granules, which accumulate in the cytoplasm (Bullesbach and Schwabe, 1994).

Over the years, problems have been reported in trying to synthesize properly folded and disulfide-linked RLN. One of the first attempts to produce recombinant RLN was reported by Stewart et al., (1983) who used a single chain construct of porcine pro-RLN expressed in bacteria E. coli. The results were disappointing because the pro-RLN was deposited in the insoluble bacterial cytoplasmic inclusion bodies. As a consequence, expensive, time-consuming and inefficient methodologies were used to extract minute amounts of pro-RLN. No detailed information was provided on the quantities, folding and purity of the final product. In the next development, the RLN B- and A-chains were expressed separately in two different bacterial culture vessels (Canova-Davis et al., 1991).

Following laborious extraction from the inclusion bodies, the A and B peptides were combined together in a highly oxidizing environment for the disulfide bond formation. In comparison with the method of Stewart et al., (1983) there was no improvement in the amount of the obtained recombinant protein.

Bullesbach and Schwabe (1991) pioneered the use of chemical synthesis for the RLN B- and A-chain peptides separately and then combined the chains in an oxidizing milieu for disulfide bond formation. Again, the amounts of protein obtained were very small and when production costs were added it proved to be prohibitively costly.

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There was one report on the expression of biologically active porcine pro-

RLN in the milk of transgenic mice. It was a part of a doctoral dissertation of Mr.

Myles Lindsay (2004) at Virginia Tech, Chemical Engineering Department -

Blacksburg, VA, USA. However, based on my reading of that report, the researcher encountered numerous technical difficulties with collecting milk from mice.

Because of that, it is likely that this approach has been discontinued. In addition, the work has not been published in a refereed journal.

In mammalian cells under normal physiological conditions proteins spontaneously fold into their native conformations and only correctly folded proteins are secreted. Any miss-folded or degraded proteins are discarded by cells and deposited in the cytoplasm for recycling. The best illustration of this process is the transition of a four-domain pre-pro-RLN (signal peptide – B-C-A – chains) into the mature B-A form of RLN. The signal and the C-peptides are enzymatically cleaved off during processing without any external templates. In addition, there is one group of enzymes present called protein disulfide isomerases (PDI) that are responsible for the creation of disulfide bonds. In bacteria, however, there is no

PDI present. Instead, in the periplasmic space, there are enzymes called foldases that are the equivalent of the mammalian PDIs (Ostenmeier et al., 1996). In the bacterial cytoplasm, disulfide bonds will not survive due to the highly reducing environment. In contrast, in the periplasm, the milieu is highly oxidative, hence supportive for foldases to create disulfide bridges.

There is a debate within the RLN research community as to whether or not pro-RLN is as active as its mature form (reviewed by Sherwood, 2004). Dschetzig

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et al., (2001) isolated an 18 kDa pro-RLN from human donors with no symptoms of congestive heart failure. In the same study, in patients suffering congestive heart failure, the levels of RLN were significantly elevated, suggesting that the pro-RLN was endogenously processed to RLN and the hormone was released into circulation in response to signals of heart failure.

There is another aspect that may perhaps be considered; RLN self- aggregation, what might be perceived by some investigators as pro-RLN. In fact,

Shire et al., (1991) reported that recombinant human RLN was prone to form dimers (two monomers of RLN). Eigenbrot et al., (1991) crystallized human RLN as a dimer and discovered that the part of the dimer interface was also involved in receptor binding. More recently, Nair et al., (2015) reported that synthetic RLN H2 covalently linked to form dimeric molecules, and retained activity equal to monomeric RLN towards the RXRP1 receptor. Moreover, the dimer RLN H2 was shown to have improved the in vitro serum stability, which could be beneficial for the creation of longer-acting forms of RLN H2.

Challenges of protein purification

In the discussed work, during protein purification stages, bacteria were processed according to the method of Neu and Heppel (1965). To extract the periplasmic proteins, 20% sucrose solution supplemented with sodium EDTA (0.5 mM, to eliminate proteases) was used. All procedures were done “on ice”, i.e., creating temperatures that were at 40C or below. After a number of experiments

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using various buffers, including Tris-HCL-NaCl-EDTA and PBS, which proved to be not effective, distilled sterile water was showed to be the best solute.

The 6His-MBP-RLN H2 fusion protein was next digested with Factor Xa and carboxypeptidases. Following purification on His-Bind columns, pure RLN was harvested. Due to the elution buffer’s high salt content, RLN solution was filtered on a 6 kDa cut-off size desalting columns. Instead of lyophilizing, I chose to use the acetone precipitation method, which minimized loss of the product.

(Furthermore, acetone is easily miscible with water. Although there is no scientific evidence to support the assumption, it could be possible that traces of acetone used for precipitation of RLN remained in the storage tube, or were bound to the polypeptide making it easier to reconstitute the protein).

The sodium acetate buffer used to rehydrate acetone precipitated RLN H2 was set to pH 5.5. Relaxin stored in this buffer at 40 C remained stable for up to 3 months, as assessed on SDS-PAGE protein gels, in in vitro and in vivo experiments. Acetone precipitated RLN was stored at -200 C and remained stable for up to 9 months with no signs of degradation. The “Toxi Sensor” Endotoxin

Assay Kit (Fisher Scientific, Nepean, ON, Canada) was used and there were no endotoxins detected in any of the experimental batches of RLN tested. For comparison, commercially available RLN (R&D Systems, Phoenix

Pharmaceuticals, and Sigma-Aldrich) is guaranteed for 12 months in lyophilized form, packaged as shipped (nitrogen filled vials) and stored at -20 to -700 C. After reconstitution, one month at 2 to 80 C and up to 3 months at -20 to -700 C.

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Production system design

As previously described in the literature review section, there are many systems available for the production of recombinant proteins. The decision-making process took into consideration the subject of this project that is RLN H2. Since the bacterial systems required no major investment of funds to purchase equipment and appeared to be easy to learn, no other systems were tested.

Summary of technical considerations

As expected, the work to develop a system that could produce recombinant

RLN H2 in the laboratory was time-consuming, frustrating and required significant repetition, trial and error, and strategy adjustments. There were a number of failed attempts to produce recombinant RLN in any form, or in quantities that can be detected, or in forms that are stable and biologically active. In the initial series of experiments pre-pro-RLN DNA fragment was cloned into the maltose binding protein based plasmid pMal-p2x directed for periplasmic expression in the supplier’s (New England Bio Laboratories) recommended TB1 strain of E. coli. This was not successful. Two additional strains of bacteria were used but there was no improvement in the results.

Maltose binding protein fusion was one of the first systems developed for expression of recombinant proteins in bacteria (Guan et al., 1987). The method uses a hybrid tac promoter that requires IPTG stimulation to initiate translation,

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leading to expression of the fusion protein. The results reported in this thesis,

Chapter 2, show that after addition of IPTG to the media, the density of cells decreased, indicating that the compound was possibly killing the bacteria and as a result, no fusion protein could be obtained. In addition, the recommended strain

TB1 of E. coli is not a specialized strain suitable for expression of proteins as complex as RLN. The bacteria have wild-type proteases that could possibly digest expressed proteins.

One more drawback with this system is the limitation of Luria-Bertani media.

The exponential growth of bacteria is arrested after reaching an OD600 = 0.3 due to the lack of utilizable carbon source (Sezonov et al., 2007). Poor amylose affinity binding purification was the final setback of the MBP fusion system. Cells grown in

LB media have substantial amounts of an amylase enzyme that interferes with binding of MBP fusion protein to amylose resin. Amylase enzyme presumably cuts the fusion of the column prematurely releasing maltose into eluate and fused protein gets lost in the process (Silhavy et al., 1975).

A novel design strategy that worked

At the end of my studies, it was possible to develop a system for production of RLN using the following approach:

Maltose binding protein itself has been shown to be one of the most useful tags enhancing the solubility of the expressed proteins (Maina et al., 1988). The

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solubility of RLN is critical for evaluation of its biological activity. As was discussed in Appendix 1, MBP-RLN fusion by itself did not yield any form of the protein. An alternative scheme was created using plasmid pET28a+. Maltose binding protein sequence was cloned into the expression cassette following 6-His fragment on one site and RLN B- and A-chains on the C-terminal of the MBP. The relaxin and MBP were joined by Factor Xa cleavage site to separate RLN during the purification process.

In mammalian cells, RLN B-chain contains 33 amino acids. The last 4 amino acids are cleaved off during cellular processing. In the present design the last 4 amino acids: K, R, S and L, of naturally occurring RLN B-chain, have been substituted by 4 amino acids: I, E, G, and R, making up the Factor Xa cleavage site. The molecular weight of this redesigned B-chain was 29 Da less than the natural B-33 chain. In addition, this new 4 amino acid sequence acted as a mini C- peptide that is necessary for proper folding of RLN. It is interesting to note that the

BAYER Corporation recently applied for and was granted a patent on a similar construct design: that is RLN B-29, Factor Xa, and A-24. In the patent description, there is also a suggestion that digestion with Factor Xa is not required to achieve biologically active RLN (Wilmen et al., 2014).

Another improvement over the initial method was the choice of Rosetta strains of E. coli pre-transformed with the pRARE plasmid necessary for expression of 6 amino acids that are rarely used by bacteria but are critically important for expression of mammalian proteins. Relaxin contains 28.3% amino acid sequences that are not frequently used by bacteria.

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Finally, to avoid the limitation of Luria-Bertani media (Sezonov et al., 2007) an ONE-TB medium was used because there was no need to use IPTG and the medium contains no salt (Studier, 2005). It is a self-inducing medium delivering balanced amounts of glucose, to tighten the T7 promoter and well-balanced amount of lactose that induces expression. Dual Tag: MBP, to enhance solubility and to direct expression into the periplasmic space and 6-His to improve purification was beneficial for RLN H2 expression.

Bio-assay for RLN H2

The cyclic adenosine monophosphate (cAMP) assay measures the levels of cAMP in culture supernatant, cell lysate, and tissue homogenate samples. It is a G-protein-coupled receptor-triggered signaling cascade used in cell communication. In a cAMP-dependent pathway, the activated Gs alpha subunit of the GPCR receptor binds to and activates an enzyme called adenylyl cyclase, which in turn, catalyzes the conversion of ATP into the cyclic AMP. At the same time, separation from the beta and gamma subunits takes place. This path can also be triggered downstream by directly activating adenylyl cyclase or protein kinase A (PKA) and/ or PK13.

Increases in the concentration of the second messenger cAMP may lead to the activation of many cascades including:

1. Activation of transcription factors, which control gene expression

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2. Stimulation of an enzyme called proteinase A (PKA)

3. Initiation of cardiac muscle contraction leading to an increase in heart rate.

Compounds that activate cAMP pathway are:

1. Forskolin – a diterpene natural product that activates adenylyl cyclase

2. Cholera toxin – increase levels of cAMP

3. Caffeine - inhibits cAMP phosphodiesterase, which degrades cAMP

leading to increased levels of cAMP

4. IBMX, 3-isobutyl-1methylxanthine, nonselective, competitive

phosphodiesterase inhibitor causing rises in intracellular cAMP.

Cyclic AMP response to human RLN H2 in cultured human endometrial cells (NHE cells) primed with 1 μM of forskolin and 50 μM IBMX, was first reported by Fei et al., (1990). Recombinant human RLN H2 (10 ng/ml) caused a time- and dose-dependent accumulation of cAMP with the half-maximal amount of 3.56 ±

0.65 ng/ml and the standard curve ranging from 0.39 to 25 ng/ml. Other hormones such as insulin and insulin growth factor-1 did not have any effects on cAMP production. Fei et al., (1990) concluded that they developed a specific and high- throughput bioassay for RLN H2 and proposed that the general format of the assay could be adapted for other cell types in which the receptors for RLN were present.

Following the procedure of Fei et al., (1990), Parsell et al., (1996) reported that relaxin H2 bound with high affinity (Kd=102pM) to a specific receptor on THP-

1 cells (human monocytic cell line). There were approximately 275 sites binding to two types of cell surface proteins of molecular weight of over 100 and 200 kDa identified on each cell and coupling of relaxin initiated intracellular signaling

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pathways involving cAMP. The doses of RLN H2 ranged from 0.2 to 6.25 ng/ml with the maximal cAMP levels of approximately 380 to 420 fmol (the equivalent of

131.6 pg/ml). Although Parsell et al., (1996) used 80,000 THP-1 cells per assay well (6.67 times more cells than 12,000 NHE cells per assay well used by Fei et al., 1990), the amounts of cAMP reported by Parsell et al., (131.6x10-12 g/ml) were significantly (27 times) less, compared with the amounts of cAMP (3.56x10-9 g/ml) reported by Fei et al., (1990), (131.6 pg = 0.1316 ng). Insulin, insulin-like growth factors 1 and 2 and relaxin-like factor were unable to displace the binding of RLN to THP-1 cells, suggesting that a distinct receptor for RLN might exist on this monocyte/macrophage cell line. Parsell et al., (1996) proposed that the reported data may involve a novel role for RLN H2 in macrophage biology, however, no suggestion was made that the THP-1 cell system could be used as a universal bioassay for the RLN H2 activity.

There were countless papers published since then reporting on relaxin and cAMP in THP-1 cell assays and many researchers used different concentrations of RLN, forskolin, and IBMX. For instance, Nguyen et al., 2003 used as much as

500 ng of RLN H2 per 1 ml. There are also discrepancies in other reports. Samuel et al. (2004) reported that using 1000 ng of RLN H2 per 1 ml produced two positive and two negative results using the same kit. On the other hand, Halls and Cooper

(2010) reported cAMP response to treatments with sub-picomolar concentrations of RLN H2. In yet another aspect, forskolin, which is commonly used to prime cells prior to treatment with relaxin, was reported to activate the adenylyl cyclase

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pathway directly, without the need for guanine nucleotide regulatory protein (G- protein coupled receptor; Seamon et al., 1981 and Yuan et al., 2007).

In general, THP-1 cells are difficult to grow in a standing flask culture and specialized equipment like rolling bottles or top magnetic stirrers are required to produce high enough concentration of cells in culture that would be sufficient for running an assay. As an added complication, the cells are sold (Millipore-Sigma,

ATCC) in frozen vials containing DMSO as a cryoprotectant, which is commonly used as a THP-1 differentiation-causing agent. Therefore, it would be logical to assume that upon restarting of the culture from DMSO preserved THP-1 cells, the revitalized cells might already be a population of macrophages. Ivell et al., (2016) commented that the various time courses and diverse amounts of cAMP accumulation in THP-1 cells depend also on the cell’s passage number, which could be indicative of their differentiation status towards macrophage-like phenotype. Ivell et al., (2016) further suggested that some of the discrepancies could be the result of direct activation of adenylyl cyclase with the omission of G- protein coupled receptors. In addition, it was suggested that effort should be directed towards studying the RLN signaling pathways using cells that possess natural RLN receptor. The receptors for RLN have been identified in various tissues and cell types (literature review section) but currently, there are no published reports on the identification of RLN receptor in THP-1 cells.

There is a new trend developing in the RLN receptor research involving either transient or stable transfection of certain cell types with vectors carrying

RXFP receptors. For instance, HEK 293T cells transfected with an RXFP1 vector

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exhibited over 20,000 receptor sites per cell (Muda et al., 2005). However, Ivell et al., (2016) indicated that the interpretation of results from such constructs in cells that normally do not express the RLN receptor should be considered with caution.

In support of Ivell et al., (2016), Marshall et al., (2016) recently used human endometrial stromal cells known to have RLN receptors, treated with recombinant human RLN H2 (10 ng/ml), to investigate the angiogenesis- and remodeling- related genes in vitro and compared with in vivo treatments using relaxin-deficient mice. Microarray analysis identified 63 genes that were differentially expressed in human endometrial cells after relaxin treatment. However, the authors concluded that endogenous RLN does not play a major role in pre-implantation angiogenesis in the mouse uterus.

In addition to all previous assessments reported in the thesis on the identity and activity of the bacterially-produced RLN H2, namely:

1. Plasmid DNA sequencing and restriction enzyme digestion

2. Western dot blot analysis

3. Mass Spectrometry, and

4. In vivo experiments using rats,

Numerous attempts were made to run cyclic AMP assays using THP-1 cells. The first vial of cells obtained from Millipore-Sigma (Canada) contained cells that were at passage 19 and were only 45% viable upon arrival. No viable culture was achieved. Upon request, the second vial of THP-1 cells was received but was it

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exactly from the same batch as the first vial. The results were the same as previously and I was not able to establish a viable culture and to grow a sufficient number of cells to run a cyclic AMP assay. Due to time constraints, this line of investigation was not pursued further.

The challenge of RLN receptor

Another unresolved issue puzzling RLN researchers is the receptor for RLN

H2 known as RXFP1 (RLN Family Peptide Receptor 1). It belongs to a large group of highly complex class A, G-protein coupled receptors (GPCR). The drawing below (Figure 28) illustrates the most recent proposed concept of RLN H2-RXFP-

1 interaction and was adapted from Sethi et al., 2015. The legend description explains it.

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Figure 28. Shown in this figure is the most recent proposed concept of RLN H2-

RXFP-1 interaction (adapted from Sethi et al., 2015).

On the cell surface, the RXFR-1 receptor presents its N-terminal low-density lipoprotein class A (LDLa) segment that has been suggested to be essential for activation (Sethi et al., 2015). The LDLa module is connected to a leucine-rich repeat (LRR) by a 32-residue linker. Sethi et al., (2015) propose that RLN binds with high affinity to the LRR domain making it possible for the LDLa module to bind and activate the transmembrane (TM) part of the receptor. Therefore, the extracellular signaling molecule, RLN, causes conformational changes within the seven helices transmembrane domain that initiate the coupling of G-proteins to activate downstream signaling cascades. To date, two possible signaling pathways have been identified: cyclic adenosine monophosphate (cAMP) in response to treatment with mature RLN, and extracellular signal-regulated kinases one/two (ERK1/2) as previously demonstrated following treatment with a truncated

B 7-33 chain RLN H2 analog (Hossain et al., 2016).

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One of the upregulated genes identified in the microarray experiments, discussed below, was identified as Gnb2l1 (guanine nucleotide binding protein, G- protein, β polypeptide 2 like 1). This gene is classified as a receptor for activated

C kinase 1 that could be considered as one of the G-protein components that become activated in response to treatment with RLN (Mouse Genome Database,

July 2016).

Scientifically and regulatorily approved methods used for characterization of recombinant forms of RLN H2.

It is important for any recombinant protein to be characterized by demonstration of its molecular identity, amino acid sequencing, biological actions and mechanisms of binding to a specific receptor. However, the binding of RLN

H2 to its cognate receptor(s) is neither simple nor well understood. The emerging evidence suggests that the binding processes are complicated because of many interactions and various cross-talks between and among different forms of RLN and their cognate receptors. These complexities and challenges may explain why there is no one scientifically accepted agonist-receptor gold standard for RLN H2.

On December 18th, 2013, Novartis Pharmaceuticals Corporation (Novartis) applied to the Food and Drug Administration (FDA) for permission to conduct a

Phase 2/3, multicenter, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of serelaxin in human subjects with acute heart failure. On May 22nd, 2014 the European Medicines Agency, Committee for

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Medicinal Products for Human Use (CHMP) released an assessment report in response to Novartis’s application (mirroring that of the FDA submission). Three variants of serelaxin were evaluated: one synthetic and two recombinant RLN H2 produced in bacteria E. coli using the double chain, and a single chain process. As part of the evaluation process of all variants, Novartis was required to present comparability data on clinical safety and clinical efficacy. In addition, physical- chemical and biological (in vitro) comparability data including reversed-phase high- performance liquid chromatography (RP-HPLC), a cell-based potency assay and a receptor binding assay. The results of the comparison of all forms of serelaxin were acceptable to the review committees. The decisions of the review panels were based on the content of the active substance and included appearance, pH, identity, quantity, purity, endotoxin and microbial count. Novartis demonstrated that all three forms of serelaxin showed expected molecular weights and physical- chemical and biological properties. The biological activity of serelaxin was measured by a cell-based bioassay (ELISA). Novartis also conducted a series of pharmacokinetic, physiological, and metabolic studies in vivo in rats (physiological and/or reproductive status not disclosed) and in two groups of rhesus monkeys: one pregnant the other not pregnant. Overall, the non-clinical pharmacokinetic parameters (clearance, bioavailability, bioequivalence and steady-state volume distribution) comparison between the three forms of serelaxin after intravenous administration in rats and rhesus monkeys (pregnant and non-pregnant) showed a statistically significant difference in all parameters in rats (approximately 40%) and in clearance in both pregnant and non-pregnant monkeys (around 50%). Both

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agencies, FDA and CHMP, noted that although the above data show some differences between responses in rats and monkeys, the conclusion from it is

“scarcely informative”.

Toxicological assessments were done in a four-week study in monkeys receiving daily intravenous injections of serelaxin of decreasing doses (from 6.5 mL/min/kg to final concentration of 3.1 mL/min/kg). In this study serelaxin induced an inflammation and induration of the injection site (highly common, also present in control animals) accompanied by a reversible increase in neutrophil count in two animals at the high dose. Diarrhea was present in all treated monkeys with the sporadical presence of blood in feces and also in urine. In human subjects, similar findings were not substantiated. This study indicates that the clinical relevance of observed diarrhea in animals is limited.

The information on the toxicological and pharmacokinetic effects of different forms of serelaxin provided by Novartis in their applications is mainly based on the publications available in public domain with almost no studies performed by the

Corporation. The FDA and CHMP agreed that sufficient relevant data is available, and repetition of animal studies is not needed (nor ethical). As indicated earlier in this section, in its applications, Novartis presented physical-chemical and biological comparability data of three forms of serelaxin including reversed-phase high-performance liquid chromatography (RP-HPLC), a cell-based potency assay

(ELISA) and a receptor binding assay. No information was disclosed as to what kind of receptor binding assay was used. Furthermore, Novartis stated that RXFP1 receptor “is believed to mediate the physiological effects of RLN H2”. Although no

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additional information on characterization of serelaxin was available from Novartis, this information was deemed satisfactory for both regulatory authorities.

Sudo et al., (2003) reported that the interaction between RLN and its receptor is a two-stage binding process. In this proposed model (Figure 29, below) the RLN initially binds to the high-affinity leucine-rich repeat site (LRR; located in the ectodomain region) to form a structure that is important for receptor-ligand binding. This interaction leads to disruption of the transmembrane region allowing for the ligand-LRR complex to be able to interact with the low-affinity site located in there. As a result, the initial high-affinity binding site directs the receptor response, but the following ligand-LRR combo association with the low-affinity site is required for full receptor activation and stabilization. A study with the transmembrane receptors only showed that although they bind radioligand, the ectodomain (i.e. LRR element) is necessary to produce the cAMP response (Halls et al., 2005).

Figure 29. Proposed model of RXFP1/RXFP2 receptor binding and activation.

A, proposed locations of the two binding sites found on both RXFP1 and RXFP2.

The primary high-affinity site is located on the ectodomain of the receptor (dashed line box), likely within the leucine-rich repeat (LRR) region. The low-affinity binding site is located within the transmembrane region (solid line box).

B, initially, the receptors are thought to reside in an inactive conformation (1).

Relaxin/INSL3 can then bind to both the primary high-affinity ectodomain site and

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the low-affinity transmembrane binding site, changing the conformation of the receptor (2). (Adapted from Halls et al., 2005).

Haugaard-Kedstrom et al., (2014) used nuclear magnetic resonance (NMR) spectrometry to study solution structure, aggregation behavior and flexibility of

RLN H2. These authors confirmed the binding model of RLN H2 to RXFP1 proposed originally by Sudo et al., (2003) and Halls et al., (2005) and further demonstrated that RLN H2 also binds to the RXFP2 receptor (cognate receptor for

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INSL3), albeit with reduced affinity. The mechanism of RLN H2 binding to RLXFP1 and RLXFP2 was similar. It was further determined that the primary ligand-binding site of both receptors is located in the LRR domain and a key motif in RLN H2,

RXXXRXXI/V, which is located in the B-chain, is projected to interact with the LRR domain. In addition, although it was confirmed that the bindings of RLN H2 to

RXFP1/2 are intimately linked, the relationship is to a certain degree promiscuous because among ten prongs in the LRR area and between three amino acids in the

RLN H2 binding cassette, there could be about 120 of possible allosteric connections. There were many attempts made to create chimeric RLN H2 analogs

(Haugaard-Kedstrom et al., 2014), including truncated B- and A-chains and/or amino acid substitutes, or synthetically created bio-mimetics of RLN H2. However, to date, not a single analogue that retains equally high-affinity binding and the ability to activate the receptor has been identified. In most cases, a loss of binding ability results in loss of activation, indicating that RLN H2 peptide binding and receptor activation are functionally linked.

The precise signaling pathways mediated by RXFP1 and RXFP2 are not well characterized to date. Most research was focused on RLN H2-mediated signaling leading to increases in cAMP in some target tissues and cell lines (Fei et al., 1990; Parsell et al., 1996). In other organs and cell lines, there were no increases in cAMP (Palejwala et al., 1998; Kompa et al., 2002) and it has been suggested that mitogen-activated protein kinase Erk1/2 could be activated without an associated increase in cAMP levels. Halls et al., (2006) showed that in human embryonic kidney (HEK) 293T cells stably transfected with RXFP1, cAMP

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accumulation was modulated by coupling to multiple G proteins. These include a

Gαs-mediated stimulation, a GαoB-mediated inhibition, and a delayed Gαi3-Gαγ- mediated stimulation via phosphoinositide-3-kinase (PI3K) and protein kinase Cζ isoform. This complex interrelationship between pathways and relative influence of pathway component expression could provide a possible explanation of the many varied responses observed after RLN H2 treatments in multiple target organs and/or cell lines (Halls et al., 2006).

To further investigate the interplay between RLN H2 and its receptor

RXFP1, Hossain et al., (2010) chemically synthesized the RLN H2 analog, B-

R13/17K H2 RLN, and named it the RXFP1 antagonist. This compound of 5907.11

Da molecular weight was characterized by HPLC, MALDI-TOF mass spectrometry and amino acid analysis. In addition, the biological activity of RXFP1 antagonist was assessed in three cell lines. In HEK 293T cells the antagonist bound to RXFP1 with 500-fold lower affinity, where it acted as a partial antagonist. In rat renal myofibroblasts and MCF-7 cancer cells, both of which express the native RXFP1 receptor, it acted as a full antagonist. The authors concluded that their newly created RLN H2 analog/antagonist can be used as a useful tool to advance understanding of the functioning of the RXFP1 receptor. Therefore, this RXFP1 receptor antagonist would have a limited use in order to characterize bench-top

RLN H2 described in this thesis. It is intriguing to note that Hossain et al., (2010) did not test their RXFP1 antagonist in a “gold standard” cell line THP-1.

Described in this thesis recombinant bench-top RLN H2 was sufficiently evaluated, assessed and compared to serelaxin as well as other forms of RLN in

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both in vitro and in vivo tests. Ideally, it would be of assistance to have unrestricted access and unlimited resources to characterize the product further. For instance, x-ray crystallography and/or nuclear magnetic resistance spectrometry would provide data for evaluation of the primary, secondary and tertiary structures of the bench-top RLN H2. However, to the best of my knowledge, University of Guelph presently has no equipment to run these test.

As discussed above and in the section on RLN H2 bioassay, the binding capabilities and mechanisms of RLN H2 interactions with the receptor(s) are poorly understood and there is a debate about a suitable receptor assay for RLN H2. With the complexities of the binding process and the resultant interactions of serelaxin, it is difficult to validate experiments that would demonstrate specific or partially specific binding activities of the bench-top RLN H2. To all intents and purposes of the current work, the bench-top RLN H2 was shown to perform in ways comparable to serelaxin. Having access to a definitive RXFP1 antagonist would most likely improve the thesis to some extent.

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Effects of RLN H2 on differentiation of P19CL6 stem cells

Microarray analyses of cDNA’s are broad ranging techniques enabling investigations of changes in levels of gene expression for thousands of genes at the same time. Presently, there are no published reports on the effects of RLN on the differentiation of stem cells and no microarray gene expression profiling approach was used to investigate the effects of RLN.

Carcinoembryonic stem cells, P19CL6, were chosen as a new model to investigate the effects of RLN H2 on differentiation in the direction of cardiomyocytes. Relaxin caused a four-day delay in the initiation of spontaneous contractions, compared to controls treated with DMSO. In addition to that delay,

80 genes were downregulated, 27 genes were upregulated and 18 genes were both, initially switched on and after day 4, switched off during the remaining period of testing. The extent of changes in gene expression levels is described and illustrated in graphs with accompanying tables in Chapter 3.

Bigazzi and Bigazzi (2005) used mature murine myoblast cells and rat cardiomyocytes obtained from adult male Wistar strain of rats of about 250-300 g body weight. Both primary cultures of mouse and rat cells were mixed together at a ratio of 3:1, respectively, and co-cultured in M199 medium supplemented with

15% of fetal bovine serum (which is 50% higher than a standard content for stem cells). Human recombinant RLN was added to co-cultures at 10 nMol/L at 24, 48,

72 and 96 hours. The co-cultures were then stimulated with specific agonists of the cardiomyocytes such as caffeine and/or isoproterenol. In addition, some

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cardiomyocytes were stimulated with mechanical impulses using the cantilever of an atomic force microscope. The conclusion of this study was that recombinant

RLN H2 caused a reduction in the proliferative rhythm of the murine myoblasts demonstrated in their delayed rate of differentiation into rat cardiomyocytes.

Microarray results show the genes affected by RLN H2

The data obtained from the microarray analysis experiments show first and foremost that commercially available serelaxin and the lab-bench produced RLN

H2 appear to affect the same genes in the same ways (Appendix 2, Tables A 2-3,

A 2-4, and A 2-5). At the cellular level, their actions are similar – in this case – differentiation of stem cells and contractions of terminally formed cell layers resembling mitochondria-rich cardiac myocytes.

Validation of microarray results by quantitive PCR analysis

In order to validate microarray data, quantitative PCR is often done to confirm the increased or decreased expression of those specific genes identified by the array. In the study above, the 125 genes in cardiomyocytes confirmed to have changed following RLN H2 treatment require confirmation using qPCR analysis. Following several attempts, this confirmation was eventually abandoned due to limited time and resources and the technical challenges associated with qPCR proved to be overwhelming.

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Microarray analysis of complementary DNA (cDNA) allows for the study of a large number of gene expression levels at the same point in time. However, the vast volume of data obtained from microarrays can also bring about considerable inconsistencies within, and between research laboratories. The differences in results could be dependent on the quality and amounts of the RNA, the source of supply of the microarray slides, experimental protocols, and quality of analysis of the data. A simple comparison of two sets of results obtained from two experiments that were run in parallel will be difficult to interpret because it is not always feasible to compare all of the results of a microarray experiment. Arbitrary choice to compare for instance only the gene transcripts that were up-regulated by 2-, 5- or

10-fold, or transcripts that were only down-regulated by 2-, 5- or 10-fold, may appear too simplistic. This approach could lead to missing or discarding of some of the valuable information from the entire set of microarray data because it limits what could be said about the remaining genes that were not selected for comparison because of the subjectively selected levels of change fold. At present, it is not known how significant a level of change in the expression of a gene would need to be to result in a similarly significant change in biological effects. For instance, in a hypothetical disease state, a small change might be critical, and in contrast, a large change that might be compensated for somewhere else that we are not aware of. Another example of this is the technical ability to detect micro- levels of metastatic cells in lymph nodes or minimal amounts of tumor-associated transcripts circulating in the blood, neither of which could be used as a definitive prognostic determining factor that could be impacting the patient. Miron et al.,

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(2006) proposed that in order to fully take advantage of the power of microarrays, it would be useful to create a quality index for the microarray study that in turn could be useful for subsequent cross-study analyses. However, at the present time, there is no such a quality index reported.

It has been proposed that real-time PCR, also referred to as quantitive PCR

(qPCR) could be used for validation of the microarray results. Currently, however, there is no consensus on how best to perform and interpret qPCR per se. Bustin et al., (2009) published a paper suggesting Minimum Information for Publication of

Quantitive Real-Time PCR Experiments (MIQE Guidelines) in which the authors describe the minimum information necessary for evaluation of qPCR experiments.

One of the fundamental prerequisites for validation of microarray result by qPCR is that the starting material, the RNA must be collected and purified at a predetermined time point in the experiment and in quantities sufficient for running both the microarray and the qPCR tests simultaneously. The next step of the qPCR, protocol is the conversion of RNA into cDNA during a process called the reverse-transcription. There are many additional correlating variables introduced at this stage: the amount of RNA to be transcribed, priming strategy, enzyme type, sample volume, temperature, efficiency, and duration of the reverse-transcription step. It is recommended that the reverse-transcription step is carried out in duplicate or triplicate and that the total RNA concentration is exactly the same in every step. Furthermore, Bustin et al., (2009) suggested that there are a number of key issues affecting the results of qPCR and these are listed below:

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1. Analytical sensitivity, typically reported as the limit of detection, refers to

the minimum number of copies in a given sample that can be measured

accurately with an assay.

2. Specificity of analysis refers to the qPCR assay detecting the intended

target sequence rather than other non-specific targets that could also be

present in the analyzed sample.

3. Accuracy pertains to the difference between levels experimentally

measured and the actual concentrations reported as fold changes or

estimated copy number.

4. Repeatability or short-term accuracy, also known as intraassay variance

that refers to the precision and robustness of the assay wherein the

same samples are repeatedly analyzed in the same assay.

5. Reproducibility or long-term accuracy is also known as interassay

variance and refers to the difference in results between separate assay

runs or between different laboratories.

Recently Marshall et al., (2016) using a single microarray analysis reported on the effects of recombinant human relaxin H2 on human endometrial stromal cells in vitro. There were a total 321 up-regulated genes (by 1.4-fold or more) and

203 down-regulated genes (by at least 30%) in human endometrial stromal cells after 24-hour relaxin treatment. No validation of the microarray results by qPCR was reported. In the same study Marshall et al., (2016) also assessed the expression of a selected group of genes associated with angiogenesis in response

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to in vivo relaxin treatment, by using qPCR on uterine samples obtained from wild- type and relaxin-deficient strains of mice. The microarray results showed that in response to relaxin treatment in human endometrial cells six angiogenesis- associated genes were up-regulated but the same genes were down-regulated in murine uterine samples as measured by qPCR. The conclusion from that study was that endogenous relaxin plays no major role in pre-implantation angiogenesis in the mouse uterus in vivo. No final assumptions referring to the microarray results of relaxin treatment of human endometrial cells in vitro were reported.

Ideally, some validation of the microarray results is required to be able to confirm RLN H2’s role in modulating gene expression in cardiomyocyte differentiation. However, based on the above-described issues associated with qPCR itself, it would be difficult to justify using a similar approach to validate the multiple microarray results evaluating the effects of commercial and /or bench-top relaxin H2 on the differentiation of P19CL6 cells reported in this thesis. In addition, qPCR could potentially add even more indeterminate data, further complicating interpretation of the microarray results. Future studies could investigate knock-out, knock-down or silencing the specific genes to determine their roles in mediating the RLN-induced effect.

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Limitations

Is DMSO-induced differentiation of P19CL6 carcinoembryonic stem cells into contracting cardiomyocytes a really equal model to mimic the normal differentiation process?

Is RLN H2-induced differentiation of P19CL6 carcinoembryonic cells into contracting cardiomyocytes a really equal model to mimic the normal differentiation process?

It appears that bench-top RLN H2 produced during this research work is at least equal to DMSO in inducing the differentiation of P19CL6 cells into cardiomyocytes. The RLN H2 effects were delayed by 4, but it is safer to use than

DMSO.

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OPPORTUNITIES FOR FUTURE RESEARCH

There are opportunities for improvements in the experimental design to make a better RLN expression system in the laboratory. One of the options could be testing a wider selection of bacterial expression hosts and to engineer simpler plasmid vector. The process is frustrating and time-consuming. It could be suggested that a faster, cell-free expression system using mammalian cells could be an option to speed up the initial small-scale testing. In fact, I have completed the preparatory work in the laboratory of Dr. Summerlee. A plasmid carrying H2

RLN B- and A-chains, linked to a green fluorescence protein (GFP) gene has been created. The plasmid is planned to be used for expression of GFP-RLN construct in a cell-free system based on the human HeLa cell line. The rationale here is that

GFP-RLN could be followed easily during all stages of production and could be observed by simple exposure of purification columns to a fluorescent light source.

As GFP is harmless to mammalian cells, GFP-RLN H2 could be monitored with ease in both, in vitro and in experiments in vivo.

The pilot study project described in this thesis, on the role of RLN H2 in the differentiation of carcinoembryonic stem cells, indicates that there are many directions in which future investigations would be warranted. At present, I’ve acquired a vial of human stem cells obtained from umbilical cord blood (Dr.

Anthony Atala, Wake Forest University, Institute for Regenerative Medicine,

Winston-Salem, NC, USA). It is anticipated that the cDNA microarray approach will be continued with modifications to include a review of a broader spectrum of gene

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expression. The very stringent criteria set out presently showed only 125 RLN affected genes. In future experiments, using a similar system, it would be probably beneficial to lower the selection regimen to potentially expose more genes. In addition, there are currently available human protein microarrays containing thousands of unique, full-length human proteins, including kinases, phosphatases, nuclear receptors, proteases and most importantly for RLN researchers, GPCRs.

I am strongly inclined to believe that expanding research into protein microarrays could potentially contribute to better understanding of RLN and GPCR’s interactions.

OVERALL SUMMARY

In conclusion, it is possible to make recombinant human RLN H2 in an average scientific laboratory. The process is not as easy as it would appear at first glance but with a lot of research and support, it is attainable. The maximum amount of dry weight RLN H2 accomplished during this project was approximately 1.53 mg from 1 liter of bacterial culture, which is below the pharmaceutical industry standards of 10 to (preferably) 100 mg/L. The quality, stability, and purity of the lab produced RLN is close to being on a par with commercially available proteins. The price tag for making RLN in the laboratory was most likely several thousand dollars. It would be interesting to know the costs of industrial production but judging by prices that the commercial suppliers charge for RLN, their costs must be considerably higher.

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This is the first experimentally confirmed report that RLN H2 can affect the differentiation of carcinoembryonic stem cells. However, the results are preliminary and provide a guide to the exciting possibility of investigating further this unique action of relaxin. As well, expanding research into human stem cell systems using protein microarrays could explore and possibly expand our understanding of the continuously evolving roles for relaxin.

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APPENDIX 1

SYNTHESIS OF RECOMBINANT HUMAN RELAXIN H2 IN E. COLI USING THE MALTOSE BINDING PROTEIN SYSTEM

Purpose

In this thesis, the literature review of the methods for creating recombinant proteins revealed that the bacterial E. coli hosts are most frequently reported to be used for these purposes. Among these systems the maltose binding protein (MBP) expression platform was described as simple to use, promising fast expression, easy purification and high yield of correctly folded recombinant proteins. As well, in a study designed to compare protein solubility enhancement properties, the E. coli MBP tag was shown to be a more effective protein solubility fusion partner than were the glutathione-S-transferase (GST) and thioredoxin (TRX) proteins.

There are two options for expression based upon the MBP fusion protein system of bacteria (Guan et al., 1987; Bedouelle and Duplay, 1988) available commercially from New England Bio labs Ltd. (Whitby, ON, Canada). The first version, pMal-c2x, is designed for cytoplasmic expression of single peptides and/or unmodified proteins. The second variety, pMal-p2x, is intended for periplasmic expression, which is recommended for proteins containing disulfide bonds.

Included within the sequence of the malE gene, at its 5’ end, is encoded the MBP signal peptide, to direct the fusion protein for export into the periplasmic space of

E. coli cells.

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The pMal plasmid vector contains the tac promoter (Donovan et al., 1996), which is inducible by lactose, or a synthetic compound isopropyl-β-D- thiogalactoside (IPTG). While not being subjected to catabolite repression, IPTG allows for control over the level of induction. The periplasmic form of pMal-p2x fusion system was used in the present series of experiments in an effort to express and purify recombinant human RLN H2, a protein containing 3 disulfide bonds. All features of the pMal-p2x vector and position of RLN cloned into it are shown in

Figures 3 and 4, below.

Materials and Methods

A bacterial plasmid vector pMal-p2x carrying the 558 base pairs cDNA coding for human H2 pre-pro-RLN (5’ signal peptide, B chain, C-peptide and an A chain, 3’) was constructed by means of cloning, at the junction between malE

(maltose) and lacZα (lactose) genes, within reading frame sequence of the plasmid vector pMal-p2x (New England Bio labs Ltd., Whitby, ON, Canada). The pre-pro-

RLN sequence was used because it has been reported that the C-peptide is required for proper folding of the mature, active form of RLN (Stults et al., 1990).

This plasmid named pMalp2x-ppRLN was used to transform the chemically competent, recombination deficient DH-10 strain of E. coli. Small scale cultures of bacteria carrying the plasmid were grown and the plasmid DNA was extracted for use in the expression capable strains.

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For the fusion protein expression experiments TB-1 strain of E. coli [F-araΔ(lac- proAB){Φ80dlacΔ(lacZ) M15}rps(StrR)thihsdR] (Guan et al., 1987) was transformed using the rubidium chloride method, as it promises the highest rate of success among chemical methods of transformation (Sambrook and Russell,

2001).

Selection of transformed bacterial colonies was done on agar plates containing ampicillin (100 µg per 1 mL of LB agar medium). A single colony from the transformation plate was selected and streaked onto a fresh agar plate containing ampicillin. A starter culture (5 mL volume of LB medium with antibiotic) was established by picking a single colony from the streaked plate and grown overnight on a shaker at 370C. A larger scale culture was then started by inoculating 10 mL of LB medium with 100 µL of the starter culture and grown on a shaker overnight at 370C. Plasmid DNA was extracted from 4.5 mL of the overnight culture using a High Pure Plasmid Isolation Kit according to the manufacturer’s protocol (Roche Applied Science, Laval, PQ, Canada). From the remaining culture, a glycerol stock was prepared and frozen for future use.

The purity and amount of obtained plasmid DNA were measured using a

Nano-Drop ND 1000 spectrophotometer (NanoDrop Technologies, Wilmington,

DE, USA). The purified plasmid DNA sample was digested with Not1 enzyme, at a site uniquely present within the ppRLN H2 sequence, and subjected to electrophoretic evaluation on 1% agarose gel (expected size of the plasmid 7245 base pairs). DNA sequencing was done to confirm proper size (558 base pairs)

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and orientation of the inserted ppRLN (5’ signal peptide, B chain, C-peptide and an A chain, 3’) within the pMal-p2x reading frame.

Large-scale trials were then done using 114 mL cultures. Protein samples obtained during purification procedures were subjected to SDS-PAGE electrophoresis. The tricine method (Schägger and von Jagow, 1987) was employed, as it is more sensitive than the glycine system (Laemmli, 1970), which can detect only proteins larger than 10 kDa. There is also a difference in gel design: glycine based gels have 1-2 centimeter stacking gel and the remainder of the gel is resolving gel. In contrast, a tricine gel consists of 3 parts: 1-2 centimeter stacking gel, 2-3 centimeter spacer gel and then small pore resolving gel. Due to the small size of RLN, approximately 6Kda, throughout the experiments described here a

10% spacer gel and 16.5% resolving gels poured in the laboratory were used.

Based on the electrophoresis results and if the pre-pro-, pro-, or RLN were detected, purification steps on amylose resin columns were followed according to the supplier’s protocol (New England Bio labs Ltd., Whitby, ON, Canada).

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Figure A1-1. A map of the pMal-p2x-ppRLN H2 vector.

All the features of the vector and the position of the pre-pro-RLN insert are indicated. Arrows show the direction of transcription. The size of this plasmid vector is 7245 base pairs.

IacI rop q

ORI Ptac

ColE1

pMalpMALp2-p2x-ppRLN RLX H2

maIE

ORI - ORI + M13 +

IacZ

RLXppRLN INSERT H2 insert R. POTERSKI et al. 2001

Figure A1-2. A fragment of the polylinker sequence illustrating the Factor Xa cleavage site at the 5’ end of the RLN insert.

Factor Xa cleavage site malE…ATC GAG GGA AGG ATG CCT CGS……TGC TGA…lacZα

Ile Glu Gly Arg ppRLN H2 cDNA

203

The human mRNA for pre-pro-RLN H2 used in experiments described in this chapter of the thesis.

GenBank: X00948.

558 bp mRNA

Reference: Hudson et al., 1984

UniProtKB/Swiss-Prot: P04090

Protein Translation:

MPRLFFFHLLGVCLLLNQFSRAVADSWMEEVIKLCGRELVRAQIAICGMSTWSK

RSLSQEDAPQTPRPVAEIVPSFINKDTETINMMSEFVANLPQELKLTLSEMQPAL

PQLQQHVPVLKDSSLLFEEFKKLIRNRQSEAADSSPSELKYLGLDTHSRKKRQL

YSALANKCCHVGCTKRSLARFC

Locations and features of cDNA sequence:

1-75 = signal peptide

76-171 = B-chain

172-483 = C-peptide

484-555 = A-chain

1 atgcctcgcc tgtttttttt ccacctgcta ggagtctgtt tactactgaa ccaattttcc

61 agagcagtcg cggactcatg gatggaggaa gttattaaat tatgcggccg cgaattagtt

204

121 cgcgcgcaga ttgccatttg cggcatgagc acctggagca aaaggtctct gagccaggaa

181 gatgctcctc agacacctag accagtggca gaaattgtgc catccttcat caacaaagat

241 acagaaacca taaatatgat gtcagaattt gttgctaatt tgccacagga gctgaagtta

301 accctgtctg agatgcagcc agcattacca cagctacaac aacatgtacc tgtattaaaa

361 gattccagtc ttctctttga agaatttaag aaacttattc gcaatagaca aagtgaagcc

421 gcagacagca gtccttcaga attaaaatac ttaggcttgg atactcattc tcgaaaaaag

481 agacaactct acagtgcatt ggctaataaa tgttgccatg ttggttgtac caaaagatct

541 cttgctagat tttgctga

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Figure A1-3. A typical bacterial culture growth characteristics (modified from

Sambrook and Russell, 2001).

During culturing of bacteria in a tube or a flask, the population of cells initially, adjusts to the new medium over the first 7-8 hours (lag phase). Next stage is a phase of exponential growth and regular dividing by means of binary fission, which lasts for up 16-17 hours. The growth of bacteria becomes then limited and the cells cease to divide. This stage is called stationary phase, which leads to loss of viability and death of cells. Optical densities (OD600, Y axis) of culture samples collected at set times (X-axis) during culturing are shown.

Typical growth curve of a bacterial culture 10 9 8 7 6 600 5

OD 4 3 2 1 0 0 10 20 30 40 50 Time (hrs)

Upon completion of culturing, bacteria were harvested and osmotic shock fractions of periplasmic proteins collected according to the procedure of Neu and Heppel

206

(1965). Polyacrylamide gel electrophoresis analyses were done to determine the presence of MBP-RLN fusion proteins.

Results

Over 100 repetitions, including a minimum of 3 combinations in each parameter were carried out. The following parameters were adjusted individually or collectively in an attempt to produce positive results:

Various bacterial strains:

TB1

JM109

HB101

Different growth temperatures:

370C or 300C

Selection of media:

LB Miller

LB Lennox

Mode of IPTG supplementation to induce synthesis of fusion protein:

One portion

Divided into 3 equal portions.

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Optical density measurements were taken prior to each treatment with

IPTG, 1 hour and 4 hours after the last portion of the inducer. Numerous attempts were made to isolate and purify MBP-RLN H2 fusion protein, however, no positive results were obtained. Shown in Table 8 below are the recorded data from a typical culture:

Table A1-1. A typical bacterial culture growth profile and treatments with IPTG as measured by optical density OD600.

Length of culturing Optical density IPTG dosage

(hrs) (OD600) (1mM final)

15 0.60 0.33

16 0.71 0.33

17 0.83 0.34

18 0.85 0.0

22 0.75 0.0

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Figure A1-4. A scanned image of a 1% agarose gel.

Shown in lane 1 is pMal-p2xppRLN H2 plasmid DNA. Lane 2 illustrates 1kb DNA molecular marker. The plasmid from lane 1 was linearized at Not1 site uniquely present within the ppRLN sequence and it is pictured in lane 3. The arrow on the right side of the image points to approximate size of the plasmid, which is 7245 base pairs and it is of correct molecular weight.

1 2 3

7245 bp

209

Figure A1-5. A scanned image of 1% agarose gel illustrating pMal-p2xppRLN plasmid DNA isolated from 3 different strains of bacteria.

Lane 1 is 1kb DNA marker. Lanes 2 and 3 show supercoiled plasmid isolated from

TB1 strain, lane 4 the same plasmid linearized with Not1 enzyme uniquely present in the ppRLN sequence. Lanes 5 (supercoiled) and 6 (linearized) show the plasmid from JM109 strain and lanes 7 (supercoiled) and 8 (linearized) plasmid from

HB101 strain.

1 2 3 4 5 6 7 8

7245 bp

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Figure A1-6. A scanned photograph of a representative SDS-PAGE protein gel.

Samples were collected during extraction of fusion protein grown in TB1 strain of

E. coli. Lanes 1 and 2 are uninduced samples, lanes 3 and 4 are induced fusion

protein samples, lanes 5 to 7 are samples of cold osmotic shock fluid (COSF)

extracts purified on an amylose resin column and eluted with 10 mM maltose.

Lanes 8 to 10 are samples seen in lines 5 to 7 that were concentrated 10 times.

The arrow on the left points to the induced MBP of the approximate size of 42.5

kDa present in lanes 3 and 4. No MBP-RLN fusion protein was detected.

1 2 3 4 5 6 7 8 9 10

42.5 kDa

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Discussion

The objective of this study was to express RLN H2 from a fusion protein

MBP-ppRLN H2. Three strains of bacteria were used in this series of experiments:

TB1, HB101, and JM109. Despite employing numerous parameters and variations in experimental designs that are: three various strains of bacteria and lowering of culture temperatures from 370C to 300C, no positive results were achieved. There could be several possible explanations, which are indicated below.

Plasmid pMal is a derivative of pBR322, which is a low copy number vector of 15-20 per cell (Bolivar and Rodriguez, 1977). Also, inserts cloned into pMal-p2x vector have about 8-16-fold reduced level of expression when compared to the same insert cloned into pMal-c2x. Therefore, the amounts of fusion proteins expressed from pMal-p2x vector could not be very high. Expressing at lower than

370C temperatures was the next thing tried here. Of course, the cells grew slower at these temperatures, and it is very likely that the cells were dying due to prolonged exposure to the inducer IPTG (Bishai et al., 1987).

Maltose binding protein has an affinity to amylose resin in the micromolar range of Kd =1.6 to 40 x 10-7 (Miller et al., 1983). The binding of MBP to amylose resin highly depends on the nature of the protein fused to the C-terminus of MBP

(Riggs, 2000). Certain fused proteins reduce or completely block binding with amylose. Another disadvantage of MBP fusion vectors is the requirement for using

Factor Xa to cleave off the MBP fusion partner after expression and purification steps. Furthermore, cells grown in LB and similar media contain substantial

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amounts of an enzyme amylase that interferes with binding, presumably by either digesting the fusion off the column or by releasing maltose that elutes the fusion from the column before expected.

Kapust and Waugh, (1999) reported that if the growth of cultures begins to slow or ceases completely within 3 to 4 hours after induction with IPTG it means that the expressed protein is toxic to its host. Please see Table 8 above to note that at 4 hours post IPTG induction (hour 22 of culturing) optical density fell considerably, indicating that there was toxicity caused by RLN.

Personal communication from Dr. Ross Bathgate, Howard Florey Institute,

University of Melbourne: “Our pilot experiments using the pMal systems have shown very low expression in cytoplasmically targeted expression but none with the periplasmic directed. It is not easy, Connetics Corporation spent years perfecting their recombinant expression”.

After my numerous inquiries with the supplier of the pMal system, it would be reasonable to conclude that RLN was toxic to the bacterial hosts used in presented experiments. It is interesting to note that New England Biolabs Ltd. discontinued the supply of this system and currently offers a modified platform for expression of recombinant proteins.

In the next stage of this research project, a modified system for the expression of RLN H2 will be designed and tested. Instead of using ppRLN, the mature form of RLN consisting of B- and A-chains, connected by a four amino acid site for Factor Xa and combined with MBP and 6-Histidine (6-His) tag will be used.

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It is anticipated that by using 6-His tag purification and yield of the new fusion protein will be improved.

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APPENDIX 2 MICROARRAY TABLES

Table A2-1. Functional descriptions of the genes with up-regulated and

differentially altered (positive at day 4 and negative at day 8 and 12) expression

levels by RLN H2 treatment. The information was obtained from the Mouse

Genomic Informatics.

UG Name of the gene Symbol Component of Function Process Chro Cluster mo some Mm Guanine nucleotide Gnb2l1 Cell soma, Protein kinase Intracellular signaling 11 5305 binding protein cytoplasm C binding; cascade; protein kinase C (G-protein) beta receptor activation; protein polypeptide 2 like 1 activity; localization; signal receptor transduction binding Mm Ribosomal protein Rpl26 Cytosolic ribosome; Structural Ribosome biogenesis 5 3229 L26 large ribosomal constituent of subunit; ribosome ribonucleoprotein complex Mm Ribosomal protein Rps4x Cytosolic ribosome; rRNA binding; Protein biosynthesis; X 66 S4, x-linked ribonucleoprotein structural ribosome biogenesis. complex; ribosome constituent of Rps4x, Ube1x, and Zfx are ribosome subject to X-inactivation in mouse but not in human, suggesting that Turner syndrome may be in part due to insufficiency in these gene products Mm Ribosomal protein Rpl27a Intracellular; Structural Protein biosynthesis 7 305750 L27a ribonucleoprotein constituent of complex; ribosome ribosome Mm Ribosomal protein Rps3a Cytosol; cytosolic Protein binding; Induction of apoptosis; 3 399829 S3a ribosome; structural protein biosynthesis; intracellular; constituent of ribosome biogenesis; nucleus; ribosome translational initiation ribonucleoprotein complex; ribosome Mm Ribosomal protein Rpl3 Intracellular; Structural Translation; this protein is 15 396243 L3 ribonucleoprotein constituent of a component of the large complex ribosome subunit of cytoplasmic ribosomes Mm Ribosomal protein Rpl23 Cytosolic ribosome; Structural Ribosomal protein import 6 391693 L23 nucleolus; constituent of into nucleus; protein ribonucleoprotein ribosome biosynthesis complex Mm Ribosomal protein Rpl6 Cytosolic ribosome; Structural Ribosome biogenesis and 5 262021 L6 intracellular constituent of assembly, translation. ribosome Specifically binds to domain c of the tax- responsive enhancer element in the long terminal repeat of htlv-I Mm Ribosomal protein Rps6 Cytoplasm; Structural Glucose homeostasis; 4 425090 S6 cytosolic ribosome; constituent of ribosome biogenesis and assembly. May play an

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ribonucleoprotein ribosome; RNA important role in complex binding controlling cell growth and proliferation through the selective translation of particular classes of mRNA. Mice with an inducible, liver-specific null mutation exhibit failure of liver regeneration and an absence of 40S ribosomes in hepatocytes. Mice with a mutation where serines are unphosphorylatable exhibit hyperinsulinemia, impaired glucose tolerance, and smaller MEFs and beta cells Mm Ribosomal protein Rpsa Intracellular; Receptor Protein biosynthesis 9 4071 SA ribonucleoprotein activity; complex; ribosome; structural small ribosomal constituent of subunit ribosome Mm Ribosomal protein Rpl18a Intracellular; Structural Protein biosynthesis; could 8 379251 L18A ribonucleoprotein constituent of play a role in regulation of complex; ribosome ribosome ribosomal protein formation during mouse myoblast differentiation Mm Ribosomal protein Rpl4 Intracellular; Protein binding; Protein biosynthesis 9 280083 L4 ribonucleoprotein structural complex; ribosome constituent of ribosome; oxidoreductase activity Mm Ribosomal protein Rpl15 Intracellular; Structural Protein biosynthesis. 14 2050 L15 ribonucleoprotein constituent of Rpl15 promotes cell complex; ribosome ribosome proliferation in gastric neoplasms Mm Ribosomal protein Rps3 Cytoplasm; RNA binding This protein has been 7 236868 S3 ribonucleoprotein implicated in the complex; small processing of DNA ribosomal subunit damage Mm Eukaryotic Eef1al Cytoplasm; GTP binding; Anti-apoptosis; translation. 2 380075 translation eukaryotic GTP ase This protein promotes gtp- elongation factor 1 translation activity dependent binding of alpha elongation factor 1 aminoacyl-tRNA to the a- complex site of ribosomes during protein biosynthesis. Homozygotes for a spontaneous mutation exhibit muscle wasting, lymphoid hypoplasia, lack of intestinal IgA plasma cells, cerebellar dysfunction, neurodegeneration, an age-dependent increase in chromosomal aberrations, and lethality around 28 days of age Mm Eukaryotic Eef2 GTP-binding; Translation This protein promotes the 10 326799 translation GTPase activity gtp-dependent elongation factor 2 translocation of the nacent protein chain from the a- site to the p-site of the ribosome Mm Eukaryotic Eef1a1 This protein 1 379939 translation promotes the gtp- elongation factor 1 dependent binding alpha 1 of aminoacyl tRNA

216

to the a-site of ribosomes during protein biosynthesis. Mm Eukaryotic Eef1g Eukaryotic Translation Probably plays a role in 19 379129 translation translation elongation anchoring the complex to elongation factor 1 elongation factor 1 factor activity other cellular components gamma complex (by similarity) Mm Solute carrier Slc8a3 Integral to Calcium: Calcium ion transport; cell 9 296560 family 38, member membrane integral sodium communication; transport. 3 to plasma antiporter Important role in the membrane activity; carrier control of calcium activity concentration in the skeletal muscle fibers and at the neuromuscular junction. Mice homozygous for disruptions in this gene display a normal phenotype Mm Solute carrier Slc14a1 Integral to Copper ion Specialized low-affinity 18 33832 family 14 (urea membrane binding; urea urea transporter; mediates transporter) transporter urea transport in member 1 activity erythrocytes. Mice homozygous for disruptions in this gene display a grossly normal phenotype although they have an inability to concentrate urea in urine Mm Histocompatibility H2-K1 External side of This protein is Mice homozygous for 17 422886 2, K1, K region plasma membrane; involved in the disruptions of this gene integral to presentation of display T-cell membrane foreign abnormalities and antigens to the abnormal susceptibility to immune various viral infections system. Mm Heat shock protein Hspa8 Nucleus ATP binding; Chaperon cofactor- 9 290774 8 ATPase activity dependent protein folding. May function as an endogenous inhibitory regulator of hsc70 by competing with the co- chaperones Mm Heat shock protein Hsp90a Mitochondrion ATP binding; Protein folding; response 17 2180 90kDa alpha b1 molecular to heat. Homozygotes for (cytosolic), class B chaperone; has a gene-trapped null member 1 ATP-ase mutation exhibit placental activity defects including failure to form a placental labyrinth and lack of expansion of allantoic blood vessels. Mutants die around mid- gestation Mm Nucleophosmin 1 Npm1 Nucleolus; nucleus DNA binding; Protein localization; 11 6343 nucleic acid regulation of DNA damage binding response; signal transductionby p53 class mediator. Associated with nucleolar ribonucleoprotein structures and binds single-stranded nucleic acids; it may function in the assembly and/or transport of ribosome. Homozygous mutation of this gene results in embryonic lethality,

217

anemia, defects in primitive hematopoeisis and abnormal brain development. Heterozygous mutation results in hematopoeisis defects and chromosomal instability Mm POU domain, class Pou5f1 Nucleus DNA binding; Germ-line stem cell 17 17031 5, transcription (Oct4) protein binding maintenance; negative factor 1 regulation of transcription; DNA dependent. Transcription factor that binds preferentially to the octamer motif (5’-atgttaat- 3’); may exert a regulatory function in meiotic events that are required for terminal differentiation of male germ cell. Homozygosity for a targeted null mutation results in peri-implantation lethality prior to the egg cylinder stage, associated with failure to develop a pluripotent inner cell mass. Conditional mutations show defects in reproduction Mm Cbp/p300- Cited1 Nucleus Transcription Embryonic placenta X 2390 interacting trans- factor activity; development; melanocyte activator with transcription differentiation. Seems to Glu/Asp-rich regulator be associated with carboxy-terminal activity pigmentation. Mutation of domain 1 this locus results in impaired embryonic growth and survival due to defects in trophoblast and placental development. Phenotypic penetrance is subject to influence by genetic background and parental inheritance Mm Aldolase 2, B AldoB Catalytic activity; Glycolysis; Has a role in metabolism 4 218862 isoform fructose- metabolic bisphosphate process aldolase activity Mm Aldolase 3, C AldoC Mitochondrion Fructose- Glycolysis; metabolism 11 7729 isoform bisphosphate aldolase activity; lyase activity; protein binding Mm Methylenetetrahydr Mthfd1 Formate- Histidine ATP binding; catalytic 12 29584 ofolate tetrahydrofolate catabolism; activity; amino acid dehydrogenase ligase activity; plays a role in biosynthesis process; folic (NADP+ formyl rapid cell acid and derivative dependent) tetrahydrofolate growth biosynthetic process methenyltetrahydro dehydrogenase folate activity; cyclohydrolase, methenyltetrahydro formyl folate tetrahydrofolate cyclohydrolase synthase activity; methylenetetrahydr ofolate dehydrogenase (NADP+) activity; nucleotide binding

218

Mm Reduced Rex2 Intracellular; Metal ion Regulation of transcription, 4 327224 expression 2 nucleus binding; nucleic DNA-dependent acid binding; zinc ion binding Mm Nucleolar protein Nol11 Nucleus Function 11 294617 11 unknown Mm Helicase-like Hltf Nucleus Matrix Possesses intrinsic ATP- 3 209650 transcription factor associated, dependent nucleosome actin remodeling activity. This dependent activity may be required regulator of for transcriptional chromatin, activation or repression of subfamily a specific target promoters (by similarity). These may include the SERPINE1 promoter, to which this protein may bind directly Mm Helicase, lymphoid Hells Centric ATP binding, Anti-apoptosis; cell cycle. 19 57223 specific heterochromatin; ATP-dependent Transcriptional co- nucleus helicase activity activator cooperating with nuclear hormone receptors to potentiate transcriptional activation. Plays an essential role in normal development and survival. Involved in regulation of the expansion or survival of lymphoid cells. Required for de novo or maintenance DNA methylation. May control silencing of the imprinted CDKN1C gene through DNA methylation. May play a role in formation and organization of heterochromatin, implying a functional role in the regulation of transcription and mitosis Mm Tubby like protein 4 Tulp4 Cytoplasm Molecular Intracellular signaling 17 28251 function cascade; biological process. Could be involved in the hypothalamic regulation of body weight (possibly associated with juvenile diabetes) Mm Poly A binding Pabpc4 Cytoplasm Poly(A) binding; Biological process. Binds 4 277091 protein, poly(U) binding to poly A tail of mRNA , cytoplasmic 4 may be involved in cytoplasmic regulatory processes of mRNA metabolism, can probably bind to cytoplasmic RNA sequences other than poly A in vivo (by similarity) Mm Phosphoglycerate Pgam1 Cytosol Bisphosphoglyc Interconversion of 3- and 19 391589 mutase 1 erate mutase 2-phosphoglycerate with activity; 2,3-bis-phosphoglycerate bisphosphoglyc as the primer of the erate reaction, can also catalyze phosphatase the reaction of ec5.4.2.4 activity (synthase) and ec3.1.3.13 (phosphatase) but with a reduced activity

219

Mm Vitamin K epoxide Vkorc1 Endoplasmic Oxidoreductase Positive regulation of 7 29703 reductase complex, reticulum; integral activity; vitamin coagulation; vitamin K subunit 1 to membrane; K epoxide biosynthesis. Involved in membrane reductase vitamin K metabolism. (warfarin Catalytic subunit of the sensitive) vitamin K epoxide activity reductase (VKOR) complex which reduces inactive vitamin K 2,3- epoxide to active vitamin K Mm RIMS binding Rimbp2 Membrane Serine-type Proteolysis 5 233996 protein 2 peptidase activity Mm Finkel-Biskis-Reilly FBR- Intracellular; Protein binding; Protein biosynthesis; 19 329631 murine sarcoma MuSV ribonuleoprotein structural protein modification virus (FBR-MuSV) Fau complex; ribosome constituent of ubiquitously ribosome expressed (fox derived) Fau Mm Carbonic Car10 Carbonate Zinc ion binding Brain development; one- 11 342160 anhydrase 10 dehydrogenase carbon compound activity metabolic process Mm PDZ domain Pdzd11 Protein binding Intracellular During embryonic X 291607 containing 11 signaling development, some cascade; isoforms are essential for protein proper neuronal targeting differentiation and organization required for cell polarity; maintenance of apicobasal polarity, plays a critical role at septate junctions in cellular growth control during development. The presence of a guanylate kinase domain suggests involvement in cellular adhesion as well as signal transduction to control cell proliferation Mm Data not found - - - - - 432028 Mm RIKEN cDNA - - - - 14 282706 6330409N04 gene Mm RIKEN cDNA - - - - 5 11311 2310057D15 gene Mm RIKEN cDNA - - - - 12 35583 4930573I19 gene Mm cDNA sequence - Undefined - - 2 343110 AK190093, similar to SUMO-1-specific protease Mm DNA fragment - Undefined - - 7 133704 Mm Coiled-coil domain Ccdc98 Undefined - - 5 221269 containing 98 Mm RIKEN cDNA - - - - 10 383824 2310011J03 gene Mm RIKENcDNA26100 - - Plays a general role in the 17 426690 36F08 gene hierarchies of gene expression leading to metamorphosis.

220

Table A2-2. Functional descriptions of the genes with down-regulated expression levels by RLN H2 treatment. The information was obtained from the Mouse Genomic Informatics.

UG Name of the gene Symbol Component of Function Process Chromo Cluster some Mm TAF11 RNA Taf11 Transcription Protein binding Regulation of 17 267998 polymerase II, factor TFIID RNA transcription TATA box binding complex; nucleus polymerase II regulation of protein (TBP)- transcription transcription DNA- associated factor factor activity; dependent. DNA binding Implicated in adipose- genesis Core TAFII present in both of the previously described TFIID species which either lack or contain TAFII30 (TFIID alpha and TFIID beta respectively) (by similarity) Mm Nuclear factor I/B Nfib Intracellular; DNA binding; DNA replication; 4 317947 nucleus transcription forebrain factor activity development. Recognizes and binds to palindromic sequence 5’- ttggcnnnnngccaa-3’ present in viral and cellular promoters and in the origin of replication of adenovirus type 2. These proteins are individually capable of activating transcription and replication. Homozygous null mutants exhibit severe lung defects and open eyelids, and die soon after birth from respiratory failure. Heterozygotes exhibit delayed pulmonary differentiation Mm Decorin Dcn Dcn Protein binding Extracellular May affect the rate of 10 56769 space; fibrils formation (by proteinaceous similarity); may be extracellular implicated in the matrix dilation of the rat cervix. Mutant mice have fragile skin and exhibit abnormal collagen morphology in skin and tendons, supporting this gene's role in regulating collagen fiber formation Mm Chemokine Ccl2 Extracellular Chemokine Chemotaxis, immune 11 290320 (C-C motif) ligand region; activity; response. 2 extracellular cytokine Chemotactic factor space activity that attracts monocytes, but not neutrophils.

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Homozygous mutation of this gene results in impaired recruitment of monocytes and macrophages following intraperitoneal thioglycollate injection and reduced production of IL-4, IL- 5, and Ifn-gamma in splenocytes Mm Integral membrane Itm2b Membrane; ATP binding; Apoptosis; regulation 14 4266 protein 2B membrane protein binding of apoptosis by fraction inducing loss of mitochondrial membrane potential Mm Protease, serine, Prss23 Hydrolase Proteolysis Prss23 mRNA levels 7 250438 23 activity; decrease transiently peptidase after ovulation activity; serine- induction and again in type the postovulatory endopeptidase period activity Mm Peripheral myelin Pmp22 Extracellular Cell cycle; cell Might be involved in 11 1237 protein space, integral to growth arrest growth regulation and membrane specific in myelination in the peripheral nervous system. Mice with one or two copies of several mutations exhibit tremors, a tendency toward seizures, and partial paralysis associated with demyelination and loss of peripheral axons. Mutants have high juvenile mortality and males are often sterile Mm Insulin-like growth Igfbp5 Extracellular Growth factor Regulation of cell 1 405761 factor binding region; binding; growth. IGF-binding protein 5 extracellular insulin-like proteins prolong the space growth factor half-life of the IGFs binding and have been shown to either inhibit or stimulate the growth promoting effects of the IGFs on cell culture; they alter the interaction of IGFs with their cell surface receptors Mm Myosin, light Myl6 Cytoskeleton; Calcium ion Cytoskeleton 10 337074 polypeptide 6, myosin complex; binding; motor organization and alkali, smooth unconventional activity; biogenesis, muscle muscle and non- myosin structural filament sliding; muscle constituent of striated muscle muscle development Mm TSC22 domain Tsc22d1 Nucleus Transcription Regulation of 14 153272 family, member 1 factor activity transcription; DNA- (Transforming dependent; growth factor transcription. beta 1 induced Transcriptional transcript 4) repressor. Acts on the C-type natriuretic peptide (CNP) promoter

222

Mm Secreted acidic Sparc Basement Calcium ion Transmembrane 11 291442 cysteine rich membrane; binding; copper receptor protein glycoprotein extracellular ion binding tyrosine kinase space signaling pathway. Appears to regulate cell growth through interactions with the extracellular matrix and cytokines; binds calcium and copper, several types of collagen, albumin, thrombospondin and cell membranes. There are two calcium binding sites: an acidic domain that binds 5-8 Ca+2 with a low affinity and a left- hand loop that binds a Ca+2 with a high affinity. Homozygotes for targeted null mutations exhibit cataracts, reduced skin collagen content, accelerated wound closure, osteopenia associated with reduced bone remodeling, and increased growth of implanted tumors Mm Protocadherin 18 Pcdh18 Integral to Calcium ion Brain development; 3 87246 plasma binding; protein calcium-dependent membrane binding cell-cell adhesion; homophilic cell adhesion. Could function as a cell- adhesion protein; acts as a tumor suppressor; required for correct morphogenesis Mm Oral-facial-digital Ofd1 Basal body Molecular Cilium axoneme X 247480 syndrome 1 gene function biogenesis. May be homolog (human) involved in differentiation of met nephric precursor cells. Hemizygous conditional deletion of this gene results in embryonic lethality during organogenesis, impaired left-right axis patterning, and malformation of Henson's node cells. Heterozygous conditional deletion of this gene results in neonatal lethality, cystic kidneys, polydactyly and cleft palate. Mm Insulin-like growth Igf2 Extracellular Growth factor Cell proliferation; 7 3862 factor 2 region; activity; organ extracellular hormone morphogenesis. The space activity IGFs possess growth-

223

promoting activity. In vitro they are potent mitogens for cultured cells; IGF2 is influenced by placental lactogen and may play a role in fetal development. Mutations that are paternally transmitted result in growth deficiency. Heterozygous mice inheriting a mutant allele from their mother appear to be phenotypically normal Mm S100 calcium S100a11 Cytoplasm; Calcium ion Cytokine which 3 280038 binding protein extracellular binding activates host A11 (calizzarin) space cytokine response activity mechanisms. Mice with disruptions in this gene display no obvious phenotype abnormalities other than reduced sperm counts in males Mm Chondroitin sulfate Cspg2 Proteinaceous Calcium ion Cell adhesion; heart 13 158700 proteoglycan 2 extracellular binding; development; matrix hyaluronic acid Homozygotes for binding insertional mutation exhibit anterior- posterior segmental defects of the heart, lack endocardial cushions of the conus and atrioventricular region, and die around embryonic day 10.5 Mm Actin alpha 2, Acta2 Actin filament; ATP binding; Cytoskeleton 19 213025 smooth muscle, cytoskeleton nucleotide organization and aorta binding; protein biogenesis. Most of binding; actins are highly structural conserved proteins constituent of that are involved in cytoskeleton; various types of cell structural motility and are molecule ubiquitously activity expressed in all eukaryotic cells. Polymerization of globular actin (G- actin) leads to a structural filament (F- actin) in the form of a two-stranded helix. Each actin can bind to 4 others. In vertebrates 3 main groups of actin isoforms, alpha, beta and gamma have been identified. The alpha actins are found in muscle tissues and are a major constituent of the contractile apparatus. The beta

224

and gamma actins coexist in most cell types as components of the cytoskeleton and as mediators of internal cell motility Mm Actin, beta, Actb Actin filament; ATP binding, Murine homozygous 5 391967 cytoplasmic cytoskeleton; nucleotide null mutants are cytosol; soluble binding; protein embryonic lethal. fraction binding Homozygotes for a structural hypomorphic targeted constituent of mutation develop cytoskeleton; normally until structural embryonic day 8.5; molecule are growth retarded activity by day 9.5 and die shortly thereafter. Dystrophin and utrophin bind actin through distinct modes of contact Mm Periostin, Postn Extracellular Heparin Cell adhesion, 3 236067 osteoblast specific space, binding, protein extracellular matrix factor proteinaceous binding organization and extracellular biogenesis, tissue matrix development. Homozygous null mice display abnormalities of the enamel, periodontal ligament, ameloblasts and incisors Mm Glutamyl Enpep Apical part of cell; Aminopeptidas Angiogenesis, cell 3 1193 aminopeptidase apical plasma e activity, migration. Appears to membrane calcium ion have a role in the binding catabolic pathway of the rennin- angiotensin system; probably plays a role in regulating growth and differentiation of early b-lineage cells Mm Lysosomal Lamp2 Integral to Aminoacyl- tRNA amino X 486 membrane membrane; late tRNA ligase acetylation for protein glycoprotein 2 endosome activity; ATP- translation. Implicated binding in tumor cell metastasis, may function in protection of the lysosomal membrane from autodigestion, maintenance of the acidic milieu of the lysosome, adhesion when expressed on the cell surface (plasma membrane), and inner- and intracellular signal transduction. The majority of hemizygous or homozygous mutant mice die prematurely displaying cardiomyopathy and accumulation of autophagic vacuoles in several tissues including liver,

225

pancreas, spleen, kidney and skeletal and cardiac muscle Mm NADH Ndufa4 Mitochondrial NADH Transfer of electrons 6 415865 dehydrogenase inner membrane; dehydrogenas from NADH to the (ubiquinone) mitochondrion e (ubiquinone) respiratory chain, the 1 alpha sub activity; NADH immediate electron complex 4 dehydrogenas acceptor for the e activity enzyme is believed to be ubiquinone (by similarity) Mm Epithelial Emp1 Integral to Cell growth Role of the adhesion 9 182785 membrane membrane, molecule, EMP-1, as protein 1 membrane a biomarker of gefitinib clinical resistance, and further suggests a probable cross-talk between this molecule and the epidermal growth factor receptor signaling pathway. GEFITINIB – marketed by Astra- Zeneca as IRESSA, an immunotherapy drug used for lung cancer. Mm Procollagen type1 Col1a2 Collagen; Extracellular Cell adhesion; 6 277792 alpha 2 cytoplasm matrix phosphate transport. structural Type I collagen is a constituent; member of group I extracellular collagen (fibrillar matrix forming collagen). structural Forms the fibrils of constituent tendons, ligaments conferring and bones. In bones tensile strength the fibrils are mineralized with calcium hydroxyapatite. Belongs to the fibrillary collagen family Mm Procollagen, Col3a1 Collagen; Extracellular Cell adhesion; 1 249555 type III, alpha 1 cytoplasm matrix phosphate transport. . structural Collagen type III constituent; occurs in most soft extracellular connective tissues matrix along with type I structural collagen. constituent Most homozygous conferring mutants die within 48 tensile strength hours after birth. Surviving mutants have reduced body size, skin lesions, enlarged intestines, and die by 6 months of age from ruptured blood vessels. Occasionally intestinal rupture also results in early death Mm Procollagen type V Col5a Basement Extracellular Cell adhesion; 2 7281 alpha 1 membrane; matrix phosphate transport. collagen structural Type V collagen is a constituent; member of group I extracellular collagen (fibrillar

226

matrix forming collagen). It is structural a minor connective constituent tissue component of conferring nearly ubiquitous tensile strength distribution. Type V collagen binds to DNA, heparan sulfate, thrombospondin, heparin, and insulin (By similarity). Transcriptionally activated by CEBPZ, which recognizes a CCAAT-like motif in the COL5A1 promoter. Homozygous mutation of this gene results in lethality around E10-11 due to cardiovascular insufficiency and lack of collagen fibril formation. Heterozygotes exhibit poorly organized and less dense fibers in the dermis and reduced skin tensile strength and are a model for Ehlers- Danlos Syndrome Mm Procollagen type Col6a3 Collagen Endopeptidase Cell adhesion. 1 7562 VI alpha 3 extracellular inhibitor Collagen typeVI is space activity; specific for basement extracellular membranes, vital for matrix embryonic structural development constituent conferring tensile strength Mm Junction Jup Cell-cell Protein binding Cell adhesion. 11 299774 plakoglobin adherens Common junctional junction; plaque protein; the cytoskeleton membrane- associated plaques are architectural elements in an important strategic position to influence the arrangements and function of both the cytoskeleton and the cells within the tissue. The presence of plaktoglobin in both the desmosomes and in the intermediate junctions suggests that it plays a central role in the structure and function of submembranous plaques. Homozygous null mutants die with severe heart defects at embryonic day 10.5-16, depending

227

on genetic background. Mutants that survive to birth exhibit skin blistering and subcorneal acantholysis associated with reduced number of desmosomes Mm OVO homolog-like Ovol1 Intracellular; DNA binding, Epidermis 19 280225 (Drosophila) nucleus metal ion development; binding mesoderm development. Putative transcription factor; involved in hair formation and spermatogenesis; may function in the differentiation and/or maintenance of the urogenital system (by similarity). Null mutant homozygotes show reduced growth, abnormal hair, and cystic kidneys. Females are sub fertile with dilated uterus and cervix, and constricted or imperforate vagina. Mutant males have small testes, with few mature germ cells Mm Tumor necrosis Tnfrsf1a Integral to Electron carrier Apoptosis; cell 6 1258 factor receptor membrane activity; heme surface receptor superfamily, binding linked signal member 1a transduction. Receptor for Tnfsf2/tnf-alpha and homotrimeric Tnfsf1/lymphotoxin alpha; the adapter molecule FADD recruits Caspase-8 to the activated receptor. The resulting death- inducing signaling complex (disk) performs Caspase-8 proteolytic activation which initiates the subsequent cascade of caspases (aspirate-specific cysteine proteases) mediating apoptosis (by similarity). Homozygotes for targeted null mutations exhibit disrupted splenic architecture, increased adult liver weights, reduced IgG immune response, deficits in some host defense and inflammatory

228

responses, LPS resistance, and reduced graft-vs-host disease. Mm Tumor necrosis Tnfrsf12 Cell surface; Protein Angiogenesis; 17 28518 factor receptor a integral to binding; apoptosis; cell superfamily, membrane; receptor adhesion; cell death; member 12a membrane; activity cell differentiation; plasma development; positive membrane; ruffle regulation of axon extension; substrate- bound cell migration; cell attachment to substrate. Receptor for TNFSF12/TWEAK (by similarity). Weak inducer of apoptosis in some cell types. Promotes angiogenesis and the proliferation of endothelial cells. May modulate cellular adhesion to matrix proteins. Highly expressed in fetal heart, intestine, kidney, liver, lung and skin, and in adult heart and ovary. Intermediate expression in adult kidney, lung and skin Mm Thrombospondin 1 Thbs1 Extracellular Calcium ion Cell adhesion; 2 4159 region; binding; inflammatory extracellular heparin response; negative space binding; protein regulation of binding; angiogenesis. structural Adhesive glycoprotein molecule that mediates cell-to- activity cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin, type V collagen and integrins alpha- V/beta-1, alpha- V/beta-3 and alpha- IIb/beta-3 Mm Thrombospondin 2 Thbs2 Extracellular Calcium ion Cell adhesion. 17 26688 region; binding; Thrombospondins extracellular heparin deployed by space binding; protein thrombopoietic cells binding; determine angiogenic structural switch and extent of molecule revascularization. activity Adhesive glycoprotein that mediates cell-to- cell and cell-to-matrix interactions. Can bind to fibrinogen, fibronectin, laminin and type V collagen. Mm Glucosaminyl Gcnt1 Integral to Acetylglucosa Forms critical 19 244825 (N-acetyl) membrane minyltransferas branches in o- transferase 1, e activity, beta- glycans. Mice core 2 1,3-galactosyl- homozygous for O-glycosyl- disruptions in this glycoprotein allele display a beta-1,6-N- grossly normal

229

acetylglucosa phenotype and are minyl- fertile. There are transferase abnormalities in white activity blood cell counts and in inflammatory response however Mm Microsomal Mgst1 Endoplasmic Glutathione Glutathione metabolic 6 14796 glutathione reticulum; transferase process. Conjugation S-transferase 1 membrane activity; of reduced transferase glutathione to a wide activity number of exogenous and endogenous hydrophobic electrophiles Mm H19 fetal liver H19 Cytoplasm The murine 7 14802 mRNA H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo Mm Heat shock factor Hsbp1 Nucleus Transcription Negative regulation of 8 358714 binding protein 1 corepressor transcription from activity RNA polymerase II promoter; Negative regulator of the heat shock response. Negatively affects HSF1 DNA-binding activity. May have a role in the suppression of the activation of the stress response during the aging process (by similarity) Mm A disintegrin-like Adamts5 Extracellular Hydrolase Integrin mediated 16 112933 metallopeptidase matrix; activity; metal signaling pathway; (reprolysin type) proteinaceous ion binding protein amino acid with extracellular prenylation. Cleaves thrombospondin matrix aggrecan, a cartilage type 1 motif 5 proteoglycan and (aggrecanase-2) may be involved in its turnover; may play an important role in the destruction of aggrecan in arthritic diseases; may play a role in proteolytic processing mostly during the peri- implantation period. Mice homozygous for one null allele exhibit a significant reduction in cartilage degradation after induction of osteoarthritis whereas those homozygous for another show no affect Mm A disintegrin-like Adamts1 Extracellular Hydrolase Biological function – 13 115970 and 6 matrix; activity; metal proteolysis metallopeptidase protinaceous ion binding;

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(reprolysin type) extracellular metalloendope with matrix ptidase activity; thrombospondin metallopeptida type 1 motif 16 se activity; molecular function; peptidase activity; zinc ion binding Mm RIKEN CDNA 4930432 Intracellular; Metal ion Regulation of 17 26540 4930432O21Rik O21Rik nucleus binding; zinc transcription, DNA- gene ion binding; dependent. It may nucleic acid play a role in renal binding development and may also be involved in the repair of the kidney after ischemia- reperfusion or folic acid administration Mm Nur77 downstream Ndg1 Cytoplasm Caspase Research by Rajpal 1 26006 gene 1 activation; et al. suggests by a induction of direct assay that apoptosis Ndg1 is in the cytoplasm and is involved in the induction of apoptosis and caspase activation Mm ATP synthase, H+ Atp51 Mitochondrial Hydrogen ion ATP biosynthesis; 9 14663 transporting, inner membrane; transporter ATP synthesis mitochondrial F0 mitochondrion; activity; coupled proton complex, subunit g proton- hydrogen- transport; ion and transporting ATP transporting proton transport synthase ATPase complex; proton- activity, transporting ATP phosphorylativ synthase e mechanism; complex, hydrogen- coupling factor F transporting (o); proton- ATPase transporting two- synthase sector ATPase activity, complex rotational mechanism; hydrogen- transporting ATPase activity, rotational mechanism; metal ion binding Mm CD24a antigen Cd24a External site of GPI anchor Neuromuscular 10 29742 plasma binding synaptic membrane transmission; synaptic vesicle endocytosis. May have a specific role to play in early thymocyte development. May have a pivotal role in cell differentiation, the triggering mechanism of signal transduction may be due to the interactions of differentiating cells with the matrix substrate via the

231

carbohydrate structure of the molecule. In this way the signal transducer can play very different roles in different cell types as a direct consequence of its glycosylation. Homozygous mutation of this gene results in slight impairment of B cell development. Mutant erythrocytes have increased tendency to aggregate Mm Serine (or Serpinf1 Neurotrophic Potent inhibitor Loss of function 11 2044 cysteine) protein; induces of results in increased peptidase inhibitor, extensive angiogenesis. microvasculature, clade F, member 1 neuronal As it does not pancreatic differentiation in undergo the S enlargement, and retinoblastoma (stressed) to R prostatic hyperplasia. cells. (relaxed) conformational transition characteristic of active serpins, it exhibits no serine protease inhibitory activity Mm Amyloid beta (A4) App Apical part of cell; Binding; Adult locomotory 16 277585 precursor protein axon; ciliary copper ion behavior; apoptosis; rootlet; coated binding; DNA axon cargo transport; pit; cytoplasm; binding; axon midline choice cytoplasmic endopeptidase point recognition; vesicle; Golgi inhibitor axon genesis; cell apparatus; activity; adhesion; collateral integral to heparin sprouting in the membrane; binding; iron absence of injury; membrane; ion binding; copper ion membrane metal ion homeostasis; dendrite fraction; binding; protein development; neuromuscular binding; serine- endocytosis; junction; neuron type extracellular matrix projection; endopeptidase organization and perinuclear inhibitor biogenesis; forebrain region; spindle activity; zinc development; G2 midzone ion binding phase of mitotic cell cycle; locomotory behavior; mating behavior; mRNA polyadenylation; neurite development; neuron remodeling; Notch signaling pathway; positive regulation of progression through mitotic cell cycle; positive regulation of transcription from RNA polymerase II promoter; regulation of balance; regulation of body size; regulation of synapse

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structure and function; regulation of translation; smooth endoplasmic reticulum calcium ion homeostasis; suckling behavior; synaptic growth at neuromuscular junction; visual learning. Mice homozygous for disruption in this gene exhibit reduced body weight, brain weight, size of forebrain commissures, locomotor activity, forelimb grip strength, and spatial learning scores. Many mice also exhibit agenesis of the corpus callosum, and extensive reactive gliosis Mm Elastin Eln Proteinaceous Extracellular Regulation of actin 5 275320 extracellular matrix filament matrix structural polymerization; stress constituent; fiber formation. Major protein binding structural protein of tissues such as aorta and nuchal ligament, which must expand rapidly and recover completely. Molecular determinant of the late arterial morphogenesis, stabilizing arterial structure by regulating proliferation and organization of vascular smooth muscle. Mice homozygous for a disruption in this gene die postnatally of an obstructive arterial disease. They exhibit a decrease in arterial diameter due to sub endothelial accumulation of arterial smooth muscle Mm Cyclin D1 Ccnd1 Cyclin-dependent Cyclin- Cell cycle; cell 7 389995 protein kinase dependent division; fat cell holoenzyme protein kinase differentiation; protein complex; cytosol; regulator amino acid nucleus activity; kinase phosphorylation; re- activity; protein entry into mitotic cell binding; protein cycle; regulation of kinase activity progression through cell cycle; unfold protein response. Homozygotes for targeted mutations exhibit reduced body

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size and viability, impaired retinal development, pregnancy-insensitive mammary glands, and resistance to breast cancer induced by neu and ras oncogenes Mm Stromal antigen 1 Stag1 Nucleus Binding Cell cycle; cell 9 42135 division; chromosome segregation; meiosis; mitosis. Component of cohesin complex, a complex required for the cohesion of sister chromatids after DNA replication. The cohesin complex apparently forms a large proteinaceous ring within which sister chromatids can be trapped. At anaphase, the complex is cleaved and dissociates from chromatin, allowing sister chromatids to segregate. The cohesin complex may also play a role in spindle pole assembly during mitosis (by similarity) Mm Tropomyosin 1 Tpm1 Cytoplasm; Actin binding, Embryonic 9 121878 alpha cytoskeleton structural development, in utero constituent of embryonic cytoskeleton development. Tropomyosin in association with the troponin complex plays a central role in the calcium dependent regulation of muscle contraction. Mice homozygous for disruptions in this gene display embryonic lethality Mm Tropomyosin 2 Tpm2 Cytoskeleton; Actin binding; Muscle contraction; 4 646 beta muscle thin protein binding; Binds to actin filament structural filaments in muscle tropomyosin constituent of and non-muscle cells. cytoskeleton Plays a central role, in association with the troponin complex, in the calcium dependent regulation of vertebrate striated muscle contraction. Smooth muscle contraction is regulated by interaction with caldesmon. In non- muscle cells is implicated in stabilizing

234

cytoskeleton actin filaments Mm Nuclear receptor Nr2f2 Nucleus DNA binding; Anterior/posterior 7 158143 subfamily 2, ligand- pattern formation; group F, member dependent blood vessel 2 nuclear morphogenesis. receptor Regulation of the activity; apolipoprotein A-I gene transcription, binds to DNA site A (by similarity). Homozygotes for a targeted null mutation exhibit impaired angiogenesis and heart development with hemorrhagic brains and hearts, and die around embryonic day 10. About 5% of heterozygotes share the hemorrhagic phenotype at embryonic day 9.5 Mm TRM5 tRNA Trmt5 tRNA processing Methyl Specifically 12 311344 methyl transferase transferase methylates 5 homolog activity; guanosine-37 in (S. cerevisiae) transferase various tRNAs. Not activity dependent on the nature of the nucleoside 5' of the target nucleoside (by similarity) Mm Intersectin 1 (SH3 Itsn1 Intracellular, Calcium ion Regulator of 16 40546 domain protein 1A) lamellipodium binding; endocytosis; guanyl- intracellular signaling nucleotide cascade. Adapter exchange protein that may activity provide indirect link between the endocytic membrane traffic and the actin assembly machinery. May regulate the formation of clathrin- coated vesicles. Homozygous disruption of this locus is embryonic lethal. Mm Cofilin 2, muscle Cfl2 Cytoskeleton; Controls It has the ability to 12 276826 intracellular; reversibly actin bind G- and F-actin in nucleus; polymerization a 1:1 ratio of cofilin to cytoplasm and de- actin. It is the major polymerization component of in a pH- intranuclear and sensitive cytoplasmic actin manner rods Mm Activated Alcam Axon; cell soma Protein binding Axon guidance; cell 16 288282 leukocyte cell adhesion molecule adhesion molecule that binds to CD6. Involved in neurite extension by neurons via heterophilic and homophilic interactions. May play a role in the binding of T- and B-cells to activated leukocytes,

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as well as in interactions between cells of the nervous system. Homozygous null mice display abnormal motor neuron and retinal ganglion cell morphology and retinal dysplasia Mm Paternally Peg3 Intracellular; Metal ion Induces apoptosis in 7 389800 expressed 3 nucleus binding; cooperation with nucleic acid SIAH1A. Acts as a binding; zinc mediator between ion binding TP53/p53 and BAX in a neuronal death pathway that is activated by DNA damage. Acts synergistically with TRAF2 and inhibits TNF induced apoptosis through activation of NF- kappa-B. Plays a role in regulating maternal behavior and offspring growth Mm Pleiomorphic Plagl1 Intracellular; DNA binding; Positive regulation of 10 287857 adenoma nucleus metal ion transcription from gene-like 1 binding; zinc RNA polymerase II ion binding promoter. Shows weak transcriptional activatory activity. Transcriptional regulator of the type 1 receptor for pituitary adenylate cyclase- activating polypeptide Mm Chemokine Cxcl12 Extracellular Chemokine Ameboidal cell 6 303231 (C-X-C motif) region activity; migration; brain ligand 12 cytokine development; activity; growth chemotaxis; germ cell factor activity development; germ cell migration; immune response; induction of positive chemotaxis; motor axon guidance; patterning of blood vessels; positive regulation of cell migration; regulation of cell migration; T cell proliferation. Chemoattractant active on T- lymphocytes, monocytes, but not neutrophils. Stimulates the proliferation of bone marrow-derived b progenitor cells in the presence of IL-7 as well as growth of the stromal cell- dependent B-cell clone DW34 cells. Homozygous null

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mice display late embryonic lethality, impaired myelopoiesis, abnormal cerebellum development, abnormal germ cell migration, abnormal angiogenesis around the stomach, and ventricular septal defects Mm Pleiotrophin Ptn Extracellular Growth factor Bone mineralization; 6 279690 space; activity learning; ossification; protinaceous regulation of extracellular progression through matrix cell cycle. Heparin binding mitogenic protein. Has neurite extension activity (by similarity). Homozygous null mice exhibit enhanced long term potentiation, an impairment of spatial learning, and increased anxiety. The brains of mutant mice are morphologically normal Mm Purine rich Purb DNA replication DNA binding; Apoptosis; cell 11 296150 element binding factor A double- differentiation; cell protein B stranded DNA proliferation; DNA binding; replication initiation; double- DNA unwinding stranded during replication; telomeric DNA negative regulation of binding; mRNA transcription DNA- binding; protein dependent; regulation binding; RNA of myeloid cell polymerase II differentiation; transcription regulation of factor activity, transcription, DNA- enhancer dependent; binding; single- transcription. Has stranded DNA capacity to bind binding; SMAD repeated elements in binding; single-stranded DNA transcription such as the purine- factor activity; rich single strand of transcription the PUR element factor binding located upstream of the MYC gene. Participates in transcriptional and translational regulation of alpha- MHC expression in cardiac myocytes by binding to the purine- rich negative regulatory (PNR) element. Modulates constitutive liver galectin-3 gene transcription by binding to its promoter. May play a

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role in the dendritic transport of a subset of mRNAs (by similarity). Plays a role in the control of vascular smooth muscle (VSM) alpha- actin gene transcription as repressor in myoblasts and fibroblasts Mm Polymerase (RNA) Polr2h Nucleus Nucleotidyltran DNA-dependent RNA 16 288730 II (DNA directed) sferase activity polymerase catalyzes polypeptide H the transcription of DNA into RNA using the four ribonucleotide triphosphates as substrates (by similarity) Mm G protein-coupled Gpr177 Integral to Receptor Protein of unknown 3 6766 receptor 177 membrane; activity function membrane Mm CDC28 protein Cks2 Cyclin-dependent Cell cycle; cell Binds to the catalytic 13 222228 kinase regulatory protein kinase division; subunit of the cyclin subunit 2 regulator activity; meiosis dependent kinases kinase activity and is essential for their biological function. Forms an homohexamer that can probably bind six kinase subunits Mm Cysteine rich Cyr61 Extracellular Growth factor Cell adhesion; 3 1231 protein 61 region; binding; chemotaxis. extracellular heparin binding Promotes cell space proliferation, chemotaxis, angiogenesis and cell adhesion. Appears to play a role in wound healing by up- regulating (in skin fibroblasts) the expression of a number of genes involved in angiogenesis, inflammation and matrix remodeling including: VEGA-A, VEGA-C, MMP1, MMP3, TIMP1, UPA, PAI-1 and integrins: ALPHA-3 and ALPHA-5. Cyr61- mediated gene regulation is dependent on heparin-binding. Down-regulates the expression of ALPHA-1 and ALPHA-2 subunits of collagen type-1. Promotes cell adhesion and adhesive signaling through integrin ALPHA-6/BETA-1,

238

cell migration through integrin ALPHA- 5/BETA-5 and cell proliferation through integrin ALPHA- 5/BETA-3. Targeted null mice die pre- or perinatally, and none survive beyond 24 hrs of birth. About 30% of embryos die by E10.5 from defects in chorioallantoic fusion, whereas 70% die from placental vascular defects, including impaired allantoic vessel bifurcation, and loss of large-vessel integrity Mm RIKEN cDNA 9530068 Integral to Not known Function not 11 291979 9530068E07 gene E07Rik membrane. determined Mm Nucleosome Nap1l5 Nucleus Nucleosome Function not 6 389747 assembly protein assembly determined 1-like 5 Mm DEP domain Depdc1a Intracellular Function unknown 3 371655 containing 1 a signaling cascade. Mm Hypothetical - - - - X 396053 protein, clone mvx2015 Mm cDNA, - - - - 11 391808 clone:Y2G0106I17 , strand: unspecified Mm RIKEN cDNA 4930473 - - Muscle contraction 4 180032 4930473A06 gene A 06Rik Mm RIKEN CDNA 3930401 - - - 13 391849 3930401B19 gene B19Rik Mm In vitro fertilized - Product: - - -- 400420 eggs cDNA, unclassifiable, full RIKEN full-length insert sequence. enriched library, clone 7420405G17 Mm RIKEN cDNA 4933427 - - - 6 281744 4933427D06 gene D06 Mm Transcribed locus - - - - 12 394798 Mm DNA segment, D0H4S1 - - - 18 407415 human D4S114 14 Mm Delta-like 1 Dlk1 - - - 12 157069 homolog (Drosophila) Mm Data not found - - - - 431975 Mm Transcribed locus, - - - - 19 410445 weakly similar to XP_912058.1 hypothetical protein XP_906965 (Mus musculus) Mm Transcribed locus - - - - 10 380595

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Table A2-3. Comparison of microarray gene expression levels down-regulated in cells treated with serelaxin and bacterially expressed RLN H2, respectively. No significant differences were found.

Unique gene Cells treated with Cells treated with bacterially- ID serelaxin derived RLN H2 Day 4 vs Day 8 vs Day 12 Day 4 vs Day 8 vs Day 12 vs control control vs control control control control Unknown -2.26 -2.62 -2.53 -2.57 -2.39 -2.44 Mm. 30153 -1.15 -1.16 -0.28 -0.72 -0.71 -1.16 Mm. 213025 -1.19 -1.32 -0.85 -1.08 -1.02 -1.25 Unknown -3.59 -3.80 -3.35 -3.57 -3.47 -3.69 Unknown -1.03 -1.06 -1.14 -1.10 -1.08 -1.04 Mm. 180032 -3.06 -2.12 -3.30 -2.71 -3.18 -2.59 Mm. 236067 -1.60 -1.68 -1.31 -1.49 -1.45 -1.64 Unknown -1.03 -1.04 -0.30 -1.03 -1.03 -1.04 Mm. 1237 -1.35 -1.52 -1.39 -1.45 -1.37 -1.43 Mm. 396053 -2.79 -2.85 -2.87 -2.86 -2.83 -2.82 Mm. 980 -1.53 -1.39 -0.37 -1.38 -1.45 -1.46 Mm. 332720 -1.03 -1.07 -0.23 -1.15 -1.13 -1.05 Mm. 153272 -1.41 -1.34 -1.21 -1.27 -1.31 -1.37 Mm. 40546 -1.30 -0.88 -1.12 -1.50 -1.21 -1.09 Mm. 1231 -1.57 -1.89 -1.11 -1.50 -1.34 -1.73 Mm. 410445 -2.71 -2.27 -1.65 -1.96 -2.18 -2.49 Unknown -1.94 -1.26 -0.57 -1.41 -1.30 -1.15 Mm. 7562 -1.19 -1.37 -1.66 -1.51 -1.43 -1.28 Mm. 200362 -1.23 -1.17 -0.42 -1.29 -1.32 -1.20 Mm. 247480 -2.31 -2.19 -1.79 -1.98 -2.05 -2.25 Mm. 340911 -1.16 -1.28 -0.37 -1.32 -1.27 -1.22 Mm. 328360 -1.22 -1.02 -0.75 -0.88 -0.98 -1.12 Mm. 407415 -1.02 -0.89 -1.03 -0.96 -1.30 -0.95 Unknown -1.14 -1.23 -1.01 -1.12 -1.07 -1.18 Mm. 3862 -1.14 -1.23 -1.37 -1.30 -1.25 -1.18 Mm. 38241 -1.20 -1.24 -0.40 -1.32 -1.30 -1.22 Mm. 330116 -1.15 -1.08 -0.32 -1.20 -1.23 -1.11 Mm. 426616 -1.16 -1.08 -0.18 -1.13 -1.17 -1.12 Unknown -2.79 -2.74 -3.08 -2.91 -2.93 -2.76 Mm. 405761 -2.00 -2.32 -1.88 -2.10 -1.94 -2.16 Mm. 239117 -1.23 -1.28 -0.18 -1.23 -1.20 -1.25 Mm. 332522 -1.21 -1.06 -0.23 -1.14 -1.22 -1.13 Unknown -1.31 -0.83 -1.18 -1.00 -1.24 -1.07 Mm. 318250 -0.13 -1.04 -1.57 -1.30 -1.35 -1.08 Unknown -1.15 -1.15 -0.38 -1.26 -1.26 -1.15 Unknown -1.03 -1.22 -0.26 -1.24 -1.14 -1.12

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Mm. 337074 -1.28 -1.16 -1.17 -1.16 -1.22 -1.22 Unknown -1.52 -1.60 -0.65 -1.62 -1.58 -1.56 Mm. 273195 -0.97 -1.67 -1.45 -1.56 -1.21 -1.32 Mm. 1193 -1.12 -1.32 -1.10 -1.21 -1.11 -1.22 Mm. 34871 -1.42 -1.28 -0.39 -1.33 -1.40 -1.35 Mm. 182785 -1.86 -1.98 -1.43 -1.70 -1.64 -1.92 Mm. 250438 -1.01 -1.04 -1.03 -1.70 -1.02 -1.02 Mm. 277792 -1.30 -1.34 -1.68 -1.51 -1.49 -1.32 Mm. 290320 -1.36 -1.41 -1.37 -1.39 -1.36 -1.38 Unknown -1.10 -1.26 -0.65 -0.95 -0.87 -1.18 Mm. 249555 -3.30 -3.31 -3.24 -3.27 -3.27 -3.30 Unknown -1.03 -1.14 -0.56 -1.10 -1.04 -1.08 Mm. 4266 -1.43 -1.37 -1.45 -1.41 -1.44 -1.40 Unknown -1.17 -1.20 -0.38 -1.29 -1.27 -1.18 Mm. 400420 -1.60 -1.80 -1.66 -1.73 -1.63 -1.13 Unknown -1.10 -1.17 -0.69 -1.18 -1.14 -1.13 Mm. 4159 -1.27 -0.94 -1.11 -1.02 -1.19 -1.10 Mm. 279690 -1.05 -0.83 -1.24 -1.03 -1.14 -0.94 Mm. 182574 -0.48 -1.22 -1.02 -1.12 -1.00 -1.10 Unknown -1.23 -1.23 -0.32 -1.27 -1.26 -1.22 Mm. 291442 -0.99 -1.09 -1.01 -1.05 -1.01 -1.04 Unknown -1.11 -1.36 -0.49 -1.17 -1.05 -1.23 Mm. 391849 -3.30 -3.56 -3.01 -3.28 -3.15 -3.43 Mm. 280038 -1.19 -1.44 -1.38 -1.44 -1.28 -1.31 Mm. 209071 -1.10 -1.04 -0.39 -1.21 -1.24 -1.07 Mm. 244825 -1.01 -1.00 -0.82 -0.91 -0.92 -1.00 Unknown -1.03 -1.09 -0.75 -0.92 -0.89 -1.06 Mm. 267998 -2.84 -2.93 -2.57 -2.75 -2.70 -2.88 Mm. 280225 -1.19 -1.47 -1.67 -1.57 -1.43 -1.33 Mm. 2044 -0.84 -1.04 -1.14 -1.09 -0.99 -0.94 Mm. 296150 -0.83 -1.23 -1.18 -1.20 -1.00 -1.03 Mm. 391808 -4.15 -3.98 -3.78 -3.88 -3.96 -3.81 Unknown -1.21 -1.38 -1.22 -1.30 -1.21 -1.29 Mm. 181959 -1.47 -1.33 -0.12 -1.22 -1.29 -1.40 Mm. 87246 -1.06 -1.15 -1.20 -1.17 -1.13 -1.10 Mm. 394798 -1.65 -1.60 -1.38 -1.49 -1.51 -1.62 Mm. 26540 -1.03 -1.06 -0.81 -0.93 -0.92 -1.04 Mm. 415865 -1.17 -1.01 -1.23 -1.12 -1.20 -1.09 Mm. 56769 -1.60 -1.71 -1.70 -1.70 -1.65 -1.66 Mm. 384634 -1.33 -0.67 -1.38 -1.33 -1.35 -1.31 Mm. 281744 -1.00 -1.11 -1.01 -1.06 -1.00 -1.05 Unknown -1.35 -1.38 -0.21 -1.29 -1.28 -1.36 Mm. 158143 -1.93 -1.44 -1.24 -1.34 -1.58 -1.68 Mm. 317947 -1.54 -1.48 -1.50 -1.49 -1.52 -1.51

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Table A2-4. Comparison of microarray gene expression levels up-regulated in cells treated with serelaxin and bacterially expressed RLN H2, respectively. No significant differences were found.

Unique gene Cells treated with Cells treated with bacterially- ID serelaxin derived RLN H2 Day 4 vs Day 8 vs Day 12 Day 4 vs Day 8 vs Day 12 vs control control vs control control control control Mm. 282706 0.94 1.21 1.21 1.20 1.07 1.08 Mm. 432028 1.37 1.56 1.08 1.32 1.22 1.46 Mm. 379939 1.31 1.60 1.17 1.38 1.24 1.45 Mm. 399372 1.24 1.06 0.63 1.09 1.18 1.21 Mm. 326799 1.39 1.54 1.10 1.32 1.24 1.46 Unknown 1.26 1.45 1.03 1.24 1.14 1.35 Mm. 133704 1.52 1.69 1.19 1.44 1.35 1.60 Mm. 6343 0.97 1.20 1.14 1.17 1.05 1.08 Mm. 396243 0.97 1.22 1.01 1.11 0.99 1.09 Mm. 22723 1.07 1.02 0.89 0.95 0.98 1.03 Mm. 5305 1.12 1.31 1.23 1.27 1.17 1.21 Mm. 245522 0.73 1.01 1.05 1.03 1.01 0.99 Unknown 1.00 1.46 0.92 1.19 0.98 1.23 Mm. 360075 1.31 1.37 1.13 1.25 1.03 1.08 Unknown 1.05 1.11 1.01 1.06 1.03 1.08 Mm. 17031 0.97 1.18 1.19 1.18 1.08 1.07 Unknown 1.10 1.16 0.96 1.06 1.03 1.13 Unknown 0.95 1.25 1.06 1.15 1.05 1.10 Mm. 399829 1.02 1.05 0.88 1.06 1.05 1.03 Unknown 1.36 1.49 0.84 1.41 1.35 1.42 Mm. 66 1.32 1.52 1.23 1.37 1.27 1.42 Unknown 1.22 1.45 1.15 1.30 1.18 1.33 Unknown 1.28 1.34 1.06 1.20 1.17 1.31 Mm. 272045 1.01 1.09 0.74 1.04 1.00 1.05 Mm. 221269 1.01 1.15 0.93 1.04 0.97 1.08 Mm. 30745 0.73 1.11 1.11 1.09 1.04 1.05 Mm. 33832 1.24 1.47 1.01 1.24 1.12 1.35 Mm. 396243 0.95 1.20 1.03 1.11 0.99 1.07

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Table A2-4. Comparison of microarray gene expression levels differentially- expressed (up-regulated at day 4, and down-regulated from day 8 forward) in cells treated with serelaxin and bacterially expressed RLN H2, respectively. No significant differences were found.

Unique gene Cells treated with Cells treated with bacterially- ID serelaxin derived RLN H2 Day 4 vs Day 8 vs Day 12 Day 4 vs Day 8 vs Day 12 vs control control vs control control control control Unknown 4.04 -1.78 -3.03 3.92 -1.65 -2.90 Unknown 3.95 -1.56 -2.88 3.83 -1.45 -2.77 Mm. 57415 3.79 -1.42 -2.45 3.84 -1.53 -2.56 Unknown 3.04 -1.07 -1.67 3.19 -1.22 -1.82 Unknown 4.13 -1.61 -2.80 4.00 -1.73 -2.60 Mm. 130054 1.15 -0.74 -1.06 1.20 -0.79 -1.11 Unknown 3.26 -1.61 -2.08 3.35 -1.77 -1.91 Mm. 126450 3.33 -1.24 -1.74 3.49 -1.40 -1.90 Mm. 335641 1.56 -0.37 -1.25 1.63 -1.15 -1.32 Unknown 3.47 -1.45 -2.11 3.64 -1.62 -1.93 Unknown 1.64 -0.66 -1.05 1.72 -0.74 -1.13 Unknown 3.90 -1.26 -2.11 3.70 -1.55 -1.91 Unknown 3.25 -1.10 -1.90 3.41 -1.26 -2.06 Unknown 3.94 -1.48 -2.68 3.74 -1.67 -2.48 Unknown 3.33 -1.07 -1.28 3.49 -1.23 -1.44 Unknown 4.04 -1.02 -2.87 3.83 -1.22 -2.67 Mm. 423302 3.03 -1.33 -1.74 3.18 -1.48 -1.89 Mm. 158650 2.40 -0.85 -1.37 2.52 -1.07 -1.49

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APPENDIX 3

Media and buffer preparation procedures:

Rubidium chloride method for transformation of competent E. coli.

Medium Psi broth (per liter)

• Bacto-yeast extract 5 g

• Bacto-tryptone 20 g

• Magnesium sulfate 5 g

Adjust pH to 7.6 with potassium hydroxide.

Tfb 1 medium (for 200 mL)

Adjust pH to 5.6 with dilute acetic acid and filter to sterilize (0.45 μM, Nalgene or

Millipore filters). Distribute into smaller size aliquots and use 1 volume of Tfb 1 per

2.5 volumes of culture.

Tfb 2 medium (for 100 mL)

Adjust pH to 6.5 with dilute NaOH or KoH and filter to sterilize (as above). Freeze the stock and use 1 volume Tfb 2 for 25 volumes of original culture.

Procedure

1. Inoculate a single colony from a rich agar plate (Luria, Luria-Bertani) into 2

mL of rich broth (RB; Luria, Luria-Bertani) in a plating tube. Shake overnight

at 37°C.

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2. Subculture the overnight growth 1:100 in 1 volume unit of Psi broth (typically

250 mL). Grow to OD 590 = 0.4-0.6 or Klett = 60 (approximately 2 to 3

hours).

3. Centrifuge at 5000 rpm for 5 minutes at 4°C.

4. Gently re-suspend cell pellet in 1/2.5 volume unit of ice cold Tfb 1.

From this point on, perform all steps on ice and use chilled pipette tips,

tubes, flasks etc.

5. Incubate on ice for 5 minutes.

6. Centrifuge 5000 rpm for 5 minutes at 4°C.

7. Re-suspend pellet in 1/25 (original culture volume) of cold Tfb II.

8. Incubate on ice for 15-60 minutes.

9. Aliquot 100μL per micro centrifuge tube for storage at -70°C. Quick-freeze

the tubes if possible.

To transform, thaw an aliquot of competent cells on ice and add plasmid

DNA or ligation reaction solution (2-20 μL). Incubate on ice for 1 hour. Heat shock in a water bath for 45-60 seconds at 37°C and transfer back onto ice for 2 minutes.

Dilute 15-fold into SOC medium with no antibiotics (to allow for phenotypic expression). Grow on a shaker with vigorous aeration (250 rpm) at 37°C for 20 minutes. Plate 100 μL of the transformation reaction culture and spread gently

(using a sterile glass spreader) on selective agar plates containing antibiotic. The plates should be covered up with aluminum foil and left on the lab bench for

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approximately 30 minutes. Next, the plates should be inverted with the agar side up and placed for overnight into a dry incubator set to 37 °C. This procedure works well with most strains of bacteria and should routinely give more than 107 colony forming units per microgram of pBR322 plasmid using K-12 E.coli derivatives.

Detailed plating techniques

Agar plates were removed from the incubator. Transformation reactions were done in 1.5 mL micro centrifuge tubes. To remove the transformation sample, tubes were gently flicked 4 – 5 times, caps opened and 100 μL volumes of transformation suspension were placed in the center of the plates. If there is less than 25 μL of the transformation, plating should be done onto a pool of 40 – 60 μL

SOC medium, which enables even colony distribution on the entire surface of the plate. For even distribution (seeding, spreading) of the dispensed transformation volumes, plating glass “hockey” spreaders were used. (Made in the laboratory using glass Pasteur pipettes formed in the flame of a propane torch to look like a hockey stick). A hockey spreader was completely immersed in ethanol and flamed to sterilize. Approximately 10 seconds after the flame was extinguished, the spreader was placed on an agar plate (away from the pool of cells in the center of the plate) to further cool the spreader. While turning the plate slowly and supporting the weight of the spreader, the pool of cells was evenly distributed on the plate. At this stage, the cells are very fragile. Pushing down the spreader to hard or

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overspreading is lethal to the cells. Seeded plates were left on the work bench, under cover of aluminum foil, for 15 – 30 minutes to allow excess moisture to absorb into the agar medium before the plates were inverted and placed into the

37 °C dry incubator for overnight. Plates were kept in the incubator for 15 – 18 hours. (Longer incubation will result in the formation of colonies overgrowth and carrying wild-type plasmids or antibiotic-resistant mutants.)

Luria Bertani (LB-M, Miller) medium

• Pancreatic digest of casein 10 g

• Yeast extract 5 g

• Sodium chloride 10 g

• Deionized water 1 Liter

All ingredients of the medium were dissolved in water using magnetic stirring bars (covered with Teflon) and a bench top stirrer-hot plate and Pyrex glass bottles. After adjusting pH to 7.0 ± 0.2 with sodium hydrochloride solution, the medium was sterilized in an autoclave for 15 minutes at 121° Celsius. When sufficiently cooled, the medium was placed in the lab fridge and kept refrigerated until needed. Antibiotics, glucose and/or maltose were always added to the medium just before planned use. Similar standard operating procedures (SOP) were implemented for preparation of and when using other kinds (listed below) of bacteriological media.

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Luria Bertani (LB-L, Lennox) medium

• Pancreatic digest of casein 10 g

• Yeast extract 5 g

• Sodium chloride 5 g

• Deionized water 1 Liter

Final pH 7.0 ± 0.2 adjusted with sodium hydrochloride solution.

Luria Bertani (LB, Luria or LB Base) medium

• Pancreatic digest of casein 10 g

• Yeast extract 5 g

• Sodium chloride 0.5 g

• Deionized water 1 Liter

Final pH 7.0 ± 0.2 adjusted with sodium hydrochloride solution.

Super broth (Select APS)

• Soy hydrolysate 12 g

• Yeast extract 24 g

• Dipotassium phosphate 11.4 g

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• Monopotassium phosphate 1.7 g

All ingredients were suspended in 1 liter of water containing 5 mL of glycerol. Final preparation was made, as described above for SOP.

SOB medium

• Tryptone 20 g

• Yeast extract 5 g

• NaCl 0.5g

• Deionized water 950 mL

Mix in all ingredients until dissolved. Add 10 mL of a 250 mM solution of

KCL. (This solution is made by dissolving 1.86 g of KCL in 100 mL of deionized water). Adjust the pH of the medium to 7.0 with 5 N NaOH (approximately 0.2 mL).

Adjust the volume of the solution to 1 Liter with deionized water. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Just before use, add 5 mL of a sterile solution of 2 M MgCl2. (This solution is made by dissolving

19 g of MgCl2 in 90 mL of deionized water. Adjust the volume of the solution to

100 mL with deionized H2O and sterilize by autoclaving as described above.)

SOC medium

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SOC medium is identical to the SOB medium (described above), except that it contains 20 mM glucose. After the SOB medium has been autoclaved, allow it to cool to below 60 º C and add 20 mL of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 mL of deionized water. After the sugar has dissolved, adjust the volume of the solution to 100 mL with deionized water and sterilize by passing it through a 0.22 μm filter).

Maltose stock solution

• Maltose 20 g

• Deionized H2O 100 mL

This formula is for a sterile 20 % solution of maltose. Dissolve maltose in water and sterilize by passing it through a 0.22 μm filter. Store the sterile solution at room temperature.

Preparation and storage of antibiotic solutions

Note: Stock solutions of antibiotics dissolved in water were sterilized by filtration through a 0.22 μm filter. Antibiotics dissolved in ethanol need not be sterilized. All stock solutions need to be stored in tight (preferably lockable) 1.5 mL centrifuge microtubes and kept in the freezer. (Avoid unnecessary exposure to light

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sources and minimize freeze-though cycles as both factors contribute to decreasing bioactivity of the antibiotics.)

Media containing agar

Liquid media prepared according to the recipes given above were supplemented with 15 g/L of Bacto Agar. The agar containing media were mixed continuously (with Teflon stirring bar) while brought to a boil on the lab hot plate stirrer. This procedure needs to be monitored to avoid boiling over. Sterilization of the media was done by autoclaving for 20 minutes at 15 psi (1.05 kG/cm2) on liquid cycle. When finished, the sterilized media were removed from the autoclave, swirled gently and allowed to cool to 50 - 60 °C on the lab bench. In the meantime plastic culture Petri dishes (here called “plates”) were prepared by means of writing on the lids: the date, kind of medium and the concentration of supplements, if any.

Cooling down of the agar-containing medium is required to allow for safe handling and for adding thermo-sensitive substances (e.g. antibiotics, glucose and maltose). To avoid producing air bubbles, the medium needs to be swirled continuously. Pouring agar plates requires working in a designated area and under the cover of the Bunsen (propane) burner, in in the flow hood. Media were then poured directly from the flask into plates. (Alternatively, 25 – 30 mL of medium per

10 cm plate can be transferred from the stock container using a sterile pipette and a pump.) When the medium hardened completely (usually overnight on the bench

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and under aluminum foil cover to protect from light), plates were inverted, stacked up, wrapped in plastic and stored at 4 °C until needed. The plates were removed from the fridge 1 – 2 hours before planned use.

Note: To avoid “sweating” (condensation of water drops on the surface of the agar and the plates) and to minimize the risk of cross-contamination, it is best to place the cold plates directly into the dry incubator pre-set to 37 °C and leave the plates until ready to use them.

Media glycerol stocks of bacterial cultures

Bacterial colonies growing on agar plates, or in liquid cultures, can be stored in aliquots of LB medium containing 30 % (v/v) sterile glycerol. Aliquots of 1 mL in

1.5 –1.8 mL cryovials should be prepared and vortexed to ensure that the glycerol is completely dispersed and mixed with the culture to be frozen. The laboratory standard operating procedure requires gradual freezing as follows: starting by placing clearly described (cryoware pens) freezing vials into the fridge for 1 – 2 hours, then overnight at – 20 °C and finally, for long-term storage in the – 80 °C freezer.

Extraction of plasmid DNA

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General Electric Healthcare product Illustra Plasmid Mini Spin Kit was used for all plasmid DNA extractions according to the supplier’s instructions:

1. Harvest 1.5 ml of fresh overnight bacterial culture into sterile Eppendorf

tubes (1.8 ml capacity) and centrifuge at 16000xg for 30 seconds. Discard

supernatant.

2. Re-suspend the cell pellet in 175 µl of lysis buffer type 7 by gently vortexing

the tube.

3. Add to the tube 175 µl of lysis buffer type 8 and mix immediately by

moderate inversion of the tube (approximately 5 times) until the solution

becomes clear and viscous. Incubate the tube for up to 5 minutes at room

temperature.

4. Neutralization – add 350 µl of lysis buffer type 9 and invert the tube carefully

for up 5 times until the precipitate is evenly dispersed.

5. Centrifuge the tube at 16000xg for 4 minutes.

6. Insert one plasmid mini column into a new sterile Eppendorf tube.

7. Carefully load the centrifuged supernatant onto the column and spin at

16000xg for 30 seconds. Discard the flow through by emptying the

collection tube.

8. Wash the column with 400 µl of lysis buffer type 9. Centrifuge as in step 7

and discard the flow-through.

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9. Wash and dry – add 400 µl of wash buffer type 1 and centrifuge at 16000xg

for 1 minute. Discard the flow-through and collection tube.

10. Transfer the plasmid mini column into a fresh micro centrifuge tube and add

100 µl of elution buffer type 4 directly onto the centre of the column.

Incubate the column for 30 seconds at room temperature.

11. Centrifuge the tube with column at 16000xg for 30 seconds to collect

purified plasmid DNA.

12. Measure purity and concentration of DNA using nano-drop apparatus and

proceed to the next step of the experiment.

His-Bind Quick 300 cartridges

Buffer preparation

1. Prepare 7 ml 1X Binding Buffer per cartridge by diluting supplied stocks to

1X with deionized water, or prepare according to buffer compositions

provided on page 4 of the manual supplied with cartridges.

2. Prepare 2.5 ml 1X Wash Buffer per cartridge by diluting supplied stocks to

1X with deionized water, or prepare according to buffer compositions

provided on page 4 of the manual.

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3. Prepare 1ml 1X Elute or Strip Buffer per cartridge by diluting supplied stocks

to 1X with deionized water, or prepare according to buffer compositions

provided on page 4, see manual.

Cartridge chromatography

Attach the cartridge to 5-10 cc syringe loaded with an appropriate buffer.

Push buffer through the cartridge at a rate of approximately 2 drops/second.

1. Wet and equilibrate column with 2 ml 1X Binding Buffer.

2. Load cell extracts onto the cartridge.

3. Wash with 5 ml 1X Binding Buffer.

4. Wash with 2.5 ml 1X Wash Buffer.

5. Elute with 1 ml 1X Elute Buffer or 1ml Strip Buffer.

His-Bind Quick Buffer Kit

• 8X Binding Buffer (8X = 4 M NaCl, 160 Mm Tris-HCl, 40 mM imidazole, pH

7.9)

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• 8X Wash Buffer (8X = 4 M NaCl, 480 mM imidazole, 160 mM Tris-HCl, pH

7.9)

• 4X Elute Buffer (4X = 4 M imidazole, 2 M NaCl, 80 mM Tris-HCl, pH 7.9)

• 4X Strip Buffer (4X = 2 M NaCl, 400mM EDTA, 80 mM Tris-HCl, pH 7.9)

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Figure A3-1. The sequence of RLN H2 DNA within plasmid 6His-MBP. The procedure was performed at the Molecular Services Laboratory (MSL), University of Guelph. The sequence was verified and confirmed to be correct by Dr. Jiping Li (MSL, University of Guelph).

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