STUDIES TOWARDS THE PRODUCTION OF PHARMACEUTICALLY IMPORTANT PROTEIN IGF-1 IN TOBACCO (Nicotina tobaccum)

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1 STUDIES TOWARDS THE PRODUCTION OF PHARMACEUTICALLY IMPORTANT PROTEIN IGF-1 IN TOBACCO (Nicotina tobaccum) M. Kajan S 9962 Dissertation submitted to the Department of Chemistry in partial fulfillment of the requirement for the B.Sc. General Degree in Biochemistry & Molecular Biology Department of Chemistry, University of Colombo, Colombo 03, Sri Lanka. [February 2014]

2 DECLARATION OF AUTHORSHIP I certify that this dissertation does not incorporate without acknowledgement any material previously submitted for a degree or diploma in any university and to the best of my knowledge and belief it does not contain any material previously published or written or orally communicated by another person except where due references is made in the text.... M. Kajan Date Department of Chemistry, University of Colombo. To the best of my/our knowledge the above particulars are correct.... Supervisor Date Dr. N. V. Chandrasekharan Senior lecturer, Department of Chemistry, University of Colombo.... Supervisor Date Dr. C. M. Hettiarachchi Senior lecturer, Department of Chemistry, University of Colombo. ii

3 ABSTRACT Human recombinant Insulin-Like Growth Factor-1 is a pharmaceutically important protein which is used to treat secondary growth failures such as growth hormone insensitivity / Laron syndrome. Human IGF-1 is a 70 amino acid long protein molecule in active form that is secreted in response to growth hormone signal in the Liver and promotes functions in the body relevant to growth and development. Recombinant Human IGF-1 is called Mecasermin and sold under the trade name Increlex or Iplex and is usually produced in Escherichia Coli. It s a FDA approved drug. IGF-1 produced by recombinant DNA technology in Escherichia Coli is expensive and a 40 mg vial of Increlex injections cost about 230 US dollars. Producing important proteins in transgenic plants makes the production fold less expensive than producing in bacteria depending on the protein structure, compatibility with the producing plant system and the types of post-translational modifications that take place to the protein structure. The aim of this research was to produce low cost recombinant IGF-1. Total RNA was extracted from human whole blood using Tricol reagent, reverse transcribed and then the IGF-1 cdna was amplified using PCR. Tobacco Ribulose-1,5- bisphosphate small subunit genes chloroplast transit peptide (CTP) was amplified using PCR from genomic DNA. Amplified CTP region was fused with the IGF-1 gene ligated to pgem-t easy vector and transformed into E.Coli JM109 strain. PCR amplified IGF-1 gene using Pfu DNA polymerase was cloned into the pet-28a vector and transformed into the E.coli JM109 bacterial strain for the expression of recombinant protein inside E.coli BL21 bacterial strain. In this study CTP, IGF-1 fused coding sequence was successfully cloned into pgem-t easy vector and then IGF-1 coding region was cloned into pet-28a vector. iii

4 ACKNOWLEDGEMENTS I would like to take the opportunity and thank all the people who spent their time and shared their knowledge to help me to complete this research. This research would not have been possible without their essential and gracious support. I wish to express my most profound gratitude to my supervisors Dr. N. V. Chandrasekharan & Dr. C. M. Hettiarachchi for their inspiring guidance, constant encouragement and support given to me so that this dissertation became a reality. Without their guidance and persistent help this dissertation would not have been possible. I also thank Mr. Palitha Harasgama and Mr. M. N. Wickramanayake of the Biotechnology Laboratory and all the other technical staff in the Department of Chemistry, University of Colombo, who helped me in my work. I take this opportunity to record my sincere thanks to all postgraduate students of Biotechnology laboratory, Department of Chemistry, University of Colombo for their friendly support & guidance they have provided whenever I struggled to accomplish any tasks. I sincerely thank my colleagues for their continuous help, support and advices which made this report a success. iv

5 TABLE OF CONTENTS COVER PAGE DECLARATION OF AUTHORSHIP ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES ABBREVIATIONS i ii iii iv v ix xi xii CHAPTER 1 INTRODUCTION Introduction to Insulin-like growth factors IGF-I IGF-II IGF Receptors IGF Binding Proteins (IGFBPs) Physiology of IGF Chemical Structure of IGF Roles of IGF Contributing to aging Neuropathy Diabetes Healing Potential Kidney Diseases Osteoporosis Osteoarthritis 13 v

6 1.4.8 Atherosclerosis Dwarfism Role in sports Transformation of foreign gens in plants Molecular Mechanism of Agrobacterium-mediated gene transfer Bacteriology, Host Range and Opines Tumor-inducing (Ti) Plasmid Transfer DNA (T-DNA) of Ti Plasmid Induction of vir Gene Expression Pili Formation T-complex, an Intermediate of T-DNA Transfer Binary Vectors of Ti Plasmid Factors to Increase Gene Expression and Transformation Efficiency Tobacco as an expression host Downstream processing Why IGF-1 in tobacco? Recombinant protein targeting into chloroplasts 26 CHAPTER 2 - INSTRUMENTS AND METHODOLOGY Instruments & Equipments Materials Methodology Designing of primers for PCR amplification Primers for amplification of Human IGF Primers for amplification of Tobacco CTP coding sequence 30 vi

7 2.3.2 Sterilization Solutions Preparation Total RNA Extraction from Blood (Mini scale) Total RNA Extraction from Blood (Large scale) Analysis of Isolated RNA Non denaturing agarose gel electrophoresis of total RNA First strand cdna synthesis PCR amplification PCR amplification of human IGF-1 gene PCR amplification of Tobacco CTP region of ribulose- 1,5-Bisphosphate carboxylase small subunit gene Fusion PCR amplification of CTP-human IGF Column Elution of PCR product using Wizard SV Gel and PCR Clean-Up System Cloning and Transformation with pgem-t easy vector Plasmid isolation and DNA sequencing of positive Cloning and transformation with pet-28 vector 47 CHAPTER 3 - RESULTS AND DISCUSSION Non denaturing agarose gel electrophoresis of total RNA PCR amplification of human IGF-1 gene PCR amplification of Tobacco CTP region of ribulose-1,5-bisphosphate carboxylase (RuBisCO) small subunit gene Fusion PCR amplification of CTP-human IGF Cloning and Transformation with pgem-t easy vector Plasmid isolation and DNA sequencing of positive pet-28a plasmid vector Isolation and Confirmation 63 vii

8 3.8 Preparing insert and vector Cloning and transformation 66 CONCLUSION 68 FUTURE WORK 69 ANNEXURE 70 REFERENCES 89 viii

9 LIST OF FIGURES Figure 1.1 Chromosomal Location of Human IGF-1 Figure 1.2 Structurally related Insulin Receptor and IGF-1 Receptor Figure 1.3 Origin of IGF-1 and its main functions in human Figure 1.4 IGF-1 and IGF-I receptors, their main downstream pathways Figure 1.5 IGF1 expression levels in normal human tissues Figure 1.6 2D Structural Comparison of IGF-1, Pro Insulin and Insulin Figure 1.7 Regions of immature IGF-1 Figure 1.8 The 3D structure of IGF-1 Figure 1.9 Insulin/IGF1 signaling pathways in the regulation of ageing Figure 1.10 Reduced IGF1 signalling is related to the classic 'rate of living' hypothesis. Figure 1.11 IGF-1 receptor targeting: cancer therapeutic strategies Figure 1.12 Octopine type Ti-Plasmid Figure 1.13 Agrobacterium-mediated Transformation Figure 3.2 Gel image of PCR amplified Human G6PD Figure 3.3 Construct of IGF-1 PCR product including elements added using primers Figure 3.4 PCR amplification of IGF-1 using rhigf-1 primers and reverse transcribed total RNA from human blood Figure 3.5 Construct of RuBisCO CTP PCR product including elements added using primers Figure 3.6 PCR amplification of RuBisCO CTP using CTP primers Figure 3.7 Construct of Fusion PCR product Figure 3.8 Gel picture of Fusion PCR product with DNA markers Figure 3.9 Grid Plate after Transformation to JM109 Figure 3.10 Gel picture of Colony PCR products Figure 3.11 Gel picture of Isolated Plasmid DNA before and after the RNase Treatment ix

10 Figure 3.12 Gel picture of EcoRI Restriction digested putative pgem-t recombinant clone Figure 3.13 Spot gel with spotted Plasmid DNA and concentration marker Figure 3.14 Gel picture of digested and undigested pet-28a vector Figure 3.15 Undigested and XbaI digested pet-28a vector Figure 3.16 Pfu DNA polymerase amplified IGF-1 and 200bp marker Figure 3.17 Prepared pet-28a vector and insert before gel elution Figure 3.18 Spot gel with spotted concentration markers, prepared vector and Insert Figure 3.19 Spread plate of transformation mixture and grid plate prepared from spread plate x

11 LIST OF TABLES Table 1.1 Characteristics of the six IGFBPs Table 2.1 Reaction setup for first strand cdna synthesis using oligo dt primers. Table 2.2 PCR reaction mixture to amplify 210bp region of Human IGF-1 cdna Table 2.3 Optimized PCR thermo cycle parameters for amplifying IGF-1 using rhigf-1 Primers Table 2.4 PCR reaction mixture to amplify 168bp region of tobacco gdna. Table 2.5 Optimized PCR thermo cycle parameters for amplifying Tobacco CTP using CTP primers Table 2.6 PCR reaction mixture to amplify 427bp Fused genes. Table 2.7 Optimized PCR thermo-cycle parameters for fusing IGF-1 and CTP Table 2.8 Constituents of Ligation mixture Table 2.9 PCR reaction mixture to amplify the insert inside the bacterial colony. Table 2.10 Optimized PCR thermo cycle parameters for Colony PCR Table 2.11 Restriction Digestion of putative pgem-t recombinant clone Table 2.12 Restriction Digestion setup for cleaving pet-28a Table 2.13 Xba1 Restriction Digestion of pet-28a Table 2.14 Blunting Xba1 Digested pet-28a Table 2.15 BamHI Restriction Digestion of Blunted pet-28a Table 2.16 PCR amplification of IGF-1 from pgem-t Clone Table 2.17 Optimized PCR thermo cycle parameters for Pfu polymerase Table 2.18 Restriction Digestion with BamHI Table 2.19 Constituents of pet-28 Ligation mixture xi

12 ABBREVIATIONS A Adenine aa Amino acids Amp r Ampicillin resistant gene ATP Adenosine triphosphate BLAST Basic local alignment search tool bp Base pairs BP Binding Protein BSA Bovine serum albumin CaMV Cauliflower Mosaic Virus C-terminal Carboxyl (COOH)-terminal CTP Chloroplast Transit Peptide cdna Complementary deoxyribonucleic acids DMSO Dimethyl sulfoxide DNA Deoxy-ribo Nucleic Acid EC Additional bases required for efficient cut EDTA Ethylene Diamine Tetra Acetic acid EGF Epidermal growth factor ELISA Enzyme Linked Immunosorbent Assay ER Endoplasmic reticulum EtBr Ethidium Bromide G Guanine GH Growth Hormone GFP Green Fluorescent Protein GUS β-1,4-glucuronidase xii

13 HPT Hygromycin Phosphotransferase IGF Insulin-like Growth Factor IGFBP Insulin-like growth factor binding protein IGFR Insulin like growth factor receptor IPTG Isopropyl β-d-thiogalactopyranoside or Isopropyl thiogalactoside kb Kilo bases LB Luria Bertani MAP Mitogen-activated protein mrna Messenger ribonucleic acids NPTII Neomycin Phosphotransferase II NSILA Non-suppressible insulin-like activity PAGE Polyacrylamide-gel electrophoresis PBS Phosphate buffered saline PCR Polymerase chain reaction rpm Revolutions per minute RT-PCR Reverse transcriptase polymerase chain reaction RuBisCO Ribulose 1, 5-bisphosphate carboxylase SD Standard Deviation SDS Sodium dodecyl Sulfate T Thymine T-DNA Transfer-DNA T-Pilus Transfer Pilus TBE Tris Boric EDTA TE Tris EDTA Ti plasmid Tumor-Inducing Plasmid xiii

14 Tra protein T-DNA transfer protein UV Ultra Violet vir gene Virulence Gene X-Gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside xiv