Expression of recombinant proteins in tobacco system

EXPRESSION OF RECOMBINANT PROTEINS IN TOBACCO SYSTEM D. TAMILSELVI NATIONAL UNIVERSITY OF SINGAPORE 2004 EXPRESSION OF RECOMBINANT PROTEINS IN TOBACCO SYSTEM D. TAMILSELVI M.Sc. (Biotechnology) Tamil Nadu Agricultural University, India A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I express my gratitude to my supervisor, A/P Sanjay Swarup, for his guidance and support throughout my Ph. D candidature. I thank sincerely, my Ph. D committee members, A/P Prakash P. Kumar and Prof. Wong Sek Man for their guidance and suggestions. I thank Jaideep Mathur for providing me tobacco BY2 cell lines, GUS and GFP clones. I am grateful to A/P R. M. Kini for his help and guidance in protein purificaiton and sequencing work. I am grateful to Dr. Ge for providing the Angiopoietin and VEGF clones. I am thankful to my lab members, for their understanding and help. My sincere appreciation is extended to the lab officers, technical staff of the department, Mdm Liew Chye Fong, Mdm Tan Lu Wee for providing instrument facility for my research during semester breaks. PPC facility is gratefully acknowledged. I express my gratitude to my parents and in-laws for their affection, prayers and support given throughout my study. I am grateful to my husband for his incessant persuasion and moral support. D.TAMILSELVI i TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii SUMMARY ix LIST OF TABLES xi LIST OF FIGURES xii LIST OF ABBREVIATIONS xv CHAPTER 1.0 INTRODUCTION CHAPTER 2.0 LITERATURE REVIEW 2.1 Production of recombinant proteins in plant systems: A brief history 2.2 Recombinant protein expression strategies 2.2.1 Extrachromosomal expression by geminiviral DNA-based vectors Geminiviruses and their replication 10 Geminiviruses as vectors for the expression of foreign genes 11 Advantages of geminivirus vectors 15 Stable transgene expression 15 Factors influencing transgene expression 17 Environmental concerns of transgenic plants 17 Transient gene expression 18 Agroinfiltration-expression using bacterial vectors 19 Expression using RNA-based viral vectors 21 2.2.2 2.2.3 ii 2.3 Alternative expression systems 22 2.3.1 Expression of recombinant proteins in cell suspension cultures 24 2.4 Sub-cellular targeting of proteins 28 2.4.1 Mechanism of protein secretion 29 2.4.2 Extracellular secretion and apoplast targeting 31 2.4.3 Retention in the endoplasmic reticulum (ER) 38 2.5 Angiogenic growth factors as a candidate for expression 42 2.5.1 Molecular structure and development of recombinant Angiopoietin (Ang1) 42 2.5.2 Molecular structure and development of recombinant human Vascular Endothelial Growth Factor (hVEGF165) 45 2.5.3 Therapeutic angiogenesis 51 CHAPTER 3.0 STUDIES ON AGERATUM YELLOW VEIN 52 VIRUS REPLICATION IN Nicotiana benthamiana AND N. tabacum L.cv BY2 3.1 INTRODUCTION 53 3.2 MATERIALS AND METHODS 57 3.2.1 Construction of plasmids 57 3.2.2 Optimization of electroporation conditions for N. benthamiana mesophyll-derived protoplasts 59 3.2.3 Transformation of N. benthamiana mesophyll-derived protoplasts with plasmid DNA and confirmation using PCR 60 iii 3.2.4 Transformation of tobacco BY2 cells by particle bombardment and PCR detection of transformed lines 60 3.2.5 Rescue of pASVNPT and pASVGUS shuttle vectors from tobacco BY2 cells in E. coli 61 3.2.6 62 Analysis of replicating DNA by Coupled Restriction Enzyme Digestion and Random Amplification PCR (CREDRA-PCR) and Southern blot analysis 3.2.7 Histological GUS assays 3.3 62 RESULTS 3.3.1 Construction of pASV plant-E. coli shuttle vectors 63 3.3.2 Optimization of electroporation conditions 65 3.3.3 Transformation of mesophyll-derived protoplasts of N. benthamiana with pASVNPT 65 3.3.4 Transformation of pASV-derived vectors in N. tabacum BY2 cells by particle bombardment 68 3.3.5 Replication studies of pASV-derived shuttle vectors 69 PCR detection of pASVNPT and pASVGUS vector in transformed calluses 69 Shuttling ability and rescue of pASVGUS from plant cells to E. coli 72 Replication studies based on DNA methylation difference 73 Expression of foreign reporter gene 75 DISCUSSION 78 3.4 iv CHAPTER 4.0 ESTABLISHMENT OF A HOST SYSTEM TO STUDY EXPRESSION OF FOREIGN PROTEINS 83 4.1 INTRODUCTION 84 4.2 MATERIALS AND METHODS 87 4.2.1 Maintenance of Nicotiana tabacum cv BY2 suspension cells 87 4.2.2 Sensitivity of BY2 cells to antibiotics 87 4.2.3 Construction of expression plasmids 87 4.2.4 Transformation of Agrobacterium tumefaciens (EHA105) 93 Preparation of electrocompetent cells and electroporation 93 4.2.5 Agrobacterium-mediated transformation of tobacco BY2 cells 94 4.2.6 Establishing batch culture of transformed tobacco BY2 cells 96 4.2.7 Cell growth studies 96 4.2.8 Intra and extra cellular protein estimation 96 4.2.9 GUS expression analysis 97 Histochemical localization for GUS 97 Luminescence spectrometry 97 GUS extraction and fluorescence determination 98 Quantitation of GUS 98 4.2.10 Analysis for GFP by microscopy 99 GFP extraction 99 Luminescence spectrometry 99 v 4.3 RESULTS 101 4.3.1 Effect of Kanamycin and G418 on tobacco BY2 cells 101 4.3.2 Reporter gene expression cassettes 103 4.3.3 Generation of transgenic cell lines 103 4.3.4 Growth pattern of cultured tobacco cells 107 4.3.5 Intracellular and secreted protein content 107 4.3.6 Quantitation of GUS in cell lines 112 4.3.7 GFP fluorescence analysis in cell lines 112 Intracellular GFP expression in cell lines 114 Extracellular GFP expression in cell lines 114 DISCUSSION 118 4.4 CHAPTER 5.0 ISOLATION OF PLANT SECRETORY SIGNAL PEPTIDES AND THEIR USE IN THE EXPRESSION OF HUMAN VASCULAR ENDOTHELIAL GROWTH FACTOR (hVEGF165) 123 5.1 INTRODUCTION 124 5.2 MATERIALS AND METHODS 128 5.2.1 Purification of secreted proteins 128 Cell growth conditions 128 Reverse phase HPLC (High performance liquid chromatography) 128 Microsequencing of secretory proteins 129 Identification of secretory proteins and their signal peptides 129 5.2.2 vi 5.2.3 Construction of hVEGF165 (human Vascular Endothelial Growth Factor) in plant expression vector 130 Construction of hVEGF165KDEL for ER retention 130 Construction of hVEGF165 for secretion 136 Cultivation of plants and Agroinfiltration 138 Preparation of Agrobacterium for infiltration 138 Infiltration of explants 139 5.2.5 Analysis of protein extracts from infiltrated leaves by SDS-PAGE and Western blot 139 5.2.6 Heparin-Sepharose purification of hVEGF165 141 5.3 RESULTS 143 5.3.1 Identification of secreted proteins from tobacco BY2 cell suspension cultures 143 5.2.4 5.3.2 Construction of hVEGF165 expression plasmids 144 5.3.3 Expression of hVEGH165 in N. benthamiana leaves 156 5.3.4 Analysis of signal peptide efficiency in the expression of hVEGF165 in N. benthamiana leaves 158 5.3.5 Purification of hVEGF165 163 5.4 DISCUSSION 165 CHAPTER 6.0 A RAPID TRANSIENT EXPRESSION SYSTEM 172 FOR ANGIOPOIETIN (Ang1) 6.1 INTRODUCTION 173 vii 6.2 MATERIALS AND METHODS 176 6.2.1 Cultivation of plants 176 6.2.2 Construction of Angiopoietin expression plasmid 176 6.2.3 Agroinfiltration of Nicotiana leaves 178 6.2.4 Preparation of protein extracts from infiltrated leaves 180 6.2.5 SDS-PAGE and Western blot analysis 180 6.3 RESULTS 181 6.3.1 Construction of the human Angiopoietin1 (Ang1) expression cassette 181 6.3.2 Transient expression of Ang1 in N. benthamiana 181 6.3.3 Comparative time course analysis of Ang1 expression in N. tabacum and N. benthamiana 185 6.3.4 Optimization of Ang1 expression from different dilutions of bacterial suspension for infiltration 188 6.4 DISCUSSION 190 CHAPTER 7.0 CONCLUSIONS AND FUTURE WORK 193 7.1 CONCLUSIONS 193 7.2 FUTURE WORK 195 REFERENCES 197 APPENDICES 248 viii Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, Davis S, Sato TN, Yancopoulos GD (1996) Requisite role of angiopoietin-1, a ligand for the tie2 receptor, during embryonic angiogenesis. 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Cell 86: 849852 Ziegler MT, Thomas SR, Danna KJ (2000) Accumulation of a thermostable endo-1, 4-β D-glucanase in the apoplast of Arabidopsis thaliana leaves. Mol Breed 6: 37-46 Zimmermann S, schillberg S, Liao YC, Fisher R (1998) Intracelluar expression of TMVspecific single-chain Fv fragments leads to improved virus resistance in Nicotiana tabacum. Mol Breed 4: 369-379 247 APPENDICES APPENDIX I A) MURASHIGE AND SKOOG (MS) MEDIUM MS medium was prepared as below, following the protocols given earlier (Murashige and Skoog 1962). MS salts (Duechfa, Netherlands) 4.4 g Sucrose 30 g Myoinositol 0.1 g KH2PO4 0.18 mg Thiamine HCL 1.0 mg MES 0.5 mg 2, 4-D (2, 4-dichlorophenoxyacetic acid) 0.22 mg The above were dissolved in Milli-Q water to make up the volume to L, and the pH adjusted to 5.6-5.8 with KOH before autoclaving. B) MG/L broth MG/L medium was prepared following the protocol given earlier (Cangelosi et al. 1994). LB 500 ml Mannitol 10.00 g Sodium glutamate 2.32 g KH2PO4 0.50 g NaCl 0.20 g MgSO4.7H2O 0.20 g Biotin 2.00 µg The above were dissolved in Milli-Q water to make up the volume to L, and the pH adjusted to 7.0 before autoclaving. 248 Appendix II A. Growth of pBinGUS transgenic tobacco BY2 cell lines (wet weight in g/25 ml culture) (Fig 4.7a) Days cg418 s1 s2 Cell lines s3 s4 s5 s6 s7 s8 10 11 12 1.000 1.234 1.397 1.623 3.597 6.367 10.246 15.780 15.561 14.341 13.845 13.356 13.005 1.000 1.000 0.945 0.945 0.945 0.932 0.895 0.834 0.829 0.807 0.757 0.754 0.754 1.000 1.246 1.372 1.634 3.272 6.045 10.572 15.894 15.45 14.891 14.456 13.083 13.034 1.000 1.235 1.345 1.674 3.045 6.069 11.042 15.764 15.35 14.117 13.478 13.291 13.103 1.000 1.257 1.343 1.598 3.143 6.132 10.563 15.590 15.561 14.345 14.025 13.304 13.231 1.000 1.256 1.399 1.601 3.399 6.967 10.907 15.790 15.567 15.134 14.345 13.237 13.134 1.000 1.245 1.307 1.611 3.107 6.468 11.671 16.130 15.950 15.567 14.368 13.452 13.231 1.000 1.267 1.356 1.675 3.656 6.969 10.996 16.672 15.476 15.101 14.104 13.496 13.023 1.000 1.280 1.347 1.626 3.247 6.018 10.356 16.342 15.454 14.234 13.421 13.023 13.003 1.000 1.235 1.374 1.621 3.374 6.625 10.376 15.891 15.578 14.267 13.567 13.113 13.045 B. Intracellular protein in transgenic GUS cell lines (mg/g of cells) (Fig 4.7b) Days cg418 s1 s2 Cell lines s3 s4 0.687 1.232 3.035 4.385 6.251 5.107 2.685 1.561 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.687 0.976 3.048 4.380 6.293 5.159 2.521 1.677 0.685 0.975 2.950 4.224 6.287 5.212 2.607 1.584 0.688 0.981 2.957 4.260 6.219 4.971 2.564 1.799 0.685 0.973 2.969 4.321 6.165 4.957 2.508 1.961 s5 s6 s7 s8 0.693 0.982 3.018 4.421 6.247 5.106 2.431 1.728 0.689 0.981 2.993 4.389 6.241 5.018 2.444 1.588 0.686 0.982 3.018 4.359 6.178 4.952 2.733 1.689 0.687 0.974 2.984 4.171 6.111 5.041 2.522 1.603 249 C. Growth of transgenic GFP cell lines (wet weight in g/25 ml culture) (Fig 4.8a) Cell lines g4 g5 Days cg418 g1 g2 g3 10 11 12 1.000 1.210 1.387 1.623 3.597 6.367 10.246 15.780 15.561 14.341 13.845 13.356 13.005 1.000 1.000 0.935 0.947 0.938 0.927 0.877 0.824 0.811 0.805 0.767 0.752 0.751 1.000 1.250 1.355 1.625 3.255 6.025 10.552 15.874 15.436 14.881 13.636 13.063 13.014 1.000 1.246 1.335 1.664 3.033 6.059 11.022 15.757 15.344 14.107 13.548 13.274 13.100 1.000 1.267 1.355 1.588 3.123 6.142 10.543 15.585 15.529 14.325 13.815 13.010 13.000 1.000 1.245 1.385 1.611 3.355 6.615 10.365 15.888 15.588 14.237 13.548 13.110 13.042 1.000 1.267 1.386 1.600 3.377 6.957 10.900 15.765 15.547 14.124 13.725 13.217 13.115 g6 g7 g8 1.000 1.275 1.317 1.621 3.100 6.448 11.641 16.120 15.955 14.557 13.648 13.290 13.220 1.000 1.25 1.346 1.665 3.646 6.949 10.985 16.048 15.447 14.100 13.710 13.060 13.012 1.000 1.265 1.337 1.635 3.227 6.011 10.336 16.033 15.444 14.214 13.618 13.013 13.000 D. Intracellular protein in transgenic GFP cell lines (mg/g of cells) (Fig 4.8b) Days cg418 g1 g2 g3 Cell lines g4 g5 g6 g7 g8 0.678 0.961 3.031 4.377 6.128 5.217 2.566 1.561 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.681 0.961 3.018 4.373 6.225 5.221 2.540 1.677 0.679 0.958 2.941 4.291 6.217 5.235 2.533 1.584 0.676 0.966 2.987 4.292 6.201 5.226 2.543 1.799 0.683 0.968 3.028 4.315 6.171 5.136 2.545 1.961 0.681 0.968 3.025 4.375 6.146 5.311 2.503 1.588 0.677 0.967 3.018 4.388 6.182 5.252 2.634 1.689 0.684 0.968 3.026 4.275 6.184 5.260 2.590 1.603 0.681 0.963 3.011 4.311 6.155 5.256 2.574 1.728 250 E. Growth for Pr1aGFP cell lines (wet weight in g/25 ml of culture) (Fig 4.9a) Cell lines p4 p5 Days cg418 p1 p2 p3 10 11 12 1.000 1.210 1.387 1.623 3.597 6.367 10.246 15.780 15.561 15.341 14.845 13.356 13.005 1.000 1.000 0.925 0.945 0.928 0.937 0.867 0.823 0.810 0.802 0.765 0.742 0.757 1.000 1.232 1.365 1.635 3.245 6.015 10.542 15.839 15.426 15.268 14.425 13.043 13.025 1.000 1.252 1.325 1.634 3.023 6.049 11.011 15.747 15.524 15.101 14.418 13.264 13.133 1.000 1.277 1.345 1.578 3.125 6.138 10.533 15.575 15.515 15.313 14.011 13.311 13.223 1.000 1.267 1.375 1.613 3.345 6.625 10.345 15.868 15.568 15.223 14.538 13.124 13.022 1.000 1.247 1.366 1.615 3.367 6.947 10.915 15.755 15.546 15.312 14.315 13.268 13.105 p6 p7 p8 1.000 1.273 1.310 1.631 3.123 6.438 11.635 15.911 15.535 15.547 14.338 13.439 13.215 1.000 1.255 1.336 1.655 3.633 6.929 10.967 15.628 15.426 15.123 14.111 13.456 13.043 1.000 1.235 1.327 1.625 3.217 6.025 10.328 15.623 15.422 15.200 14.422 13.023 13.013 F. Intracellular protein in transgenic Pr1aGFP cell lines (mg/g of cells) (fig 4.9b) Days cg418 p1 p2 p3 p4 0.683 0.973 3.005 4.337 6.215 5.338 2.572 1.561 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 1.099 0.993 3.022 4.361 6.178 5.277 2.614 1.677 0.689 0.965 3.061 4.302 6.161 5.265 2.521 1.584 0.686 0.968 2.956 4.328 6.207 5.294 2.557 1.799 0.685 0.968 3.018 4.341 6.169 5.291 2.567 1.961 Cell lines p5 0.684 0.964 2.964 4.385 6.173 5.292 2.591 1.728 p6 p7 p8 0.689 0.995 3.004 4.371 6.209 5.273 2.6000 1.588 0.690 0.970 3.011 4.294 6.175 5.337 2.550 1.689 0.686 0.969 2.925 4.372 6.209 5.296 2.513 1.603 251 G. Extracellular total protein from transgenic Pr1aGFP cell lines (mg/l) Cell lines p4 Days cg418 p1 p2 p3 12.487 12.959 24.201 53.942 68.811 56.138 45.194 27.916 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 12.487 13.013 24.247 52.286 66.937 55.490 43.611 28.204 12.487 14.496 24.179 53.258 68.523 54.302 42.279 30.112 12.487 14.496 24.172 52.754 65.063 55.382 45.302 27.700 12.487 12.984 24.208 54.878 67.441 55.850 42.062 29.392 p5 p6 p7 p8 12.487 12.999 24.262 52.933 65.496 54.086 42.567 30.400 12.487 13.024 24.089 52.718 67.658 55.454 42.062 29.608 12.487 12.995 24.255 54.086 65.027 55.310 43.323 29.572 12.487 12.995 24.075 52.862 64.919 54.122 43.071 29.428 252 H. GUS expression levels in transgenic tobacco BY2 cell lines over a growth period (i) percentage of GUS in total soluble protein (Fig 4.11a) Cell lines s4 Days g418 s1 s2 s3 0.007 0.008 0.008 0.008 0.008 0.008 0.009 0.008 0.007 0.008 0.008 0.007 0.008 0.007 0.008 0.008 0.035 0.091 0.902 1.456 2.168 1.771 0.468 0.089 0.037 0.093 0.596 1.092 1.536 1.482 0.456 0.091 0.041 0.119 0.921 1.853 2.153 1.980 0.543 0.107 0.036 0.113 0.983 1.849 2.241 2.083 0.585 0.081 s5 s6 s7 s8 0.036 0.084 0.598 1.135 1.446 1.289 0.305 0.087 0.032 0.126 1.021 2.177 3.161 2.348 0.771 0.079 0.037 0.145 2.141 3.191 5.829 4.211 2.087 0.113 0.020 0.057 0.478 1.132 1.534 1.349 0.146 0.077 s6 s7 s8 0.218 1.232 30.548 109.243 197.277 117.806 10.507 1.254 0.252 1.428 64.599 158.012 363.180 259.0127 103.911 10.907 0.137 0.558 14.266 57.060 93.717 67.977 3.337 1.245 (ii) GUS levels in (µg/g) wet weight of the cells (Fig 4.11b) Days g418 s1 s2 s3 Cell lines s4 s5 0.047 0.093 0.240 0.395 0.484 0.395 0.255 0.132 0.034 0.037 0.038 0.037 0.042 0.037 0.040 0.039 0.241 0.889 27.501 75.130 136.425 91.358 7.872 1.496 0.256 0.909 17.572 56.915 96.604 77.239 7.226 1.441 0.281 1.166 27.235 92.135 133.904 98.413 8.911 1.930 0.247 1.101 29.177 91.647 138.190 103.250 11.493 1.594 0.249 0.823 18.043 57.981 90.309 65.834 5.704 1.496 253 I. Ratio of intracellular fluorescence level in GFP transformed cell lines (Fig 4.12a) Days 1.888 1.861 1.944 1.688 1.666 1.666 1.000 1.611 cg418 1.083 1.222 0.944 1.166 0.944 0.944 0.972 1.055 Cell lines g4 g1 g2 g3 1031.194 1740.889 2487.111 4506.361 4995.528 4948.139 3448.333 1832.389 323.916 494.388 656.194 1032.556 1323.528 1200.111 781.944 364.833 2911.389 3242.556 4073.250 7865.056 8192.722 7989.167 7255.833 5035.694 619.944 739.916 1240.000 2062.111 3045.167 2323.806 1739.861 1273.000 g5 1656.222 2073.500 3992.222 7839.750 7987.139 7868.222 7089.056 5197.944 g6 203.888 288.944 406.500 614.805 998.694 698.722 481.444 157.861 g7 g8 411.666 494.222 823.194 1576.444 1695.889 1588.917 1197.861 422.666 824.500 1119.222 1639.333 3148.139 3364.833 3322.750 2056.194 1198.528 p7 p8 87.636 189.363 531.484 678.181 1282.091 1128.212 855.000 536.545 131.575 281.121 671.636 813.424 1628.970 1170.848 962.575 586.545 Taken the lowest value in control as and worked the ratio of fluorescence level in cell lines J. Ratio of extracellular GFP fluorescence levels in transgenic Pr1aGFP cell lines (Fig 4.13a) Days 1.030 1.151 1.242 1.000 1.696 1.303 2.121 1.272 cg418 1.696 1.060 1.060 1.060 1.060 1.060 1.060 0.969 p1 p2 p3 104.969 217.939 673.151 1625.240 1991.939 1799.636 1263.788 1080.455 293.242 784.454 1645.000 3522.242 4210.636 4113.879 3443.606 2261.697 211.030 356.636 720.424 1628.424 1714.121 1628.182 1129.030 675.727 Cell lines p4 148.666 277.757 814.121 1357.212 1580.394 1443.727 980.030 491.666 p5 368.030 873.878 1779.879 3506.394 3845.061 3632.727 2884.606 1900.515 p6 63.090 131.909 452.787 536.363 900.393 807.363 534.393 218.666 Taken the lowest value of relative fluorescence in control as and worked the ratio of fluorescence level in cell lines The values in all the above table (B, D, F-J) represent the mean of three independent extract 254 [...]... Molecular farming is rapidly gaining wide acceptance as a cheap and safe technology to produce recombinant therapeutic proteins in plants This study was conducted to help better understand the conditions affecting molecular farming of recombinant proteins The expression of foreign proteins was studied in a tobacco system with four major objectives In the first part, a plant-E coli shuttle vector system was... Factor165 4) To investigate the feasibility of production of a commercially important human angiogenic therapeutic protein namely, the full length Angiopoietin1, using a rapid transient expression system in tobacco 7 CHAPTER 2 LITERATURE REVIEW 2.1 Production of recombinant proteins in plant systems: A brief history Modern biotechnology is extending the use of plants in medicine well beyond its original boundaries... 1999; Ma et al 2003) These include vaccines, antibody fragments, secretory IgA, blood substitutes, biological effectors including interleukins, milk proteins, protein polymer collagen, industrial enzymes and proteins 2.2 Recombinant protein expression strategies There are currently four types of plant transformation strategies leading to foreign protein expression in plants: 1) plant cells for extrachromosomal... cloning VEGF165 with ER-retention signal in binary vector pBI121 137 Figure 5.5 Agroinfiltration protocol for the expression of recombinant proteins 140 Figure 5.6 Extracellular protein profiles of different isolation methods from tobacco BY2 cell suspension culture 145 Figure 5.7 Fractionation and purification of secreted proteins from tobacco BY2 cell suspension culture 146 Figure 5.8 Alignment of. .. agroinfiltrated leaves 160 Figure 5.20 Purification of hVEGF165 from agroinfiltrated leaves using affinity chromatography 164 Figure 6.1 Strategy for cloning Angiopoietin1 in a plant expression vector 177 Figure 6.2 Workflow for the transient expression system of recombinant Ang1 in plants 179 Figure 6.3 Restriction digestion of Ang1 recombinant clones 182 Figure 6.4 DNA sequence verification of 5’... possible toxicity of the recombinant proteins to non-target organisms like herbivores, pollinating insects and microorganisms in the rhizosphere are being addressed All recent trials take into account such measures to reduce biosafety risks (Commandeur et al 2003) There is also concern that plant material that contains recombinant proteins could inadvertently enter the food 17 chain (Ma et al 2003)... and pharmaceutical uses in a field now popularly known as “molecular farming” Advantages of plant systems for molecular farming include low cost of growing plants on large acreage; the ease in scaleup (increase of planted acreage); the availability of natural protein storage organs; and the established practices for their efficient harvesting, transporting, storing and processing (Whitelam et al 1993)... plants offer include: 1) the elimination of the purification requirement when the plant tissue containing the recombinant protein is used as a food or feed supplement; 2) the possibility to target recombinant proteins to different organelles and secretory pathway that reduce degradation and, therefore, increase stability; 3) being free from known human pathogens; 4) ability to synthesize proteins from... for optimization of downstream processing Significant progress has been made in optimizing the rate of transcription and translation in plant cells (Outchkourov et al 2003) However, the yield of proteins expressed in heterologous systems is still a limiting factor in most plant-based systems described A generally adopted approach to increase heterologous protein accumulation levels in plants is to change... chloroplasts intracellularly and to apoplast / extracellular secretion aided by targeting signals Retaining recombinant proteins within the distinct compartments of the cell preserves integrity, protects proteins from proteolytic degradation and thereby increases accumulation levels Several signal peptides from plant and non-plant sources can function in plants, however, the efficiency of secretion . understand the conditions affecting molecular farming of recombinant proteins. The expression of foreign proteins was studied in a tobacco system with four major objectives. In the first part, a plant-E LIST OF ABBREVIATIONS xv CHAPTER 1.0 INTRODUCTION 1 CHAPTER 2.0 LITERATURE REVIEW 8 2.1 Production of recombinant proteins in plant systems: 8 A brief history 2.2 Recombinant protein expression. Expression using RNA-based viral vectors 21 iii 2.3 Alternative expression systems 22 2.3.1 Expression of recombinant proteins in cell suspension cultures 24 2.4 Sub-cellular targeting