Table Of ContentMolecular Farming of Plants and Animals for
Human and Veterinary Medicine
Molecular Farming of Plants
and Animals for Human
and Veterinary Medicine
Edited by
L. Erickson
University of Guelph,
Department (~f Plant Agriculture,
Geulph, Ontario, Canada
W.-J. Yu
Syngenta Biotechnology Inc.,
Research Triangle Park,
North Carolina, U.S.A.
J. Brandle
Research Branch,
Agriculture and Agri-Food Canada,
London, Ontario, Canada
and
R. Rymerson
Research Branch,
Agriculture and Agri-Food Canada,
London, Ontario, Canada
SPRINGER·SCIENCE+BUSINESS MEDIA, B.V.
A C.I.P. Catalogue record for this book is available from the Library of Congress.
ISBN 978-90-481-6110-2 ISBN 978-94-017-2317-6 (eBook)
DOI 10.1007/978-94-017-2317-6
Printed on acid-free paper
All Rights Reserved
© 2002 Springer Science+Business Media Dordrecht
Originally published by Kluwer Academic Publishers in 2002
Softcover reprint of the hardcover 1s t edition 2002
No part of this work may be reproduced, stored in a retrieval system, or transmitted
in any form or by any means, electronic, mechanical, photocopying, microfilming, recording
or otherwise, without written permission from the Publisher, with the exception
of any material supplied specifically for the purpose of being entered
and executed on a computer system, for exclusive use by the purchaser of the work.
Contents
Preface XVll
1. TOBACCO, A PLATFORM FOR THE PRODUCTION OF
RECOMBINANT PROTEINS 1
R. T. Rymerson, R. Menassa and J.E. Brandle
1. INTRODUCTION 1
2. AGRICULTURAL PRODUCTION OF BIOMASS 2
3. RECOMBINANT PROTEIN EXPRESSION IN
TOBACCO 4
3.1 Production in Seeds 4
3.2 Organelle Targeting 5
3.3 Chloroplast Transformation 6
3.4 Secretion to the Apoplast 7
3.5 Transient Expression 8
4. EXAMPLES 10
4.1 Antibodies 10
4.2 Biopharmaceuticals 12
4.3 Industrial Proteins 19
4.4 Vaccines 21
5. CONCLUSION 24
2. ALFALFA: AN EFFICIENT BIOREACTOR FOR
CONTINUOUS RECOMBINANT PROTEIN
PRODUCTION 33
M.-A. D'Aoust, U. Busse, M. Martel, L. Faye, D. Levesque
and L.P. Vezina
1. INTRODUCTION 33
2. EXPRESSION CASSETTES 34
3. TRANSFORMATION METHODS 36
3.1 In vitro Culture 36
3.2 Transformation with Agrobacterium 37
3.3 Direct DNA Transfer 38
3.4 Other Methods 39
4. POPULATION RAMP-UP 40
5. EXTRACTION AND PURIFICATION 41
v
VI Contents
5.1 Extraction and Purification 41
6. N-GLYCOSYLATION OF ALFALFA PROTEINS 43
7. CASE STUDY 43
7.1 Characteristics of an Anti-human IgG Produced in
Alfalfa 43
3. THE USE OF VIRAL VECTORS FOR THE
PRODUCTION OF RECOMBINANT PROTEINS IN
PLANTS 49
G.P. Lomonossoff
1. INTRODUCTION 49
2. CHOICE OF VIRUS FOR VECTOR DEVELOPMENT 50
3. TYPES OF EXPRESSION SYSTEMS 51
4. EXAMPLES OF THE EXPRESSION OF PEPTIDES
AND PROTEINS USING VIRAL VECTORS 52
4.1 Tobacco Mosaic Virus 52
4.2 Cowpea Mosaic Virus 57
4.3 Tomato Bushy Stunt Virus 60
4.4 Plum Pox Virus 62
4.5 Other Potyviruses 63
4.6 Potato virus X 63
4.7 Tobacco Rattle Virus 65
4.8 Subviral Systems 65
5. CONCLUSIONS 66
4. CONTROL OF THE N-GL YCOSYLATION OF
THERAPEUTIC GLY COPROTEINS PRODUCED IN
TRANSGENIC PLANTS: A NEW CHALLENGE FOR
GLY COBIOLOGISTS 73
P. Lerouge, M. Bardor, S. Pagny, V. Gomord, A.-C. Fitchette
andL. Faye
1. PLANT N-LINKED GL YCANS AND THEIR
DIFFERENCES WITH THEIR MAMMALIAN
COUNTERPARTS 74
1.1 Structures of Plant N-linked Glycans 74
1.2 Processing of N-linked Glycans in Plants 77
1.3 N-glycosylation and Folding of Proteins 82
1.4 Complex N-glycans Are Ubiquitous among the Plant
Kingdom 83
1.5 Structural Analysis of Plant N-glycans 84
2. N-GLYCOSYLATION OF THERAPEUTIC
GLY COPROTEINS PRODUCED IN TRANSGENIC
Contents Vll
PLANTS: CURRENT ASPECTS AND FUTURE
TRENDS 89
2.1 N-glycan Patterns of Therapeutic Glycoproteins
Produced in Transgenic Plants 90
2.2 Immunogenicity and Allergenicity of Plant N-
glycans 91
2.3 Metabolic Engineering of the Glycosylation in Plants
to Obtain a Perfect Copy of Mammalian N-glycans 92
3. FUTURE PERSPECTNES 100
5. PRODUCTION AND COMMERCIALIZATION OF
BIOPHARMACEUTICALS FROM MILK 111
M.G.A. Peters and M.F. Brink
1. INTRODUCTION 111
2. THE RISING COST OF PHARMACEUTICALS 112
3. ADVANCES IN BIOTECHNOLOGY 113
4. LIVESTOCK VERSUS BACTERIAL
FERMENTATION 115
5. TRANS GENESIS 116
6. TRANSGENIC MILK TECHNOLOGY 117
7. GENERATING TRANSGENIC ANIMALS THROUGH
MICRO- INJECTION 119
8. NEW DEVELOPMENTS: NUCLEAR TRANSFER
TECHNOLOGY 120
9. PHARMING'S TRANSGENIC CATTLE PLATFORM 122
10. EFFECTNENESS OF THE TRANSGENIC
PLATFORM 122
10.1 Human Lactoferrin 123
10.2 Human Fibrinogen 123
11. CONCLUSION 124
6. A SEED-DERNED ORAL SUBUNIT VACCINE FOR
HUMAN CYTOMEGALOVIRUS 127
E.S. Tackaberry, K.E. Wright, A.K. Dudani, I. Altosaar
and P.R. Ganz
1. PLANTS AS EXPRESSION SYSTEMS FOR
BIOTHERAPEUTICS 128
2. HUMAN CYTOMEGALOVIRUS 130
3. CONCERNS AND CHALLENGES 131
4. PREVIOUS WORK 132
5. TRANSFER OF THE HCMV Gb GENE TO TOBACCO 132
5.1 Vector Constructs and Transfer to Plants 132
V111 Contents
5.2 Molecular Analysis of Plants Containing the HCMV
gBGene 134
6. Immunological Detection of gB and Quantification in
Seed Protein Extracts 135
6.1 Extraction of Soluble Seed Proteins 135
6.2 gB-Specific ELISA 136
7. Antigenic Comparability of Seed-Derived gB vs. gB
Produced in Infected Human Cells 137
8. Subcellular Localization of gB in Endosperm Tissue of
Mature Transformed Seeds 140
9. GENERAL DISCUSSION 142
9.1 Cloning, Transfer and Expression of gB Protein 143
9.2 Sub-cellular Localization of gB in Seed Endosperm 144
9.3 Sorting of gB in Endosperm Cells 145
10. SUMMARY 146
7. USE OF PLANT VIRUS-BASED EXPRESSION
SYSTEMS FOR THE PRODUCTION OF HIV VACCINES 155
G.G.Zhang
1. INTRODUCTION 156
2. CANDIDATE PEPTIDES AND PROTEINS FOR HIV
VACCINES 157
3. EXPRESSION OF HIV VACCINES USING RNA
PLANT VIRUS- BASED EXPRESSION VECTORS 160
3.1 Presentation of the Kennedy Epitope from HIV-l
gp41 on the Surface of Cowpea Mosaic Virus 160
3.2 Expression of V3 Loop from Hiv-l Gp120 Using
Tobacco Mosaic Virus 164
3.3 Expression of an Epitope from V3 Loop of Hiv-l
Gp120 and the Full-length P24 Using Tomato Bushy
Stunt Virus 168
4. SUMMARY AND FUTURE PROSPECTS 171
8. SUPPRESSION OF AUTOIMMUNE DIABETES BY THE
USE OF TRANSGENIC PLANTS EXPRESSING
AUTOANTIGENS TO INDUCE ORAL TOLERANCE 179
S. Ma and A.M. Jevnikar
1. INTRODUCTION 179
2. ORAL TOLERANCE AND THE TREATMENT OF
AUTOIMMUNE DISEASES 181
3. UNIQUE ADVANTAGES OF USING TRANSGENIC
PLANTS FOR ORAL TOLERANCE INDUCTION 183
Contents ix
4. TRANSGENIC PLANTS EXPRESSING GAD 185
5. ORAL IMMUNOGENICITY OF THE PLANT
DERIVED GAD PROTEIN 186
6. EXPRESSION OF CHOLERA TOXIN B SUBUNIT
(CTB)-INSULIN FUSION PROTEINS 189
7. EXPRESSION OF HUMAN GAD65 190
8. CONCLUSIONS AND FUTURE PROSPECTS 191
9. THE PRODUCTION AND DELIVERY OF
THERAPEUTIC PEPTIDES IN PLANTS 197
L. Erickson, W.-J. Yu, J. Zhang, C.F.M. deLange,
B .McBride and S. Du
1. PLANT SCIENCE IN THE CONTEXT OF
VETERINARY AND MEDICAL HEALTH 197
2. SIGNIFICANCE OF INTESTINAL DISEASE 198
2.1 Human 198
2.2 Animal 199
3. INTESTINAL DISEASE AND THE ROLE OF THE
EPITHELIUM 199
4. GROWTH FACTORS AND THE EPITHELIUM 200
4.1 Epidermal Growth Factor 200
4.2 Glucagon-like Peptide-2 201
5. ANTIMICROBIAL PEPTIDES AND INTESTINAL
HEALTH 202
6. PLANTS ENGINEERED TO CONTAIN NOVEL
THERAPEUTIC PEPTIDES 203
6.1 Medicinal Plants 203
6.2 Expression of Xenoproteins in Plants: Problems and
Strategies 204
6.3 Expression of Mammalian Proteins in Plants 207
7. LEAF-BASED PRODUCTION SYSTEMS:
ADVANTAGES AND DISADVANTAGES 212
8. DELIVERY OF THERAPEUTIC PEPTIDES TO THE
INTESTINAL TRACT 214
9. REGULATORY CONSIDERATIONS 215
10. AN ORAL VACCINE IN MAIZE PROTECTS AGAINST
TRANSMISSmLE GASTROENTERITIS VIRUS IN
SWINE 223
J.Jilka
1. INTRODUCTION 223
x Contents
2. GENE CONSTRUCTS AND TRANSFORMATION OF
MAIZE VIA AGROBACTERIUM 225
3. PROTOCOLS FOR FEEDING AND CLINICAL
TRIALS 226
3.1 Transgenic Grain Production 226
3.2 Swine Feeding Trials 226
3.3 Vaccination of Feed Test Groups 226
3.4 Virus Challenge 227
3.5 Data and Sample Collection 227
3.6 Data Analysis 228
3.7 Swine Feeding Trial #1 (TGEV-1) 229
3.8 Swine Feeding Trial #2 (TGEV -2) 229
4. RESULTS FROM CLINICAL TRIALS 229
4.1 Observations of Clinical Symptoms for TGEV- 1 229
4.2 Observations of Clinical Symptoms for TGEV- 2 230
5. GENERAL DISCUSSION 232
11. PRODUCTION OF ANTIBODIES IN ALFALFA
(MEDICAGO SATIVA) 237
U. Busse, V. Levee, S. Trepanier and L. Vezina
1. INTRODUCTION 238
1.1 Antibodies in Human and Veterinary Medicine 238
1.2 Antibody Production: from Fermentors to Molecular
Farming 239
2. MOLECULAR FARMING USING ALFALFA 241
3. PRODUCTION STRATEGIES IN ALFALFA 242
3.1 Genetic Programming 242
3.2 Gene Transfer 243
3.3 Production of Biomass 245
3.4 Recovery of Recombinant Molecules 247
3.5 Disposal of Biomass 248
4. CASE STUDY 249
5. FUTURE DEVELOPMENTS 251
6. CONCLUSION 253
12. THE PRODUCTION OF RECOMBINANT ANTIBODIES
IN PLANTS AND PLANT CELLS 259
R. Fischer, N. Emans and S. Schillberg
1. INTRODUCTION 259
2. TRANSGENIC PLANTS AS BIOREACTORS FOR
RECOMBINANT PROTEIN PRODUCTION 262
2.1 Antibody Engineering 263
Contents xi
2.2 Antibody Production in Transgenic Plants 263
3. PROTEIN EXPRESSION IN PLANTS 267
3.1 Agroinfiltration 268
3.2 Viral Vectors 268
3.3 Expression in Stably Transformed Plants 270
3.4 The Ideal Crop for Production of Recombinant
Antibodies 270
4. PROTEIN PRODUCTION IN PLANT SUSPENSION
CULTURED CELLS 271
4.1 Suspension Cell Transformation 273
5. DOWNSTREAM PROCESSING OF RECOMBINANT
ANTmODIES FROM TRANSGENIC PLANT CELLS 274
6. APPLICATIONS OF RECOMBINANT ANTmODIES
EXPRESSED IN PLANTS 275
6.1 The Safety of Plant Produced Recombinant
Antibodies 275
6.2 USING RECOMBINANT PLANT-DERIVED
ANTmODIES IN HUMAN HEALTH CARE 276
7. PERSPECTIVES 277
13. IMMUNOTHERAPEUTIC POTENTIAL OF ANTmODIES
PRODUCED IN CHICKEN EGGS 287
Y. Mine and J. Kovacs-Nolan
1. INTRODUCTION 288
2. PHYSIOLOGY OF CHICKEN EGG FORMATION 289
3. CHICKEN EGGS AS AN ALTERNATIVE SOURCE
OF ANTmODIES 291
3.1 STRUCTURAL CHARACTERISTICS OF
CHICKENIGY 293
3.2 Production and Isolation of Chicken IgY 295
4. IMMUNOTHERAPEUTIC POTENTIAL OF CHICKEN
IGY297
4.1 Application of IgY for the Prevention of Human
Rotavirus Infection 297
4.2 Application of Ig Y for the Prevention of Yersinia
ruckeri in Rainbow Trout 301
4.3 Application of Ig Y for the Prevention of
Enterotoxigenic Escherichia coli Infection 302
4.4 Application of IgY for the Prevention of
Salmonellosis 306
4.5 Other Applications of IgY 308
5. CONCLUSION AND FUTURE STUDIES 309
