Expression of recombinant proteins in tobacco system

TABLE OF CONTENTS Page 2.1 Production of recombinant proteins in plant systems: 8 A brief history 2.2.1 Extrachromosomal expression by geminiviral DNA-based vectors 9 Geminiviruses as

EXPRESSION OF RECOMBINANT PROTEINS

IN TOBACCO SYSTEM

D TAMILSELVI

NATIONAL UNIVERSITY OF SINGAPORE

2004

EXPRESSION OF RECOMBINANT PROTEINS

IN TOBACCO SYSTEM

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

TABLE OF CONTENTS Page

2.1 Production of recombinant proteins in plant systems: 8

A brief history

2.2.1 Extrachromosomal expression by geminiviral DNA-based vectors 9

Geminiviruses as vectors for the expression of foreign genes 11

Factors influencing transgene expression 17 Environmental concerns of transgenic plants 17

Agroinfiltration-expression using bacterial vectors 19

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

2.4.2 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 42

2.5.2 Molecular structure and development of recombinant 45

human Vascular Endothelial Growth Factor (hVEGF165)

VIRUS REPLICATION IN Nicotiana benthamiana AND N tabacum

3.2.3 Transformation of N benthamiana mesophyll-derived protoplasts 60

with plasmid DNA and confirmation using PCR

3.2.4 Transformation of tobacco BY2 cells by particle bombardment 60

and PCR detection of transformed lines

3.2.5 Rescue of pASVNPT and pASVGUS shuttle vectors from 61

tobacco BY2 cells in E coli

3.2.6 Analysis of replicating DNA by Coupled Restriction Enzyme 62

Digestion and Random Amplification PCR (CREDRA-PCR)

and Southern blot analysis

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 65

N benthamiana with pASVNPT

3.3.4 Transformation of pASV-derived vectors in N tabacum 68

BY2 cells by particle bombardment 3.3.5 Replication studies of pASV-derived shuttle vectors 69

PCR detection of pASVNPT and pASVGUS vector in transformed 69

calluses

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

CHAPTER 4.0 ESTABLISHMENT OF A HOST SYSTEM TO STUDY

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

4.2.8 Intra and extra cellular protein estimation 96

Histochemical localization for GUS 97

4.3 RESULTS 101

4.3.1 Effect of Kanamycin and G418 on tobacco BY2 cells 101

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

CHAPTER 5.0 ISOLATION OF PLANT SECRETORY SIGNAL

PEPTIDES AND THEIR USE IN THE EXPRESSION OF HUMAN

VASCULAR ENDOTHELIAL GROWTH FACTOR (hVEGF 165 ) 123

Reverse phase HPLC (High performance liquid chromatography) 128

5.2.3 Construction of hVEGF165 (human Vascular Endothelial Growth Factor) 130

Construction of hVEGF165KDEL for ER retention 130

5.2.4 Cultivation of plants and Agroinfiltration 138

Preparation of Agrobacterium for infiltration 138

5.2.5 Analysis of protein extracts from infiltrated leaves by SDS-PAGE 139

5.2.6 Heparin-Sepharose purification of hVEGF165 141

CHAPTER 6.0 A RAPID TRANSIENT EXPRESSION SYSTEM 172

FOR ANGIOPOIETIN 1 (Ang1)

6.2 MATERIALS AND METHODS 176

6.2.2 Construction of Angiopoietin 1 expression plasmid 176

6.2.3 Agroinfiltration of Nicotiana leaves 178

6.2.4 Preparation of protein extracts from infiltrated leaves 180

6.3.1 Construction of the human Angiopoietin1 (Ang1) 181

expression cassette

6.3.2 Transient expression of Ang1 in N benthamiana 181

6.3.3 Comparative time course analysis of Ang1 expression in N tabacum 185

6.3.4 Optimization of Ang1 expression from different dilutions of 188

SUMMARY

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 developed based on a geminivirus replicon from Ageratum yellow vein virus (AYVV) to study the expression

of a foreign gene exrachromosomally in Nicotiana benthamiana and N tabacum cv BY2

cell suspension cultures The vectors can replicate extrachromosomally in actively

dividing cells of N tabacum cv BY2 but not in non-dividing cells of N benthamiana

Several lines of evidence such as methylation-based PCR assay, GUS expression and plasmid rescue showed the presence of replicating DNA of the geminiviral plant vectors over a culture period of four months This is the first report of extrachromosomal replication of monopartite begomovirus vectors with stability and foreign gene expression in long-term cell cultures In the second part, the suitability of the BY2 culture system for intracellular versus secreted proteins was addressed Stable transgenic suspension cell lines of tobacco BY2 culture were established and evaluated for intracellular (GUS and GFP) and extracellular GFP accumulation targeted for secretion

by the PR1a signal peptide Time course analysis of GUS accumulation in cell cultures showed 0.02-5.8 % of TSP over the growth period studied Maximum GUS accumulation was 363 µg/g of cells The level of reporter proteins, GUS and GFP, was directly related

to increase in cell mass during the exponential growth phase of the cell culture The intracellular accumulation of GFP protein was twofold higher compared to extracellular

accumulation in suspension cell cultures However, the secretion system of production could still be advantageous due to its ease of protein purification The third part of this study was carried out to evaluate signal peptide (SP) strengths in the expression of the human Vascular Endothelial Growth Factor (hVEGF165) Three secretory proteins were identified from cell-free medium of tobacco BY2 cultures; the NT PRp27, lipid transfer protein (LTP), and a new member of systemic acquired resistance protein (SAR8.2) The signal peptides from the latter two were studied due to their novelty Secretory and non-secretory versions of hVEGF165 were expressed with these SPs in the absence or presence

of endoplasmic reticulum (ER) retention signal All other conditions were kept constant

by using an agroinfiltration-based transient expression system The two plant secretory signal peptides led to seven- to eight-fold higher level of VEGF expression compared to the expression level with native VEGF signal peptide The apoplast yield (0.06-0.15 % TSP) was higher compared to its accumulation in ER (0.01-0.02 % TSP) Both secreted and non-secreted proteins of VEGF were glycosylated The secretory system developed would be suitable for the production of certain classes of growth factors and antibodies The last part of the study was to express a second protein involved in vascular maturation, the full length human Angiopoietin1 (Ang1) For therapeutic purposes, mostly the variant Ang1* is applied, as the native form was difficult to be produced in sufficient quantities The transient expression system was used successfully to produce a

full length version of Ang1 in N benthamiana with yields of 0.01 % of TSP Hence, the

plant system designed by us is suitable for molecular farming of Ang1

LIST OF TABLES Page

Table 2.1 Geminiviral vectors for replication and foreign gene expression 13

Table 2.2 Recombinant protein production in plant suspension cell culture 25

Table 2.4 Targeting of recombinant proteins to endoplasmic reticulum (ER) 39

Table 2.5 Production system, purification and biological assay of hVEGF165 47

Table 4.1 Effect of Kanamycin and G418 on tobacco BY2 cells 102

Table 4.2 Ratio of intracellular GFP fluorescence in Pra1GFP transgenic 117

cell lines Table 5.1 List of primers used for PCR amplification 133 Table 5.2 Signal peptide used in the present study 149 Table 5.3 Quantification of hVEGF165 expression 161 Table 7.1 Comparison of different expression strategies in tobacco system 195

LIST OF FIGURES Page

Figure 3.1 AYVV genome and cloning strategy of AYYV-derived 58

plant-E coli shuttle vector

Figure 3.2 Cloning of GUS cassette in pASV82 64

Figure 3.3 Electroporation efficiency of mesophyll protoplasts 66

Figure3.4 PCR detection of pASVNPT DNA in N benthamiana protoplasts 67

Figure 3.5 Selection of transgenic tobacco BY2 calluses 30 days after 70

bombardment with pASV vectors

Figure 3.6 PCR detection of pASVGUS in tobacco BY2 transformed 71

calluses

Figure 3.7 Rescue of pASVGUS shuttle vectors from transformed BY2 74

plant cells in E coli

Figure 3.8 Verification of pASVGUS shuttle vector replication in tobacco 76

cells using CREDRA-PCR Figure 4.1 Cloning strategy for the construction of pBinGUS vector 90

Figure 4.2 Cloning strategy for the construction of pBinGFP vector 91

Figure 4.3 Cloning strategy for the construction of pBinPr1aGFP vector 92

Figure 4.4 Restriction digestions of pBinGUS, pBinGFP, pCKPR1aGFP and 104

pBinPR1a clones Figure 4.5 DNA sequence verification of PR1a SP in pCKPR1aGFP clone 105

Figure 4.6 Selection of transgenic tobacco BY2 calluses on selection 106

medium Figure 4.7 Time course analysis of GUS-transformed BY2 cell lines 108

Figure 4.8 Time course analysis of GFP-transformed BY2 cell lines 109

Figure 4.9 Time course analysis of PR1aGFP-transformed BY2 cell lines 110

Figure 4.10 Time couse analysis of extracellular protein content in 111

transgenic PR1aGFP cell lines

Figure 4.11 Intracellular GUS expression levels in transgenic tobacco 113

BY2 cell lines over a growth period Figure 4.12 Intracellular fluorescence level in GFP-transformed cell lines 115 Figure 4.13 Extracellular fluorescence level in PRa1GFP transformed cell lines 116 Figure 5.1 Strategy for cloning hVEGF165 in pBluescript 131 Figure 5.2 Strategy for cloning LTP signal peptide with VEGF165 in 134

pBluescriptSK Figure 5.3 Strategy for cloning SAR signal peptide with hVEGF165 in 135

tobacco BY2 cell suspension culture Figure 5.7 Fractionation and purification of secreted proteins from 146 tobacco BY2 cell suspension culture

Figure 5.8 Alignment of the amino acids of NtBY2 secretory proteins 147 Figure 5.9 Screening of recombinant hVEGF165 clone 149 Figure 5.10 DNA sequence verification of pBIVK clone 150 Figure 5.11 DNA sequence verification of pBILVK clone 151 Figure 5.12 DNA sequence verification of pBISVK clone 152 Figure 5.13 DNA sequence verification of pBIVS clone 153 Figure 5.14 DNA sequence verification of pBILVS clone 154 Figure 5.15 DNA sequence verification of pBISVS clone 155

Figure 5.16 Transient expression of hVEGF165 (pBISVK clone) in 157

N benthamiana leaves using agroinfiltration

Figure 5.17 Transient expression of hVEGF165 in N benthamiana leaves 157 with controls

Figure 5.18 Accumulation of hVEGF165 in N benthamiana leaves using 159

various signal peptide and ER retention signals

Figure 5.19 Expression level of recombinant hVEGF165 in 160

agroinfiltrated leaves Figure 5.20 Purification of hVEGF165 from agroinfiltrated leaves using 164

affinity chromatography

Figure 6.1 Strategy for cloning Angiopoietin1 in a plant expression 177

vector Figure 6.2 Workflow for the transient expression system of recombinant 179

Figure 6.3 Restriction digestion of Ang1 recombinant clones 182 Figure 6.4 DNA sequence verification of 5’ Ang1 in pCKAng1 clone 183 Figure 6.5 DNA sequence verification of 3’ Ang1in pCKAng1 clone 184 Figure 6.6 Transient expression of human Angiopoietin1 in N benthamiana 186

leaves 5 days post infiltration (dpi) Figure 6.7 Comparison of human Angiopoietin1 from 3-5 dpi in 187

N benthamiana and N tabacum leaves

Figure 6.8 Expression of human Angiopoietin1 with different concentrations 189

of agrobacterial suspension in N benthamiana leaves on 3 dpi

LIST OF ABBREVIATIONS

ORF open reading frame

N-terminal amino terminal

C-terminal carboxy terminal

CLV1 cassava latent virus1

CAT chloramphenicol acetyl transferase

NPT noemycin phosphotransferase

GUS β-glucuronidase

GFP green fluorescent protein

OD optical density

PCR polymerase chain reaction

SDS sodium dodecyl sulfate

SDS-PAGE SDS polyacrylamide gel electrophoresis

xg relative centrifugal force

CHAPTER 1 INTRODUCTION

Traditional drugs produced by the pharmaceutical industry are mostly small molecules in nature However, biotechnology-based drugs or biotherapeutics are becoming highly popular in the genomics era This is mostly due to the identification of new protein-based drugs, drug targets and effective monoclonal antibodies Until recently, the only biologically based therapeutic agents available were blood products, vaccines and a limited number of hormonal and enzyme preparations All of these substances were derived from natural sources such as blood donations or animal tissue Insulin, for example, was produced exclusively from pancreatic tissue of slaughterhouse animals, while blood products were obtainable only from blood In many such instances the natural level of protein production is often low Large quantities of the source material are required to prepare appreciable amounts of the protein product (Walsh and Headon 1994)

With the advent of recombinant DNA technology, recombinant protein production

systems utilized today range from prokaryotic systems such as Escherichia coli and

Bacillus, to eukaryotic systems such as yeast, Aspergillus, mammalian and insect cell

cultures, transgenic animals and plants Most recently, the moss Physcomitrella patens (Decker and Reski 2004) and the unicellular green alga Chlamydomonas reinhardtii

(Franklin and Mayfield 2004) have also emerged as potential systems for molecular farming

Each host system has its own strengths and limitation for the production of recombinant

proteins Although E coli is the workhorse of the biotechnology industry, it is not well

suited for expressing eukaryotic genes, particularly if the protein product must be glycosylated and terminally processed In addition, the recombinant protein can be toxic

to bacteria, form inclusion bodies or be degraded by proteases (Kudo 1994) Yeast cells, unlike bacteria, possess subcellular organelles and are capable of carrying out posttranslational modifications of proteins, but these modifications differ significantly

from those in animal cells Humanization of the glycosylation pathway in Pichia pastoris

to secrete human glycoprotein is also possible now (Hamilton et al 2003) Mammalian and insect cell cultures are currently used for producing many important pharmaceutical proteins because of their ability to perform glycosylation and to process the recombinant protein similar to that of the native host (Jenkins et al 1996) However, the nutritional requirements of animal cells are most complex compared to various production systems Foetal calf serum is added as a source of non-defined essential nutrients, which is expensive and also risks contamination with blood-borne pathogens Animal cells grow slower than their microbial counterparts; therefore, greater numbers of animal cells are required to seed the reactors effectively (Walsh and Headon 1994) Transgenic animals have emerged as promising systems for producing human proteins in milk, because mammary glands are capable of performing correct posttranslational modifications including complex glycosylation and γ-carboxylation (Velander et al 1997) Transgenic animals, as a source of recombinant antibodies, are currently limited by legal and ethical constraints

Transgenic plants have emerged as potentially one of the most economical systems for large-scale production of recombinant proteins for industrial 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 scale-

up (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) Other potential advantages that 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 eukaryotes with correct folding, glycosylation, and other posttranslational events (Goodijn and Pen 1995) and 5) the presence of sialyated endogenous glycoconjugates in plant cells (Shah et al 2003)

Many companies have emerged over last several years to produce molecular farming products, which are largely based in the USA, Canada and France Corn-derived products such as avidin, GUS (β-glucuronidase) and trypsin are currently being marketed by Sigma-Aldrich In addition, at least nine products are thought to be close to reaching markets in the next five years (Horn et al 2004) At least six types of plant-derived recombinant antibodies have progressed to the preclinical testing stage, with the most advanced product now undergoing phase II clinical trials, namely, CaroRx a chimeric

secretory IgA/G antibody that prevents recolonization of Streptococcus mutans, the oral

pathogen that is responsible for tooth decay in humans (Ma et al 2003) Large Scale Biology Corp., Vacaville, California, has completed phase I trials for 38C13 scFv antibody that recognizes unique markers on the surface of any malignant B cells and offers an effective therapy for human diseases such as non-Hodgkins lymphoma

The production of recombinant proteins in plants is successful mainly because of the

efficiency of plant transformation using Agrobacterium tumefaciens, availability of direct

gene transfer technologies and RNA-based viral vectors These vectors allow stable and transient expression of recombinant protein in plants To date, however, DNA-based viral vectors have not been used extensively in plants to express foreign proteins These viral vectors have not been shown to generate stable transformants via integration or transmission through the germline However, simple genomic organization, nuclear replication and broad host range of geminiviruses makes them attractive candidates for vector development (Timmermans et al 1994) There are only a few reports to date on the expression of foreign proteins by geminivirus-based gene amplification systems In the present study, the first objective was to develop a geminiviral vector based on the

monopartite begomovirus, Ageratum yellow vein virus (AYVV) and study its replication

and expression of foreign proteins

Various systems are available for the in vitro cultivation of plant cells, such as hairy

roots, immobilized cells and free cell suspensions The latter is generally regarded as most suitable for large-scale applications in the biotechnology industry due to more

systems like bacteria, yeast and mammalian cell cultures, the number of applications of

plant cell cultures for recombinant protein production is still relatively low In the present

study, the second goal was to develop plant cell suspension cultures as a host system to

express foreign proteins

Several strategies can be implemented nowadays for higher levels of protein expression

and 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 their

compartmentalization to the endoplasmic reticulum (ER) and 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 varies (Fischer and Emans 2000; Schillberg et al 2003) The third objective in this study was, therefore, to study the effects of plant secretory

signals by identifying and isolating new sources of plant secretory signal peptides and to

exploit their potential to target foreign proteins to the endoplasmic reticulum (ER) and

apoplast

To capitalize on the advantage of plant-based systems in upstream production, the downstream purification of the recombinant product should be accomplished economically The downstream processing cost has been drastically reduced or eliminated for industrial enzymes not requiring high degree of purity or when used as feed and food supplement For the purification of other classes of proteins, several strategies such as those based on affinity tag, purification of viral particles, fusion proteins and oil bodies expressing foreign protein are rapidly gaining attention (Cramer et al.1999) Hence, depending on the crop, tissue, subcellular targeting of protein and method of transformation, each plant-recombinant protein system has to eventually be addressed and optimized separately Specific classes of proteins need to be chosen for the production in plants These criteria include low cost production of high quality biologically active human / mammalian proteins that need to undergo posttranslational modifications, passing through the secretory pathway in plants Based on these criteria, human angiogenic proteins, Angiopoietin1 and Vascular Endothelial Growth Factor (hVEGF165) were chosen for the expression study In the fourth objective, a rapid transient expression system was used to test the feasibility of the production of angiogenic proteins in a tobacco system

Taken together, the suite of tools developed and the validations carried out in this study, will help establish the development of a molecular farming programme in plants

In summary, the present investigation was carried out with the following specific objectives:

1) To develop geminiviral extrachromosomal vectors from Ageratum yellow vein

virus (AYVV) and study their replication in Nicotiana benthamiana and N tabacum cell suspension cultures for the expression of foreign proteins

2) To establish stably transformed N tabacum cell suspension cultures in order to

investigate the relative efficiency of intra and extracellular accumulation of foreign proteins

3) To isolate novel signal peptides from proteins secreted by N tabacum suspension

cells and evaluate their use for secretion or endoplasmic reticulum (ER) retention

of a model foreign protein, the human Vascular Endothelial Growth 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

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 Plants are now a source of industrial enzymes (Hood et al 2003; Bailey et al 2004), pharmaceutical proteins, such as mammalian antibodies (Hiatt et al 1989; Conrad and Fiedler 1998; Fischer et al 1999a), blood substitutes (Magnuson et al 1998) and vaccines (Mason and Arntzen 1995; Walmsley and Arntzen 2000) The milestones on the production of recombinant proteins in plant systems can be broadly categorized into four phases as given below In phase I, with the first report on stably transformed plants, in the early 1980’s (Fraley et al 1983; Horsch et al 1984), the potential of using plants as a production system for recombinant pharmaceuticals was established between 1986 and

1990 In phase II, expression of a human growth hormone fusion protein (Barta et al 1986), an interferon (De Zoeten et al 1989) and human serum albumin (Sijmons et al 1990) was reported In phase III, a crucial advance came with the successful expression

of functional antibodies in plants (Hiatt et al 1989; During et al 1990) This was a significant breakthrough as it showed that plants had the potential to assemble complex functional human glycoproteins with several subunits In phase IV, the structural authenticity of plant-derived recombinant protein was confirmed in 1992, when plants were used for the first time to produce an experimental vaccine: the hepatitis B virus (HBV) surface antigen (Mason et al 1992) The same group later showed that the vaccine produced in plants induced the expected immune response in mice (Thanavala et al

proteins for human and animal health care (for reviews, see Cramer et al 1996; Hood and Jilka 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 vectors, 2) stable nuclear transformation of a crop species that will be grown in the field or a greenhouse, 3) transient transformation of plants / plant cells and 4) stable plastid transformation of a crop species The first three strategies are briefly reviewed in this section

2.2.1 Extrachromosomal expression by geminiviral DNA-based vectors

Geminiviruses are very small plant viruses infecting a large variety of plant species The geminiviruses studied so far are found almost exclusively in the nuclei of the infected plant cells It is believed that their DNA replication occurs in nuclei based on electron

microscope studies of nuclei of Nicotiana benthamiana cells infected with cassava latent

virus (CLV) which showed nuclear inclusions called ‘fibrillar rings’ and from maize streak virus (MSV) infected nuclei in maize which exhibited paracrystalline virus-like arrays (Davies 1987) Recent advances have identified genes involved in replication, spread of virus and insect transmission (Davies and Stanley 1989) Gene replacement experiments suggest that useful plant gene expression vectors can be constructed from

these viruses (Timmermans et al 1994) Studies so far with these vectors are only with expression of reporter genes

Geminiviruses and their replication

Geminiviridae is a large family of plant viruses, whose members possess twinned or geminate virions and have small, single-stranded circular genomes of 2.5-3 kb in size There are four genera within the Geminiviridae namely Mastrevirus, Curtovirus, Topocuvirus and Begomovirus, that are classified based on their genetic organization, plant host and insect vector (van Regenmortel et al 1997; Briddon et al 1996) Members

of the Mastrevirus group (Maize streak virus, MSV; Wheat dwarf virus, WDV; Digitaria

streak virus, DSV) have a monopartite genome, infect generally, monocot species and are

transmitted by a variety of leaf hoppers Some Mastreviruses have adapted to infect dicot

hosts (Bean yellow dwarf virus, BeYDV; Tobacco yellow dwarf virus, TYDV; among others) Members of the Curtovirus (Beet curly top virus; BCTV) have a monopartite

genome, but they infect dicot species and are transmitted by leaf hoppers The

Topocuvirus members (Tomato pseudo-curly top virus, TPCTV) also have a monopartite

genome, infect dicotyledonous plants and transmitted by treehoppers Examples of

Begomovirus members are Bean golden mosaic virus, BGMV; Tomato golden mosaic

virus, TGMV; African cassava mosaic virus, ACMV; Squash leaf curl virus, SqLCV; Tomato yellow leaf curl virus, TYLCV and others They have bipartite genomes, infect

dicot species and are transmitted by white flies (Gutierrez 2000; Briddon et al 1996) Relatively few begomoviruses have been described to possess a monopartite genome

(Tomato leaf curl virus, TLCV; Ageratum yellow vein virus, AYVV; and Cotton leaf curl

virus, CLCuV) (Dry et al 1993; Tan et al 1995; Briddon et al 2000)

Members of the geminivirus group have circular ssDNA genomes, encoding only a few proteins; therefore, their DNA replication cycle relies largely on the use of cellular DNA replication proteins The ssDNA molecules replicate as minichromosome-like ds replicative form (RF)-DNA which are transcriptionally active templates in plant cell

nuclei (Palmer et al 1999; Pilartz and Jeske 1992) Only one viral gene (Rep) is required

for replication; the Rep protein is a sequence-specific DNA binding protein which recognizes the viral origin of replication and initiates rolling circle replication from a nick (within the 9 nt invariant sequence TAATATT↓AC) The Rep protein induces a nick in the loop sequence of a conserved stem-loop structure of the intergenic region (Laufs et al 1995).The ssDNA molecules replicate to high copy numbers in the nuclei Geminiviruses have, therefore, attracted wide interest for their potential use as plasmid-like DNA amplification systems for transient or stable transformation in plant cells (Timmermans et

al 1994)

Geminiviruses as vectors for the expression of foreign genes

Members of the Geminiviridae infect an extremely broad range of plant species, which implies that geminivirus-based vectors could potentially be used to express foreign genes

in virtually any agronomically important crop species Expression systems employing geminivirus replicons are of replacement type vectors In monopartite geminiviruses only intergenic and complementary strand ORFs are necessary for replication (Lazarowitz et

al 1989; Kammann et al 1991; Ugaki et al 1991) In bipartite viruses, DNA A encodes all the viral functions required for replication and encapsidation of viral DNA, while the

B component is required for symptom development and viral movement (Rogers et al 1986; Townsend et al 1986) Because the coat protein of these viruses is not required for replication (Stanley and Townsend 1986; Gardiner et al 1988), this gene is replaced with foreign sequences The types of vector constructed can be divided into two groups In one group, the inserted coding sequences are fused to the virion-sense promoter and in the other; the gene to be expressed is placed under the control of a foreign promoter (Mullineaux et al 1992)

Replication-competent vector molecules can be obtained from cloned geminivirus sequences in several ways: (1) Viral copies are cloned as tandem repeats into a plasmid

or T-DNA (Elmer et al 1988; Hayes et al 1989) Upon delivery into plant cells, monomeric viral DNA is released either through recombination between repeated sequences or, if the viral strand origin is duplicated, through rolling-circle replication (Elmer et al 1988); (2) Direct repeats of a non-viral sequence flanking viral copy can release a replicating molecule by recombination (Lazarowitz et al 1989; 1992); (3)

Transfecting a monomeric, linear viral DNA released from an E coli plasmid by

restriction enzymes, produces replicating vectors by circularization with the cellular ligase activity (Laufs et al 1990; Matzeit et al 1991); (4) A shuttle-vector with

geminiviral and E coli replicon replicates in both plant cells and E coli (Kammann et al

1991; Ugaki et al 1991; Timmermans et al 1992) Details of replication and replacement

Table 2.1 Geminiviral vectors for replication and foreign gene expression

method

N plumbaginifolia CLV 1 PEG mediated

transformation with mesophyll protoplasts

DNA A component Viral DNA replication is linked

to the cell division of the host cell

Townsend et al

1986

N benthamiana ACMV Agroinfection CAT CAT activity in systemically

infected leaves for 4 weeks (80 U/mg protein)

Monomeric and dimeric constructs

Mutation into viral coat protein did not affect DNA replication, but required for systemic infection

Woolston et al

1989

and endosperm cells

Gene replacement vector with NPTII, CAT,

β-galactosidase

Initiated de novo synthesis of the

viral genome with 20 fold amplification of marker gene

Viral DNA replilcation coincides with cell division

Matzeit et al

1991

Maize WDV Transfection of

endosperm protoplast

more

Ugaki et al 1991

Digitaria setigera DSV Agroinfection DSV replicative forms were

20-fold more abundant in S-phase nucleic and replication seems to synchronize with host DNA replication

Accotto et al

1993

non-coding region of the genome

Virus replication led to 5-10 fold increase in the mean number of GUS spots

Shen and Hohn

Black Mexican

Sweet corn

MSV Biolistic

transfection of suspension cells

Virion sense gene replacement of MSV with 1)‘long’p35S-bar

2) ‘short’p35S-bar 3) 35S –Ώ-AdhI intron-bar 4) 35S- Ώ-AdhI intron-bargor (bialaphos resista nce, glutathione reductase gene fusion)

38-60 % cell lines contained replicating viral episomes

Replicons were structurally stable, with copy number over 500/haploid genome for 1 year

Advantages of geminivirus vectors

Geminiviral vectors allow gene expression to be studied without the variation resulting from chromosomal location or chromosome region-specific DNA methylation that is encountered frequently in stable transformants (Timmermans et al 1992) Such vectors can accumulate to very high copy numbers; levels of up to 30,000/cell and 500/haploid chromosome set have been reported in the inoculated cells (Timmermans et al 1992; Palmer et al 1999) Biological containment problems for these vectors are alleviated by the lack of seed transmission and by the coat protein requirement for insect transmission Viral vectors with movement protein and coat protein, permit the characterization of genes in differentiated tissues without the need for tissue culture or somatic embryogenesis, using the ability of viruses to spread systemically through the plant (Timmermans et al 1994)

2.2.2 Stable transgene expression

Stable transformation is defined by the integration of a target gene into the host plant genome The generation of transgenic plants uses two principal technologies;

Agrobacterium-mediated gene transfer to dicots and monocots (Horsch et al 1985; Hiei

et al 1994; Hiei et al 1997 ), or biolistic delivery of genes to monocots, such as wheat and corn (Christou 1993) For transforming plants, the gene of interest is cloned into a

binary vector that can be moved between E coli and Agrobacterium The transformed

Agrobacterium itself delivers the target gene into the host cell genome Transformation is

followed by selection for cells with stably integrated copies of the target gene This is done by following a selectable resistance gene that is introduced via the T-DNA along

with the foreign gene Transgenic plants can then be grown in fields The main advantages of this method include yield, economical scalability in the field, establishment

of permanent lines expressing the protein of interest and longevity of transgenic seeds as

storage propagules

Stable transformation of plants depends on the plant variety and it can take 3-9 months for plants to be available for testing the expressed protein When long-term production of recombinant proteins at a low cost is necessary, stable transgenic plants are the most attractive strategy The quantity of recombinant protein that can be harvested is only limited by the number of hectares that can be planted with transgenics High intensity agriculture can produce large amounts of biomass Several examples are now available to establish the economy of scale For example intensive cultivation of tobacco plants can produce approximately 170 metric tones of biomass per hectare (Cramer et al 1996) Assuming that the levels of production seen on the laboratory scale could be at least kept constant in the field and that for every 170 tonnes harvested plant material, 100 tonnes are harvested leaves: a single hectare could yield 50 kg of secretory IgA (Ma et al 1995)

or 100 kg of recombinant glucocerebrosidase (Cramer et al 1996) The company, Planet Biotechnology (Mountain View, CA) is concentrating on using plants to produce the

Streptococcus mutans specific Guy’s-13 antibody, which prevents dental caries (Ma et al

1995; Ma et al 1998) Similar approaches are now underway to produce other antibodies and recombinant proteins by Prodigene Inc., USA (Horn et al 2004)

Factors influencing transgene expression

Transgene expression is influenced by several factors that cannot be controlled precisely through construct design This leads to variable transgene expression and, in some cases, its complete inactivation (Plasterk and Ketting 2000) Factors affecting transgene expression include: (1) the position of transgene integration; (2) the structure of the transgenic locus; (3) gene-copy number and (4) the presence of truncated or rearranged transgene copies (Ma et al 2003) Several strategies have been adopted in an attempt to minimize variation in transgene expression These include the use of viral genes that suppress gene silencing (Anandalakshmi et al 1998) The ability to integrate single-copy transgenes into precise locations in the plant nucleus can also eliminate position effects and the problems that are associated with variable locus structure Several laboratories are, therefore, investigating ways to improve the efficiency of gene targeting in plants (Wilde et al 2000; Britt and May 2003)

Environmental concerns of transgenic plants

The biosafety aspects of transgenic plants are covered by established and emerging regulations Environmental biosafety issues, such as the potential for transgene spread by pollen dispersal, seed dispersal and horizontal gene transfer, and the 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

chain (Ma et al 2003) Identity preservation and tracking are, therefore, important parts

of the regulatory procedure for the production of pharmaceuticals in transgenic plants

2.2.3 Transient gene expression

There are four major transient expression systems available to deliver genes to plant cells: (1) delivery of projectiles coated with naked ‘DNA’ by particle bombardment, (2) delivery of DNA by electroporation, (3) infiltration of intact tissue with recombinant agrobacteria (agroinfiltration), and (4) infection with modified viral vectors (Fischer et al 1999a) The overall level of transformation varies among these systems Particle bombardment and electroporation usually reach only a few cells and for transcription, the DNA has to reach the cell nucleus (Christou 1996; Ugaki et al 1991) Though it can be used to test recombinant protein stability before stable transformation, transient expression methods are unsuitable for the expression of larger amounts of foreign proteins Particle bombardment is more popularly used in the regeneration of transgenic cereal crops The method of agroinfiltration targets many more cells than particle bombardment and the T-DNA harboring the gene of interest is actively transferred into the nucleus with the aid of several bacterial proteins (Kapila et al 1997) A viral vector can systemically infect most cells in a plant and transcription of the introduced gene in RNA viruses is achieved by viral replication in the cytoplasm, which transiently generates many transcripts of the gene of interest

Agroinfiltration – expression using bacterial vectors

In agroinfiltration, cells of Agrobacterium carrying the expression vector are delivered

into leaf tissue by vacuum infiltration or by injection with a syringe Agrobacterial proteins then catalyze the transfer of the gene of interest into the host cells and protein expression can be detected three days after infiltration As in conventional methods for generating transgenic plants, genes of interest are cloned into binary vectors that are

transferred into suitable Agrobacterium strains A bacterial suspension is then used for

vacuum infiltration of leaves (Kapila et al 1997) and no selection method to identify transformed cells is required since the leaf tissue is only used for transient protein production, which permits the use of smaller plasmid vectors

In agroinfiltration, the transferred T-DNA does not integrate into the host chromosome but is present in the nucleus, where it is transcribed thus leading to transient expression of the gene of interest (Kapila et al 1997) A major advantage of this technique is that multiple genes present in different populations of agrobacteria can be simultaneously

expressed Thus, the assembly of complex multimeric proteins can be tested in planta

(Vaquero et al 1999) For transgenic plants, this can only be achieved by time consuming crossing experiments with individual transgenic plant lines each expressing a single component of the multimer

Using agroinfiltration, transient expression of scFvs, individual heavy and light chains as well as full size mouse-human chimeric anti-carcinoembryonic antigen (CEA) antibodies

in plant leaves were reported (Vaquero et al 1999) For full-size chimeric antibody

expression, the mouse-human chimeric heavy and light chain genes were integrated into

two vectors in two separate recombinant Agrobacterium strains and these two strains

were simultaneously infiltrated into leaves Functional full size chimeric antibodies were

assembled in vivo by simultaneous expression of both genes in the host cells This

technique is also suitable for the expression of scFv-fusion proteins, diabodies and component protein complexes (Schillberg et al 2003) Transient expression of GUS was reported in ripe fleshy fruits of apple, pear, tomato, peach, strawberry and orange by infiltration of fruits (Spolaore et al 2001) Recently, reports have described how this agroinfiltration process could be scaled-up more efficiently Researchers at Medicago Inc Canada have described agroinfiltration of alfalfa leaves, which can be scaled up to 7,500 leaves per week, producing micrograms of recombinant protein each week Similarly, up to 100 kg tobacco of leaves could be processed by agroinfiltration, resulting

multi-in the production of several hundred milligrams of protemulti-in (Fischer et al 2004) Further, this technique was coupled to MALDI-TOF-MS to validate the impact of differential targeting on structure and activity of a recombinant therapeutic glycoprotein, gastric lipase produced in tobacco plants (Issartel et al 2003)

As described above, agroinfiltration is rapid and yields sufficient quantities of protein for initial characterization of protein stability and protein function More importantly, agroinfiltration can be scaled up to produce tens of milligrams of recombinant protein and may even prove suitable for pre-clinical trials without the need for production of stably transformed plants (Fischer and Emans 2000) Also integration-deficient mutant

Expression using RNA - based viral vectors

RNA based viral vectors (Scholthof et al 1996) share several advantages with agroinfiltration Here, the gene of interest is cloned into the genome of a viral plant pathogen under the control of a strong subgenomic promoter Infectious recombinant viral transcripts are used to infect plants and produce target proteins Target genes are expressed at high levels because of the high level of multiplication during virus replication (Hendy et al 1999) Plant viruses that have a wide host range, are easily transmissible by mechanical inoculation and can spread from plant to plant, enable large number of plants to be rapidly infected with recombinant viruses Plant RNA viruses can multiply to very high titers in infected plants, which makes them ideal vectors for protein expression (Verch et al 1998) For vector construction, viral RNA genomes are

reverse-transcribed in vitro and cloned as full-length cDNAs in transcription vectors in

vitro or in vivo The cloned viral genomes can be manipulated with standard DNA

techniques There are different strategies for the insertion of foreign genes into plant viral genomes Gene replacement (Takamatsu et al 1987; Chapman et al 1992), gene insertion (Kumagai et al 1993) and gene fusion strategies (Turpen et al 1995; Fitchen

et al 1995) have been used in RNA-viral vectors

For inoculation of plants, recombinant viral vectors are usually transcribed in vitro and

the synthesized RNA is inoculated mechanically onto plants by gently rubbing the leaves with a mild abrasive Extracts from these infected plants can also be used for the

subsequent inoculation of a large number of plants

RNA virus-infected plants have been used to produce several pharmaceutical proteins, including vaccine candidates and antibodies (Streatfield and Hovard 2003) Tobacco mosaic virus (TMV) was the first rod-shaped virus to be exploited for use as a vector in

1987 Other viruses, namely barley stripe mosaic virus (BSMV), potato virus X (PVX), cowpea mosaic virus (CPMV) have also been used extensively Based on the gene

insertion method, Large Scale Biology Corp, (Vacaville, California, USA), employed

TMV-based vector to express a sequence encoding α–trichosanthin under the control of the U1 strain of TMV CP subgenomic (sg) promoter, while TMV CP synthesis was directed by a sg CP promoter from odontoglossum ringspot virus (ORSV) (Kumagai et

al 1993) The same company has also produced the 38C13 ScFv antibody that has completed phase-I clinical trials This strategy is well-suited for the rapid and small-scale production that is required to treat individual patients with unique antibodies (Ma et al 2003)

2.2 Alternative plant expression systems

Intact plants are not the only expression system available for plant-based molecular farming There are options for the production of proteins in seeds, plant suspension cells, phyllosecretion and by rhizosecretion from engineered plant roots

For production of recombinant proteins in seeds, such as expression of avidin in corn, the protein can be collected and extracted from the kernels (Hood et al 1997; 1999) Cereal crops have the advantage for recombinant protein production in seeds as they have well-