115

Pharming and transgenic plants

David Lienard1, Christophe Sourrouille1, Veronique Gomord andLoıc Faye�

Universite de Rouen, CNRS UMR 6037, IFRMP 23, GDR 2590, Faculte des Sciences, Bat. Ext.

Biologie, 76821 Mont-Saint-Aignan cedex, France

Abstract. Plant represented the essence of pharmacopoeia until the beginning of the 19thcentury when plant-derived pharmaceuticals were partly supplanted by drugs produced by theindustrial methods of chemical synthesis. In the last decades, genetic engineering has offeredan alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for theproduction of therapeutic proteins. More recently, molecular farming has rapidly pushedtowards plants among the major players in recombinant protein production systems. Indeed,therapeutic protein production is safe and extremely cost-effective in plants. Unlike microbialfermentation, plants are capable of carrying out post-translational modifications and, unlikeproduction systems based on mammalian cell cultures, plants are devoid of human infectiveviruses and prions. Furthermore, a large panel of strategies and new plant expression systemsare currently developed to improve the plant-made pharmaceutical’s yields and quality.Recent advances in the control of post-translational maturations in transgenic plants willallow them, in the near future, to perform human-like maturations on recombinant proteinsand, hence, make plant expression systems suitable alternatives to animal cell factories.

Keywords: glycosylation, molecular farming, plant-made pharmaceutical, recombinantprotein, transgenic plant, therapeutic protein.

Introduction

From 60,000 BC to the 19th century, plants were the main source for humandrugs. For instance, when sick and obliged to stay in his cave, the Nean-derthal man already used centaury to fight his fever. The first known text onmedicinal plants, the Pen Tsao, was written more than 4,500 years ago underthe direction of emperor Shen-Nung in China, and describes 365 medicinalplants, including opium, ephedra and hemp. More recently, around 1500 BC,the Ebers papyrus describes 700 remedies made from plants, includingmandrake, castor bean and hemp, illustrating that plants had a major placein Egyptian medicine. In the Middle ages, places such as Salagon abbayebecame famous for their specialization in the culture of medicinal plants anduniversities were created in Montpellier or Salerne to improve plant thera-peutics, extraction and characterization.There was a great turn in medicament history, starting at the beginning of

the 19th century until the early 1970s, when pharmacy turned to be dominated

BIOTECHNOLOGY ANNUAL REVIEWVOLUME 13 ISSN 1387-2656DOI: 10.1016/S1387-2656(07)13006-4

r 2007 ELSEVIER B.V.ALL RIGHTS RESERVED

�Corresponding author: Tel: 33-2-35-14-66-92. Fax: 33-2-35-14-67-87.E-mail: lfaye@crihan.fr (L. Faye).1David Lienard and Christophe Sourrouille have equal contributions to this work

116

by scientific chemistry with both the development of more and more sophis-ticated processes for extraction, purification and the synthesis of activepharmaceutical compounds. The 20th century became a triumph for drugsproduced at an industrial level by chemical synthesis. This evolution pro-bably started with the production of aspirin, a synthetic analogue of salicylicacid previously extracted from willow bark. In parallel, more and more so-phisticated extraction and purification procedures were developed resulting,for example, with the first extraction of morphine from poppy in 1815 orextraction of insulin from pig pancreas in 1922.As a complement of synthesis and extraction chemistry, modern biology

enters the world of pharmaceutical industry with the development of geneticengineering in the early 1970s, allowing biosynthesis of complex moleculestoo difficult to extract and purify from living material and inaccessible tosynthesis chemistry. In the last decades, genetic engineering has offered analternative to chemical synthesis and extraction procedures with the produc-tion of therapeutic molecules in transgenic bacteria, yeast and animal cells.After a temporary decrease in interest, plants are rapidly moving back intohuman pharmacopoeia, with the recent development of plant-based recom-binant protein production systems offering a safe and extremely cost-effectivealternative to microbial and mammalian cell culture. In this short review,we will illustrate that current improvements of plant expression systemsfor biopharmaceutical production are making them suitable as alternativefactories for the production of either simple or highly complex therapeuticproteins.

Plants have a high potential for pharmaceutical protein production

The need for cheap and efficient production systems emerges as a criticalfactor in therapeutic protein production. To satisfy the more and morerigorous industrial standards of performance, a heterologous system ofproduction has to fulfill several requirements. Pharmaceutical industry needslarge-scale methods using simple and inexpensive purification techniques toobtain recombinant proteins with high-production rates, reproducible qualityand for a low cost. Moreover, the system of production must be able to carryout co- and post-translational modifications (PTMs), including signal peptidecleavage, pro-peptide processing, protein folding, disulfide bond formationand glycosylation [1].Currently, no heterologous expression system of production satisfies all

of these requirements. For instance, complex therapeutic proteins producedin prokaryotes are not always properly folded or processed to provide thedesired degree of biological activity. Consequently, microbial expression sys-tems have generally been used for the expression of relatively simple thera-peutic proteins that do not require folding or extensive post-translationalprocessing to be biologically active such as insulin, interferon or human

117

growth hormone [2]. Due to the limitations of prokaryotes for productionof complex therapeutic proteins, the pharmaceutical industry had focusedefforts towards optimization of two main eukaryotic expression systems,yeasts and mammalian cell cultures. These production systems, however,suffer from many disadvantages such as inappropriate PTMs for yeast, orhigh operating costs, difficulties in scaling up to large volumes and potentialcontamination by virus or prion for cultured mammalian cells.Altogether, the biochemical, technical and economic limitations on existing

prokaryotic and eukaryotic expression systems, the growing clinical demandfor complex therapeutic proteins and the lack of bioreactor capacity havecreated substantial interest in developing new expression systems for theproduction of therapeutic proteins. To that end, plants have emerged in thepast decade as a suitable alternative to the current production systems oftherapeutic proteins and today their capacity in low-cost production of highquality, much safer and biologically active mammalian proteins is largelydocumented (for recent reviews see [1–4]).For instance, the use of transgenic plants could be a solution to the need

for a rapid increase in production capacity of therapeutic antibodies. Indeed,even with relatively low expression levels for therapeutic proteins [5,6], theproduction capacity of recombinant antibodies in transgenic plants is almostunlimited, as it only depends on the surface dedicated to the plant culture. Aplant ‘‘bioreactor’’ will allow the production of recombinant proteins up to20 kg/ha, regardless of the plant material considered: tobacco, corn, soybeanor alfalfa [7,8].Another major advantage of transgenic plants over other production sys-

tems available for large-scale and low-cost production, such as E. coli oryeasts, is their ability to perform most PTMs required for therapeutic pro-tein’s bioactivity and pharmacokinetics [4,9]. This is illustrated from theircapacity to produce complex functional mammalian proteins includingplasma proteins, antigens, growth factors, hormones, cytokines, enzymes andantibodies (Tables 1 and 2). The vast majority of therapeutic proteins under-goes several PTMs, which are the final steps in which genetic informationfrom a gene directs the formation of a functional gene product. The termPTM covers covalent modifications of individual amino-acid residues (e.g.,glycosylation, phosphorylation, methylation, ADP–ribosylation, oxidationand glycation); proteolytic processing and non-enzymatic modifications, suchas deamidation and racemization. Most therapeutic proteins require atleast proteolytic cleavage(s), oligomerization and glycosylation for their bio-activity, pharmacokinetics, stability and solubility.The production of immunoglobulins in plant cells is a good illustration of

plant capacity to produce complex proteins. Indeed, transgenic plant cellsare able to correctly synthesize, mature and assemble, via disulfide bridges,the light and heavy polypeptide chains constitutive of an antibody. Sincethe first production of a functional antibody in plant [5], many antibodies, or

Table 1. Therapeutic proteins produced in plants.

Products Proteins Transgenicplants

References

Blood and plasmaproteins

Albumin Potato, tobacco [10–12]Aprotinin Maize [13]Collagen I Tobacco [14]Encephalin Tobacco [11]Hemoglobin Tobacco [15–17]Human a1 antitrypsin Rice [18,19]

Vaccines Bet v 1 Tobacco [20]Cholera toxin Bsubunit

Potato [21]

Glycoprotein B fromhumancytomegalovirus(CMV)

Tobacco [22]

Cholera toxin Bsubunit-insulinfusion protein

Potato [23]

D2 peptide offibronectin bindingprotein B of S.aureus

Black bean [24]

VP1 Medicago sativa,Black bean

[25–27]

VP2 Arabidopsisthaliana

[28]

VP4 Medicago sativa [29]Hemagglutinin Tobacco [30,31]Hepatitis antigen Tobacco and

Potato[32,33–35]

gp41 glycoprotein Soybean [36]Enterotoxine B of E.coli

Potato, tobacco [37]

Cholera toxine B of V.cholera

Potato [38]

Epitope of P.falciparum

Tobacco [39]

Norwalk virus capsid Tobacco, potato [40,41]G protein of rabiesvirus

Tobacco,spinach,tomato

[42]

Autoantigene Potato [33]Hormones, cytokinsand growthfactors

GM-CSF(GranulocyteMacrophage-ColonyStimulating Factor)

Tobacco, candycane

[43–46]

Interferon b Tobacco [47]

118

Table 1 (Continued )

Products Proteins Transgenicplants

References

Interferon a Tobacco [48]Interferon g Rice [49]Somatotropin hGH(human GrowthHormon)

Tobacco(chloroplasts)

[50]

Erythropoitin Tobacco (cells) [51]Epidermal GrowthFactor (EGF)

Tobacco [11]

Vascular endothelialgrowth factor(VEGF)

Moss [52]

Interleukin 2 Potato [53]Human interleukin 6 Tobacco [54]Interleukin 10 Tobacco [55]Interleukin 12 Tobacco [56]Insulin like Growthfactor (IGF)

Rice [57]

TobaccoEnzymes Converting enzyme of

angiotensinTobacco, tomato [58]

Protein c (sericproteas)

Tobacco [59]

Glucocerebrosidase Tobacco (plantand seed)

[59,60,61]

Alpha-trichosantin Tobacco [62]Humanacetylcholinesterase

Tomato [63]

Dog gastric lipase Tobacco [64]Humantransglutaminase

Tobacco [65]

Others Hirudin Tobacco, [66,67]Colza

Endostatin Tobacco [68]Human lactoferrin Tobacco, rice [69–71]

119

antibody fragments, have been produced for therapeutic or diagnostic pur-poses in various plant expression systems (Table 2).Antibodies produced in plants are correctly assembled, proteolytically

matured and glycosylated. Indeed, antibodies produced in plants bear bothhigh-mannose and biantennary complex type N-glycans [72,73]. The high-mannose-type N-glycans have the same structure in plant and mammalianglycoproteins. But complex-type N-glycans are structurally different in plantsand mammals.

Table 2. Recombinant antibodies produced in transgenic plants. Adapted from [1].

Antigen Type of antibodies Indications Transformed plant Targeting signal Reference

Human carcinoembryonic

antigen

Mouse/human chimeric

IgG1 antibody

(cT84.66)

Antibody-mediated cancer

therapy (colon cancer,

breast cancer and tumour

with epithelial origin)

N. tabacum cv petit Havana

SR1 (transient expression)

MSP [secreted]

MSP+KDEL [ER]

MSP [secreted]

[74]

scFv T84.66 N. tabacum cv petit Havana

SR1

Plant codon optimized SP

[secreted]

T84.66/G68 diabody SP+KDEL [ER] [75]

Triticum aestivum MSP [secreted]

L. cv bobwhite MSP+KDEL [ER] [76]

scFvT84.66 O. sativa L. Indica

cv M12 and Bengal

Rabies virus protein Monoclonal antibody

(mAb SO57)

Rabies virus neutralization N. tabacum cv Xanthi MSP+KDEL [ER] [77]

Human IgG monoclonal

antibody

C5-1 IgG Anti-human globulin reagent

for phenotyping and

cross-matching red blood

cells of receivers and

donors

M. sativa MSP [secreted] [7]

Streptococcal surface antigen

SAI/II

Guy’s 13 IgG Tooth decay N. tabacum MSP [secreted] [78]

IgA/G

slgA/G [79]

Colon cancer surface antigen CO-17 A IgG Antibody-mediated cancer

therapy

N. benthamiana MSP [secreted] [80]

MSP+KDEL [ER]

HSV-2, protein from herpes

simplex virus (HSV)

IgG, IgA, DigA or slgA Immunoprotection against

genital herpes and

transmission of HSV

vaginal

O. sativa [81]

IgG1 G. max [82]

Fab and F(ab’)2

Zearalenone (mycotoxin) scfv Passive humanization of

animals in their feed

A. thaliana (ecotype

Columbia)

No SP [cytosol] Plant

PR1-b SP

[83]

120

Human creatine kinase-MM MAK33 IgG1 Cardiac disease,

mitochondrial disorders,

inflammatory myopathies,

myasthenia, polymyositis,

McArdle’s disease

A. thaliana A. thaliana 2S2 [seed

storage protein SP]

[84]

Fab fragment NMJ disorders, muscular

dystrophy, ALS, hypo-

and hyperthyroid

disorders

N. tabacum [85]

MAK33 scFv Central core disease, acid

maltase deficiency,

myoglobinuria,

rhabdomyolysis, motor

N. tabacum SR1 No SP [cytosol] [86]

A. thaliana 2S2 [seed

storage protein SP]

MAK33 Fab fragment Neurone diseases, rheumatic

diseases, and others that

create elevated or reduced

levels of creatine kinases

A. thaliana A. thaliana 2S2 [seed

storage protein SP]

[87]

Tumour’s surface Ig 38C13 scFv B-cell lymphoma treatment N. benthamiana Rice a-amylase SP

[secreted]

[88]

Herpes simplex virus (HSV)

glycoprotein D

HSV8 lsc (large single

chain of IgA)

Antibody-mediated herpes

therapy

Chlamydomonas reinhardtii [Chloroplastic

transformation]

[89]

Hepatitis B virus scFv Immunoaffinity purification

of recombinant HBsAg

N. tabacum cv petit Havana

SR1

No SP [cytosol] Sweet

potato Sporamin SP

[secreted] Sporamin

SP+PP [PSV]

Sporamin SP+KDEL

[ER]

[90]

Human CD30 mAb Treatment of Hodgkin

lymphoma and anaplastic

large cell lymphoma

Lemna minor [91]

KDEL, endoplasmic reticulum retention signal; MSP, murine signal peptide; SP, signal peptide; PP, propeptide; symbol [ ], organs or sub-

cellular compartments targeting of the recombinant protein.

121

122

Despite these differences in the N-glycan structures, antibodies produced inplants have similar antigen-binding capacity as their homologs producedin mammalian cells. Furthermore, an antibody half-life in the bloodstream aswell as its ability to be recognized by Fc receptors, which are both determinedby heavy chains N-glycosylation are not strongly affected when a plant-N-glycan is present instead of a mammalian N-glycan [7,12,92].

Which are the current limitations of the plant expression system?

Current limitations of plant expression systems are the low yields observedfor some therapeutic proteins and the impact of non-mammalian glyco-sylation on the activity, immunogenicity and allergenicity of glycosylatedplant made pharmaceuticals (PMPs). To achieve higher yields, differentstages of therapeutic protein expression in plants can be optimized fromtranscription to protein stability.

Adaptation of codon usage

Beside a long quest for stronger constitutive or inducible promoters, with upto now little success, it seems that in many cases where low therapeuticprotein expression levels are observed, adaptation of codon usage could in-crease the yields. The genetic code which defines a mapping between aminoacids and nucleotides is redundant: 18 of the 20 amino acids are codedby several codons known as synonyms. Most of the time, organisms usesynonymous codons in a non-random way, i.e., some synonymous code beingmore frequently used than others. Codon usage is thus biased, and this biasvaries according to the species and genes within a same species [93].

Thus each organism has a subset of synonymous code mainly used: thecodon bias. The codon bias is adapted to the abundance of ARNt for a moreeffective translation of the ARNm. Among different ARNt iso-acceptors forone amino acid, one is more abundant, this is the main ARNt. The use of themain ARNt by a gene allows its faster translation rate and higher fidelity [94].According to this model, the more a gene is expressed, the more it undergoesthe selection for the effectiveness of his translation. This brings to a pre-ferential use of optimal codon, causes a strong bias in the use of synonymouscodons and results in a low number of synonymous substitutions per site [95].So, in order to improve the rate and fidelity of translation in a plant ex-pression system, depending on its origin, it can be important to adapt thecoding sequence of the gene of interest to the codon bias of the host plant.Few information are yet available in plants, but a 5–100 times increasein protein expression was observed after codon optimization [96–99]. As anexample, expression in tobacco and tomato of a bacterial insecticide gene(cryIA) partially modified and (3% nucleotide difference) fully modified(21% nucleotide difference) were compared. Plants transformed with either

123

the partially or fully codon optimized gene respectively expressed 10 and100 times more insecticidal protein than plants transformed with the wild-type gene [96].

Suppressor of RNA silencing

RNA silencing was discovered by Andrew Hamilton and David Baulcombe[100]. This is an evolutionarily conserved control system that occurs in manyeukaryotic organisms, and was named RNA interference in animals, quellingin fungi and post-transcriptional gene silencing (PTGS) in plants. RNAsilencing plays a key antiviral defence role by influencing virus replication incells. Viruses, in turn, produce proteins capable of suppressing host cell RNAsilencing [101,102].

This mechanism of defence can be initiated not only by the presence ofvirus RNA but also by the presence of exogenous genes. In transformedplants, RNA silencing is targeted against transcripts of the transgene and anysimilar endogenous genes, so that corresponding gene products accumulateat a low level [103]. This phenomenon can be avoided by expressing simul-taneously the gene of interest and a suppressor of silencing. This providesa new tool for molecular farming in plants to obtain high-level expression oftransgenes.

Each plant-virus seems to produce its own suppressor of silencing and thecharacterization of a large number of suppressors such as HC-Pro, 2b, p25 iscurrently in progress. Today, the better-characterized suppressor is the p19protein, encoded by Tomato Bushy Stunt Virus (TBSV) [104]. This suppressorwas co-expressed with recombinant proteins and it was shown to dramati-cally enhance expression of a broad range of these proteins, allowing up to50-fold increase in yield [103,105].

Targeted expression of recombinant proteins

Targeted expression of PMPs into specific organs and subcellular compart-ments represent a plant-specific strategy to increase yields and simplifythe first steps of purification. In this way, different plant organs (leaves,seeds, root) and plant cell compartments (endoplasmic reticulum, chloro-plast, vacuole and oil body) have been efficiently used to express many thera-peutic proteins (Tables 2 and 3) [106,107].

Generally, recombinant proteins are targeted into plant organs, whichallows high-biomass yield. For example, in plants with large foliage volumesuch as tobacco, alfalfa and some other legume plants, expression is per-formed in leaves, whereas, in potato, corn, rapeseed, safflower, soybean,wheat or rice, the production and accumulation of recombinant proteinsoccur in tubers or in seeds [108,109]. Both systems have their own advantagesand drawbacks. Leaves present an active and complex metabolism which

Table 3. Examples of plant-based expression systems used for pharmaceutical protein production [110].

System Protein Expression Companya References

Stable nuclear transformation systemsWhole plant (cytosolic) HbsAg, vaccine 0.007% TSP AltaGen Bioscience Inc.

(potato)[111]

Collagen 1mg/gDW CropTech Corp. (tobacco) [14]Medicago Inc. (alfalfa)Meristem Therapeutics(tobacco)

scFv, hepatitis B 0.032% TSP PlantGenix Inc. (notreported)

[90]

Cellular compartment VacuolesIgA/G Not reported [112]

ER scFv, cutinase 1% TSP Novoplant GmbH(tobacco)

[113]

scFv, T84.66 29 mg/g FW [114]scFv, ABA 6.8% TSP [115]

Apoplast IgG1 1.3% ISP Epicyte PharmaceuticalInc. (tobacco)

[5]

IgA/G 500 mg/g FW [6]IgG1, Fab 13% ISP [84]

Tissue-specificity Seed avidin 6% TSP ProdiGene Inc. (corn) [116]hirudin 1% FW SemBioSys Genetics Inc.

(canola)[66]

124

Applied Phytologics Inc.(rice)

Epicyte PharmaceuticalInc. (corn)

IPT, Monsanto (corn)Meristem Therapeutics(rape)

Tuber scFv, oxalozone 2% TSP Meristem Therapeutics(potato)

[117]

Root IgM, RKN 0.003% TSP [118]Fruit RSV-F protein not reported [119]

Exudate human SEAP 20 mg/g Phytomedics Inc. [120]DW/day (tobacco roots)

human SEAP 2.8% TEP Phytomedics Inc. [121](tobacco leaves)Biolex Inc. (duckweed)

Stable plastid transformation systemChloroplast somatotropin 7% TSP [122]Transient transformation systemViral a-trichosanthin 2% TSP Large Scale Biology Corp.

(tobacco)[62]

DW, dry weight; ER, endoplasmic reticulum; FW, fresh weight; ISP, intercellular soluble protein; TEP, total exuded protein; TSP, total soluble

protein.aCompanies sharing a row with a protein and reference are sources of this information.

125

126

offers many possibilities, but they also contain significant protease activitieslimiting the accumulation of some PMPs [1]. The low-water content of theseeds allows accumulation of recombinant proteins into compact biomass sitefor long periods of time at relatively high concentrations [123].

Beside organ-specific storage of PMPs, many subcellular compartmentsare available for accumulation of large amounts of recombinant therapeuticproteins and thus greatly simplifying their purification.

Targeted expression of therapeutic proteins into the secretory pathwayIn plants and animals, the endoplasmic reticulum (ER) compartment allowsentry of proteins into the secretory pathway and ensures folding and correctassembly of newly synthesized secretory and resident proteins [124]. Mostrecombinant proteins produced so far in plants have been secreted into theintercellular space or apoplast [125,126]. This targeting is only dependent onthe presence of an N-terminal signal peptide cleaved during the co-transla-tional insertion of the nascent protein in the ER [1] (see Table 2). It has beenshown in many plant expression systems and for many PMPs that plant andhuman signal peptides are recognized with the same efficiency.

Interestingly, recombinant proteins targeted to the secretory pathway,can be secreted by the roots in the culture medium (rhizosecretion) and itwas shown that these proteins were accumulated in this medium in higheramounts than in the root tissues [120,127]. This technology, which avoidscropping, brings a great simplification to the purification process [128].Recently, multimeric proteins such as immunoglobulins have been producedin their active form by rhizosecretion in transgenic tobacco [129,130].

While soluble protein secretion in the extracellular compartment is a de-fault pathway only depending on the presence of a signal peptide, targeting toother compartments of the secretory pathway such as ER or vacuoles, needsadditional signals. Many examples illustrate that the H/KDEL-mediatedprotein retention in the ER could strongly increase the stability and con-sequently the yield of recombinant proteins as compared with secretion[32,77,87,90,131–136]. For instance, accumulation level of the pea vacuolarstorage protein, vicilin, was increased by up to 100-fold in transgenic alfalfaleaves when the ER-retention signal, KDEL, was fused to its C-terminus[137]. Likewise, fusing the ER-retention signal HDEL in C-terminal ontosporamin, a storage protein from sweet potato showing antitrypsin activity,significantly increased its accumulation level, presumably by preventing itsprogression to the vacuole [138]. Interestingly, as developed below, retentioninto the ER also prevents addition of complex N-glycans on plant-madeproteins, which are potentially immunogenic in humans.

The protein storage vacuole (PSV) is an intracellular organelle whereproteins are stored in seed cells and also in many different types of plant cells,including leaf and root cells [139,140]. Compared to vegetative vacuoles, seedPSVs exhibit slightly higher pH and lower hydrolytic activity. So that, the

127

PSV is an attractive compartment for recombinant protein accumulation[141]. For example, when expressed in the endosperm of rice seeds, humanlysozyme (a naturally secreted protein) was stored under a biologically activeform in PSVs [142–144]. Another secreted protein; human serum albumin hasbeen expressed and delivered into the PSVs of wheat endosperm where itshows a good stability [145]. In the objective of a better exploitation of PSVsas a storage compartment for therapeutic proteins, a better knowledge of thesignals and mechanisms responsible for protein targeting to these organelleswill help further investigation on the advantages and limitations of storage inPSVs [146].

Oilseeds accumulate lipids to supply the energy required for seedlingdevelopment in organelles arising from the ER: the oilbodies. Seed oilbodiesare limited by a protein-rich phospholipids monolayer. Oleosins, the majorproteins at the periphery of oilbody membrane, are anchored by their hydro-phobic domain exposing their N- and C-terminal ends to the cytoplasm.Targeting to oilbodies enables both high levels of expression and cost-effective recovery of recombinant therapeutic proteins. In this technology,therapeutic proteins are covalently targeted to oilbodies as oleosin fusions[147]. By combining this fusion with an expression targeted to the seed,researchers have established a simple expression/purification system in whichthe recombinant protein is recovered with oilbodies from other seed com-ponents by liquid–liquid phase separation. This mild process reduces thenumber of chromatography steps required to obtain a purified PMP andthereby significantly reduces their purification cost. This strategy has beenused for the production of hirudin, an anticoagulant from leach salivaryglands and antibodies in different oilseed plants [66,67,148]. Recently humaninsulin expressed Arabidopsis seed oilbodies was recovered as an activemolecule at commercially relevant levels [149].

Production of therapeutic proteins in chloroplastsPMP(s) expression in the chloroplasts also offers several advantages includ-ing very high yield. Each cell from higher plants leaves contains as many ashundred chloroplasts with up to hundred chloroplast genome, resulting atotal of about 10,000 genome copies per cell. The transgene is introducedinto leaf chloroplasts by particule bombardment and directly integrated intothe chloroplast genome by homologous recombination [60]. This stabletransformation of chloroplasts allows amplification of transgene copies andaccumulation of large amounts of recombinant proteins [150–152]. Forexample, transgenic tobacco chloroplasts produce 300-fold higher amountsof human somatotropin than their nuclear transgenic counterparts [122].Resulting from high expression levels and low proteolytic activity, a proteinexpressed in this organelle could represent up to 20% of total leaf proteins[153]. In some cases, concentration of recombinant proteins expressed inchloroplasts is so high that they could form inclusion bodies thus, simplifying

128

purification and increasing resistance to proteolysis of these foreign proteins[154]. However, the need for refolding these therapeutic proteins, aftersolubilization from inclusion bodies could significantly increase their over-all cost.

With a limited protein maturation capacity, the chloroplast looks parti-cularly well adapted for production of simple molecules [155], but quite sur-prisingly, tobacco chloroplasts are also capable to properly fold complexproteins with disulfide bridges, such as human somatotropin [122] and evenfull-size antibodies [156]. Ancestral plants like algae are also able to producefunctional antibodies in their chloroplasts [89]. However, expression inthe chloroplasts cannot be considered as a panacea for PMPs expressionin planta, as a number of clinically useful proteins necessitate extensive post-translational processing. For instance, oligosaccharides attached to poly-peptide chains by N- or O-glycosylation, in particular, have a strong impacton the activity of several therapeutic proteins and unfortunately chloroplastsdo not have the capacity to glycosylate proteins.

Recently, an alternative pathway that mediates post-translational deliveryof proteins to the chloroplast via the secretory pathway was described inA. thaliana [157]. This pathway provides new opportunities for complement-ation of the chloroplast protein maturation machinery with chaperonesneeding ER and/or Golgi typical maturations such as N-glycosylation fortheir biological activity or using chloroplasts as a storage compartment forglycoproteins [152].

Optimization of plant production platforms for lower proteolytic activity and

humanized glycosylation

In plants, like in any other heterologous expression system, recombinantprotein yield not only depends on an efficient expression rate of the trans-gene, but also on the stability of the resulting protein during the wholeexpression/recovery process [158].

Transgenic plants with reduced protease activity

Proteases found in the different compartments of plant cells may dramati-cally alter the stability of foreign proteins either in vivo, or in vitro duringtheir recovery from plant tissues [158,159]. Vacuolar proteases active inmildly-acidic conditions, in particular, were readily identified as potentiallydamaging for the integrity of recombinant proteins expressed in vegetativeorgans of transgenic plants. As described above, targeting strategies based onthe fusion of appropriate targeting signals to the therapeutic proteins havebeen used to avoid unwanted proteolysis in vivo by directing their accumu-lation in compartments such as ER [113,137,138] or chloroplasts where pro-teolytic activity is low [160].

129

Transgenic plant lines with reduced protease activity levels in vivo couldalso help to maximize protein yields by slowering cellular hydrolytic proc-esses. In particular, recent evidence in the literature suggests that hinderingendogenous protease activities in planta with recombinant protease inhibitorscould help enhance protein levels in vegetative organs without compromisinggrowth and development of the host plant. The rice cysteine proteinase in-hibitor, oryzacystatin I, for instance, was shown to increase total solubleprotein levels by 40% in leaves of transgenic tobacco lines expressing thisinhibitor in the cytosolic compartment [161]. Similarly, transgenic potatolines ectopically expressing the aspartate proteinase inhibitor, tomato ca-thepsin D inhibitor, exhibited total leaf protein levels up to 35% higher thanthose of control plants, while showing no visible sign of altered growth ordevelopment [162]. More recently, transgenic lines of potato expressing eithertomato cathepsin D inhibitor or bovine aprotinin, both active against trypsinand chymotrypsin, show a decrease in Rubisco hydrolysis by 30–40% relativeto control plants [163]. Based on current knowledge and progress to come onplant cell proteolytic processes, the design of transgenic plant lines deficientin specific protease activities in the secretory pathway could provide plantproduction platforms optimized for the production of complex proteins in‘‘mild’’ cellular environments.

Current strategies to humanize glycans N-linked to PMPs

Many therapeutic proteins are glycoproteins and glycosylation is oftenessential for their stability, solubility, folding and biological activity. Whena mammalian glycoprotein is produced in a plant expression system it isglycosylated on the same Asn residues as it would be in mammals, but itsN-glycan structures are different from that of its native counterpart.

For instance, plant-made antibodies bear both high-mannose (Man5–Man9glycans) and biantennary complex type N-glycans [72,73]. The high-mannose-type N-glycans have the same structure in plant and mammalian glyco-proteins. But complex-type N-glycans are structurally different in plants andmammals. For instance, in plants, the proximal N-acetylglucosamine of thecore is substituted by an a1,3-fucose in place of an a1,6-fucose in mammals,and the b-mannose of the core is substituted by a bisecting b1,2-xylose inplants, in place of a b1,4-N-acetylglucosamine in mammals. In addition, b1,3-galactose and fucose a1,4-linked to the terminal N-acetylglucosamineof plant N-glycans form Lewis a oligosaccharide structures instead of b1,4-galactose combined with sialic acids in mammals (Fig. 1).

Together with Lewis a, bisecting b1,2-xylose and core a1,3-fucose residuesare constitutive of three glycoepitopes described on complex plant N-glycans.Indeed, plant complex N-glycans are immunogenic in most laboratory mam-mals and elicit glycan-specific IgE-and IgG-antibodies in humans [164–167].So that, as observed for any other eukaryotic system currently used for

Fig. 1. Addition and processing of N-linked glycans in the endoplasmic reticulum (ER) and Golgi apparatus of plant andmammalian cells. A precursor oligosaccharide assembled onto a lipid carrier is transferred on specific Asn residues of the nascentgrowing polypeptide. The N-glycan is then trimmed off with removal of glucosyl and most mannosyl residues. Differences in theprocessing of plant and mammalian complex N-glycans are late Golgi maturation events.

130

131

therapeutic protein production such as yeasts, insect cells or mammalian-cultured cells, because of their structural differences with human N-glycans,glycans N-linked to PMPs would be immunogenic in humans when deliveredparenterally.

To fully exploit the potential of plants for the production of recombinanttherapeutic glycoproteins, it is necessary to control the maturation of plant-specific N-glycans and thus prevent the addition of immunogenic glyco-epitopes onto PMPs. One of the most drastic approaches is to preventN-glycosylation, by inactivating N-glycosylation sites through the mutationof Asn or Ser/Thr residues. Generally, this strategy neither influences IgGfolding and assembly in the plant ER nor the antigen-binding activity of anantibody [168]. However, many pharmaceuticals, including antibodies usedfor Fc-dependent functions require glycosylation for in vivo activity andlongevity. This is why most efforts in glycoengineering of plant expressionsystems were focused on the production of glycosylated therapeutic proteinsbearing non-immunogenic N-glycans. One of these strategies is based onthe inhibition of plant-specific Golgi glycosyltransferases to prevent theaddition of glyco-epitopes to PMPs. Knock-out a1,3 fucosyltransferase andb1,2-xylosyltransferase genes, to eliminate the plant-derived glyco-epitopeswas successful in several plant expression systems using either insertionalmutation in Arabidopsis mutants [169,170] or targeted gene inactivation inthe moss Physcomitrella patens [171]. RNA interference was also used for aknock-out of a1,3-fucosyltransferase and b1,2-xylosyltransferase in twoplant expression systems; Lemna minor and Medicago sativa [91,172]. Thevery high efficiency of this strategy has allowed the production in L. minor ofa monoclonal antibody cumulating the advantages of homogeneousglycosylation with a single and non-immunogenic N-glycan species.

In plants as in other eukaryotic cells, proteins that reside in the lumen ofthe plant ER contain high-mannose type N-glycans with structures commonto mammals. We have recently shown that antibodies expressed in tobaccoplants with a KDEL ER retention signal fused at the C-terminal ends of theirheavy and light chains contain exclusively non-immunogenic high-mannosetype N-glycans [173,174]. These different studies illustrate that several plantexpression systems are already available for production of glycosylatedPMPs without immunogenic glyco-epitopes. In addition to approachesinvolving glycosyltransferase inactivation, another attractive strategy tohumanize plant N-glycans is to express mammalian glycosyltransferases inplants, which would complete and/or compete with the endogenous machin-ery of N-glycan maturation in the plant Golgi apparatus. As part of thesecomplementation strategies, it has been shown that the human b1,4 gala-ctosyltransferase, expressed in plant cells, transfers galactose residues ontothe terminal N-acetylglucosamine residues of plant N-glycans [73, 175–177].These results are very promising and several laboratories are currentlyworking to increase the performance of heterologous glycosyltransferases

132

through better control of their targeting in the Golgi cisternae. Indeed, theanalysis of several plant glycosyltransferases is currently providing a panel ofspecific signals sufficient for a targeted expression of heterologous glyco-syltransferases within the different Golgi subcompartments of a plant cell[178,179].

The presence of sialic acid residues at the termini of N-glycan antennae isvery important for the clearance of many mammalian plasma proteins ofpharmaceutical interest. The absence of such residues on circulating proteinsresults in their rapid elimination from the blood by interactions with galac-tose-specific receptors on the surface of hepatic cells. Sialic acids are notdetectable and thus more probably absent from plant glycoproteins [180,181].The production of sialylated N-glycans is feasible in plants as previouslyshown in insect cells [182]. Indeed most of this complex biosynthetic pathwaylocated both in the Golgi lumen and in the cytosol of mammalian cells wasalready rebuilt in plants with the expression of mammalian a2,6 sialyltransf-erase [183], human CMP-N-acetylneuraminic acid synthetase, CMP-sialicacid transporter [184] and recently two catalytically active microbial N-acetylneuraminic acid synthesizing enzymes [185]. The next and last steps toget PMP sialylation in planta will be to express an epimerase able to convertendogenous D-GlcNAc or UDP-GlcNAc into D-ManNAc in order to supplythe heterologous N-acetylneuraminic acid synthesizing enzymes with theappropriate amino sugar, and to simultaneously express these different genesin a same plant expression system.

Emerging plant expression systems for molecular farming

As illustrated in Tables 1 and 2, tobacco, A. thaliana, maize, rice and alfalfawere very frequently used for therapeutic proteins production. However,some emerging plant expression systems, like Lemna minor, Physcomitrellapatens, Chlamydomonas reinhardtii or higher plant cell suspension culturesare offering new opportunities for molecular farming. Indeed these expres-sion systems have in common to be much more consistent with publicdemand for high containment of genetically modified plants and also morecompliant with regulatory issues for the production of therapeutic proteinssince they are grown in a completely controlled environment.

Lemna

Lemna gibba and Lemna minor, commonly named duckweeds, are free float-ing plants, which develop on water and are found all over the world. Withtheir naturally simple growth conditions, duckweeds are well adapted forintensive culturing methods. Duckweed allows very high rates of biomassaccumulation per unit of time – it can double in size every 24–48 h by aprocess. Recombinant proteins produced in duckweeds after Agrobacterium

133

tumefaciens-mediated or by biolistic transformation can be extracted andpurified or the plant containing the protein can be used directly, dry or fresh.As for other plant expression systems, secretion into the extracellular mediais dependent on the presence of a signal peptide. Lemna-recognizing plantand human signal sequences with the same efficiency [186].

The high capacity of this expression system for production of therapeuticproteins was recently illustrated by Cox et al., with the production of ahuman monoclonal antibody in a glycoengineered lemna. This antibody ex-hibited a single major N-glycan species without any detectable plant-specificN-glycans and shows a higher antibody-dependent cell-mediated cytotoxicityand effector cell receptor-binding activities than its homologs expressed incultured Chinese hamster ovary (CHO) cells [91].

Moss

Mosses are higher multicellular eukaryotes and therefore perform extensivepost-translational processing of proteins including disulfide bridge formationand glycosylation. Transgenic Physcomitrella patens are generated via thepolyethyleneglycol-mediated transfection of protoplasts. Generation of sta-ble transgenic plants take about eight weeks after transformation [187,188]and cultivation of this moss in glass bioreactors is well established. Asillustrated in Table 1, a therapeutic protein (grow factor VEGF) has beenalready produced in this expression system [53].

P. patens is unique among all multicellular plants analyzed to date inexhibiting a very effective homologous recombination process in its nuclearDNA. This allows targeted knock-outs and knock-in of genes, a highlyattractive tool for production of strains designed for PMP production[187,189].

N-glycosylation in P. patens is very similar as in higher plants [190]. Butthis moss is currently the most advanced plant expression system for glyco-engineering due to the ease with which knock-out and knock-in of glyco-sylation enzyme genes can be performed by homologous recombination inthis system [171]. Thus, P. patens has been engineered to produce a strain thatdoes not add b1,2 xylose or a1,3 fucose, but produces PMPs bearing a coreheptasaccharide identical to that of a human IgG [171].

Algae

Algae are currently emerging as alternative system for production of recom-binant therapeutic proteins. Unicellular eukaryotic green algae, such asChlamydomonas reinhardtii, Phaeodactylum tricornutum, Tetraselmis suecica,Odontella aurita, can produce a significant amount of recombinant proteins[191]. Freshwater algae C. reinhardtii is the best-studied for recombinantprotein production via chloroplast transformation [192]. C. reinhardtii

134

contains a single large chloroplast that occupies approximately 40% of thecell volume and its transformation was first realized in 1988. Unlike nucleartransformation, plastid transformation occurs via homologous recombina-tion. Hence, integration events can be targeted precisely to any region inthe chloroplast genome that contains a so-called silent site for transgeneintegration [191]. The chloroplast contains its own genome, which is a cir-cular molecule of approximately 200Kb, and each chloroplast containsapproximately 80 identical copies of the genome. As a consequence, stabletransformation of the chloroplast requires that all 80 copies convert to therecombinant form [193].

C. reinhardtii can be grown in a cost-effective manner at a large scale, in500,000-l containers. Compared to land plants, it grows at a much faster rate,doubling its cell number every 8 h [191]. Purification of recombinant proteinsshould be simpler in algae than in terrestrial plants. Indeed, the cellularpopulation of algae is uniform in size and type, hence there is no gradient ofrecombinant protein distribution, which simplifies purification and reducesthe loss of biomass. C. reinhardtii has also the ability to produce secretedproteins, a pathway which could still cut down the production costs [193]. Ahuman mAb produced in transgenic algae was correctly assembled and hasthe same capacity to bind herpes virus proteins as its mammalian homolog[89]. But chloroplast-encoded proteins are not glycosylated and this mAb hasshown no evidence for glycosylation required for the Fc-dependent functions.In addition, codon bias in algae constitutes an additional difficulty for for-eign protein expression in this system due to the need of a extensive opti-mization of gene sequences.

Higher plant suspension-cultured cells

Higher plant cell cultures offer many advantages over field grown plants oreven plants grown in greenhouses for PMP production. Among these ad-vantages, plant cells are grown in highly contained and sterile in vitro con-ditions. Some plant cells grow very fast, for instance BY2 tobacco cellsnumber is doubling every 12 h in optimal growth conditions, thus rapidlyproviding an important biomass. Many therapeutic proteins have alreadybeen successfully expressed in suspension-cultured plant cells. The potentialof plant cells for biopharmaceutical production was recently illustrated withthe use of suspension-cultured tobacco cells to synthesize correctly maturedand highly immunoreactive recombinant house dust mite allergens that couldbe used for allergy diagnostic and immunotherapy [194]. This work perfectlyexemplifies the high potential of plant suspension-cultured cells as bio-reactors for the production of therapeutic proteins under controlledand environmentally safe conditions. In addition this production systemallows for an efficient secretion of PMPs into an inorganic culture mediumoffering substantial cost advantages in downstream purification. This could

135

counterbalance an increased production cost due to the use of fermentor forproduction instead of field- or green-house production with whole plants.

Another advantage for down-stream processing is that plant cells are uni-form in size and types, which leads to a low PMP heterogeneity as comparedto production in whole plants. For instance, it was reported that glyco-sylation patterns of an antibody expressed in tobacco plants, differ fromyoung to old leaves [195]. In contrast, glycan patterns are reproducible frombatch to batch in BY2 tobacco cell cultures and interestingly complement-ation of the culture medium could strongly reduce N-glycan heterogeneity(Faye et al., unpublished results).

Conclusion

Plants offer a safe and extremely cost-effective alternative to microbial ormammalian expression systems for the production of biopharmaceuticals.Current strategies to improve plant expression systems will rapidly result inincreased yield and simplification of down-stream processing of plant-madetherapeutic proteins. These promising results, together with the fastprogresses in glycan humanization and reduced heterogeneity of PMPs arecurrently moving plants among the major expression systems, particularlywhen large quantities of multimeric recombinant proteins are required.

References

1. Gomord V, Sourrouille C, Fitchette A-C, Bardor M, Pagnt S, Lerouge P and Faye L.

Production and glycosylation of plant-made pharmaceuticals: the antibodies as a

challenge. Plant Biotechnol J 2004;2:83–100.

2. Walsh G and Jefferis R. Post-translational modifications in the context of therapeutic

proteins. Nat Biotechnol 2006;24:1241–1252.

3. Twyman RM, Stoger E, Schillberg S, Christou P and Fischer R. Molecular farming in

plants: host systems and expression technology. Trends Biotechnol 2003;21:570–578.

4. Gomord V and Faye L. Posttranslational modification of therapeutic proteins in plants.

Curr Opin Plant Biol 2004;7:171–181.

5. Hiatt A, Cafferkey R and Bowdish K. Production of antibodies in transgenic plants.

Nature 1989;342:76–78.

6. Ma JK, Hiatt A, Hein M, Vine ND, Wang F, Stabila P, van Dolleweerd C, Mostov K

and Lehner T. Generation and assembly of secretory antibodies in plants. Science

1995;268:716–719.

7. Khoudi H, Laberge S, Ferullo JM, Bazin R, Darveau A, Castonguay Y, Allard G,

Lemieux R and Vezina LP. Production of a diagnostic monoclonal antibody in

perennial alfalfa plants. Biotechnol Bioeng 1999;64:135–143.

8. Austin S, Bingham ET, Koegel RG, Mathews DE, Shahan MN, Straub RJ and Burgess

RR. An overview of a feasibility study for the production of industrial enzymes in

transgenic alfalfa. Ann NY Acad Sci 1994;721:234–244.

9. Gomord V, Chamberlain P, Jefferis R and Faye L. Biopharmaceutical production in

plants: problems, solutions and opportunities. Trends Biotechnol 2005;23:559–565.

136

10. Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJ and

Hoekema A. Production of correctly processed human serum albumin in transgenic

plants. Biotechnology (NY) 1990;8:217–221.

11. Goddjin OJM and Pen J. Plants as bioreactors. Trends Biotechnol 1995;13:379–387.

12. Farran I, Sanchez-Serrano JJ, Medina JF, Prieto J and Mingo-Castel AM. Targeted

expression of human serum albumin to potato tubers. Transgenic Res 2002;11:337–346.

13. Zhong GY, Peterson DJ, Delaney D, Bailey M, Witcher DR, Register JC, Bond D, Li

CP, Marshall L, Kulisek E, Ritland D, Meyer T, Hood EE and Howard J. Commercial

production of aprotinin in transgenic maize seeds. Mol Breeding 1999;5(4):345–356.

14. Ruggiero F, Exposito JY, Bournat P, Gruber V, Perret S, Comte J, Olagnier B, Garr-

one R and Theisen M. Triple helix assembly and processing of human collagen pro-

duced in transgenic tobacco plants. FEBS Lett 2000;469:132–136.

15. Dieryck W, Pagnier J, Poyart C, Marden MC, Gruber V, Bournat P, Baudino S and

Merot B. Human haemoglobin from transgenic tobacco. Nature 1997;386:29–30.

16. Halling-Sorensen B, Nors Nielsen S, Lanzky PF, Ingerslev F, Holten Lutzhoft HC

and Jorgensen SE. Occurrence, fate and effects of pharmaceutical substances in the

environment – a review. Chemosphere 1998;36:357–393.

17. Dieryck W, Gruber V, Baudino S, Lenee P, Pagnier J, Merot B and Poyart C. Expres-

sion of recombinant human hemoglobin in plants. Transfus Clin Biol 1995;2:441–447.

18. Terashima M, Murai Y, Kawamura M, Nakanishi S, Stoltz T, Chen L, Drohan W,

Rodriguez RL and Katoh S. Production of functional human alpha 1-antitrypsin by

plant cell culture. Appl Microbiol Biotechnol 1999;52:516–523.

19. McDonald KA, Hong LM, Trombly DM, Xie Q and Jackman AP. Production of

human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor.

Biotechnol Prog 2005;21:728–734.

20. Krebitz M, Wiedermann U, Essl D, Steinkellner H, Wagner B, Turpen TH, Ebner C,

Scheiner O and Breiteneder H. Rapid production of the major birch pollen allergen

Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo

characterization. Faseb J 2000;14:1279–1288.

21. Arakawa T, Yu J and Langridge WH. Food plant-delivered cholera toxin B subunit for

vaccination and immunotolerization. Adv Exp Med Biol 1999;464:161–178.

22. Tackaberry ES, Dudani AK, Prior F, Tocchi M, Sardana R, Altosaar I and Ganz PR.

Development of biopharmaceuticals in plant expression systems: cloning, expression

and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in

seeds of transgenic tobacco. Vaccine 1999;17:3020–3029.

23. Arakawa T, Chong DK, Merritt JL and Langridge WH. Expression of cholera toxin B

subunit oligomers in transgenic potato plants. Transgenic Res 1997;6:403–413.

24. Brennan FR, Bellaby T, Helliwell SM, Jones TD, Kamstrup S, Dalsgaard K, Flock JI

and Hamilton WD. Chimeric plant virus particles administered nasally or orally induce

systemic and mucosal immune responses in mice. J Virol 1999;73:930–938.

25. Wigdorovitz A, Perez Filgueira DM, Robertson N, Carrillo C, Sadir AM, Morris TJ

and Borca MV. Protection of mice against challenge with foot and mouth disease virus

(FMDV) by immunization with foliar extracts from plants infected with recombinant

tobacco mosaic virus expressing the FMDV structural protein VP1. Virology

1999;264:85–91.

26. Tacket CO and Mason HS. A review of oral vaccination with transgenic vegetables.

Microbes Infect 1999;1:777–783.

27. Dus Santos MJ, Wigdorovitz A, Trono K, Rios RD, Franzone PM, Gil F, Moreno J,

Carrillo C, Escribano JM and Borca MV. A novel methodology to develop a foot and

137

mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine

2002;20:1141–1147.

28. Wu H, Singh NK, Locy RD, Scissum-Gunn K and Giambrone JJ. Expression of

immunogenic VP2 protein of infectious bursal disease virus in Arabidopsis thaliana.

Biotechnol Lett 2004;26:787–792.

29. Wigdorovitz A, Mozgovoj M, Santos MJ, Parreno V, Gomez C, Perez-Filgueira DM,

Trono KG, Rios RD, Franzone PM, Fernandez F, Carrillo C, Babiuk LA, Escribano

JM and Borca MV. Protective lactogenic immunity conferred by an edible peptide

vaccine to bovine rotavirus produced in transgenic plants. J Gen Virol

2004;85:1825–1832.

30. Beachy RN, Fitchen JH and Hein MB. Use of plant viruses for delivery of vaccine

epitopes. Ann NY Acad Sci 1996;792:43–49.

31. Huang Z, Dry I, Webster D, Strugnell R and Wesselingh S. Plant-derived measles virus

hemagglutinin protein induces neutralizing antibodies in mice. Vaccine 2001;19:

2163–2171.

32. Sojikul P, Buehner N and Mason HS. A plant signal peptide-hepatitis B surface antigen

fusion protein with enhanced stability and immunogenicity expressed in plant cells.

Proc Natl Acad Sci USA 2003;100:2209–2214.

33. Ma SW, Zhao DL, Yin ZQ, Mukherjee R, Singh B, Qin HY, Stiller CR and Jevnikar

AM. Transgenic plants expressing autoantigens fed to mice to induce oral immune

tolerance. Nat Med 1997;3:793–796.

34. Richter LJ, Thanavala Y, Arntzen CJ and Mason HS. Production of hepatitis B

surface antigen in transgenic plants for oral immunization. Nat Biotechnol 2000;18:

1167–1171.

35. Kong Q, Richter L, Yang YF, Arntzen CJ, Mason HS and Thanavala Y. Oral

immunization with hepatitis B surface antigen expressed in transgenic plants. Proc Natl

Acad Sci USA 2001;98:11539–11544.

36. Buratti E, McLain L, Tisminetzky S, Cleveland S, Dimmock N and Baralle F. The

neutralizing antibody response against a conserved region of human immunodeficiency

virus type 1 gp41 (amino acid residues 731–752) is uniquely directed against a con-

formational epitope. Virology 1999;13:930–938.

37. Mason HS, Haq TA, Clements JD and Arntzen CJ. Edible vaccine protects mice

against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic

LT-B gene. Vaccine 1998;16:1336–1343.

38. Arakawa T, Yu J, Chong DK, Hough J, Engen PC and Langridge WH. A plant-based

cholera toxin B subunit-insulin fusion protein protects against the development of

autoimmune diabetes. Nat Biotechnol 1998;16:934–938.

39. Turpen T, Reinl S, Charoenvit Y, Hoffman S, Fallarme V and Grill L. Malarial

epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology

(NY) 1995;13.

40. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM and Arntzen CJ. Human

immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. J

Infect Dis 2000;182:302–305.

41. Huang Z, Elkin G, Maloney BJ, Beuhner N, Arntzen CJ, Thanavala Y and Mason HS.

Virus-like particle expression and assembly in plants: hepatitis B and Norwalk viruses.

Vaccine 2005;23:1851–1858.

42. McGarvey PB, Hammond J, Dienelt MM, Hooper DC, Fu ZF, Dietzschold B,

Koprowski H and Michaels FH. Expression of the rabies virus glycoprotein in

transgenic tomatoes. Biotechnology (NY) 1995;13:1484–1487.

138

43. James EA, Wang C, Wang Z, Reeves R, Shin JH, Magnuson NS and Lee JM. Pro-

duction and characterization of biologically active human GM-CSF secreted by geneti-

cally modified plant cells. Protein Expr Purif 2000;19:131–138.

44. Lee JS, Choi SJ, Kang HS, Oh WG, Cho KH, Kwon TH, Kim DH, Jang YS and

Yang MS. Establishment of a transgenic tobacco cell suspension culture system for

producing murine granulocyte-macrophage colony stimulating factor. Mol Cells 1997;

7:783–787.

45. Robinson A. Harvesting blood proteins from grain. Can Med Assoc J 1995;153:

427–429.

46. Wang ML, Goldstein C, Su W, Moore PH and Albert HH. Production of biologically

active GM-CSF in sugarcane: a secure biofactory. Transgenic Res 2005;14:167–178.

47. Edelbaum O, Stein D, Holland N, Gafni Y, Livneh O, Novick D, Rubinstein M and

Sela I. Expression of active human interferon-beta in transgenic plants. J Interferon Res

1992;12:449–453.

48. De Zoeten GA, Penswick JR, Horisberger MA, Ahl P, Schultze M and Hohn T. The

expression, localization, and effect of a human interferon in plants. Virology

1989;172:213–222.

49. Chen TL, Lin YL, Lee YL, Yang NS and Chan MT. Expression of bioactive human

interferon-gamma in transgenic rice cell suspension cultures. Transgenic Res 2004;

13:499–510.

50. Cramer CL, Boothe JG and Oishi KK. Transgenic plants for therapeutic proteins:

linking upstream and downstream strategies. Curr Top Microbiol Immunol

1999;240:95–118.

51. Matsumoto S, Ikura K, Ueda M and Sasaki R. Characterization of a human glyco-

protein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol 1995;

27:1163–1172.

52. Baur A, Reski R and Gorr G. Enhanced recovery of a secreted recombinant human

growth factor using stabilizing additives and by co-expression of human serum albumin

in the moss Physcomitrella patens. Plant Biotechnol J 2005;3:331–340.

53. Park Y and Cheong H. Expression and production of recombinant human interleukin-2

in potato plants. Protein Expr Purif 2002;25:160–165.

54. Kwon S, Yang Y, Hong C and Pyun K. Expression of active human interleukin-6 in

transgenic tabacco. Mol Cells 1995;5:486–492.

55. Menassa R, Kennette W, Nguyen V, Rymerson R, Jevnikar A and Brandle J. Sub-

cellular targeting of human interleukin-10 in plants. J Biotechnol 2004;108:179–183.

56. Gutierrez-Ortega A, Avila-Moreno F, Saucedo-Arias LJ, Sanchez-Torres C and

Gomez-Lim MA. Expression of a single-chain human interleukin-12 gene in transgenic

tobacco plants and functional studies. Biotechnol Bioeng 2004;85:734–740.

57. Panahi M, Alli Z, Cheng X, Belbaraka L, Belgoudi J, Sardana R, Phipps J and Altosaar

I. Recombinant protein expression plasmids optimized for industrial E. coli fermen-

tation and plant systems produce biologically active human insulin-like growth factor-1

in transgenic rice and tobacco plants. Transgenic Res 2004;13:245–259.

58. Hamamoto H, Sugiyama Y, Nakagawa N, Hashida E, Matsunaga Y, Takemoto S,

Watanabe Y and Okada Y. A new tobacco mosaic virus vector and its use for the

systemic production of angiotensin-I-converting enzyme inhibitor in transgenic tobacco

and tomato. Biotechnology (NY) 1993;11:930–932.

59. Cramer CL, Weissenborn DL, Oishi KK, Grabau EA, Bennett S, Ponce E, Grabowski

GA and Radin DN. Bioproduction of human enzymes in transgenic tobacco. Ann NY

Acad Sci 1996;792:62–71.

139

60. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V,

Schlittler M, Carroll JA, Spatola L, Ward D, Ye G and Russell DA. High-yield pro-

duction of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol

2000;18:333–338.

61. Reggi S, Marchetti S, Patti T, De Amicis F, Cariati R, Bembi B and Fogher C.

Recombinant human acid beta-glucosidase stored in tobacco seed is stable, active and

taken up by human fibroblasts. Plant Mol Biol 2005;57:101–113.

62. Kumagai MH, Turpen TH, Weinzettl N, della-Cioppa G, Turpen AM, Donson J, Hilf

ME, Grantham GL, Dawson WO, Chow TP, Piatak M and Grill LK. Rapid, high-level

expression of biologically active alpha-trichosanthin in transfected plants by an RNA

viral vector. Proc Natl Acad Sci USA 1993;90:427–430.

63. Mor TS, Sternfeld M, Soreq H, Arntzen CJ and Mason HS. Expression of recombinant

human acetylcholinesterase in transgenic tomato plants. Biotechnol Bioeng 2001;75:

259–266.

64. Gruber V, Berna P, Arnaud T, Bournat P, Clement C, Mison D, Olagnier B,

Philippe L, Theisen M, Baudino S, Benicourt C, Cudrey C, Bloes C, Duchateau N,

Dufour S, Gueguen C, Jacquet S, Ollivo C, Poncetta C, Zorn N, Ludevid D,

Van Dorsselaer A, Verger R, Doherty A, Merot B and Danzin C. Large-scale

production of a therapeutic protein in transgenic tabacco plants: effect of subcellular

targeting on quality of a recombinant dog gastric lipase. Mol Breeding 2001;7(4):

329–340.

65. Sorrentino A, Schillberg S, Fischer R, Rao R, Porta R and Mariniello L. Recombinant

human tissue transglutaminase produced into tobacco suspension cell cultures is active

and recognizes autoantibodies in the serum of coeliac patients. Int J Biochem Cell Biol

2005;37:842–851.

66. Parmenter DL, Boothe JG, van Rooijen GJ, Yeung EC and Moloney MM. Production

of biologically active hirudin in plant seeds using oleosin partitioning. Plant Mol Biol

1995;29:1167–1180.

67. Boothe JG, Saponja JA and Parmenter DL. Molecular farming in plants: oilseed

as vehicles for the production of pharmaceutical proteins. Drug Dev Res 1997;42:

172–178.

68. Hong SH, Kim KI, Chung HY, Kim YJ, Sunter G, Bisaro DM and Chung IS.

Expression of recombinant endostatin in Agrobacterium-inoculated leaf disks of

Nicotiana tabacum var. Xanthi Biotechnol Lett 2004;26:1433–1439.

69. Salmon V, Legrand D, Slomianny MC, el Yazidi I, Spik G, Gruber V, Bournat P,

Olagnier B, Mison D, Theisen M and Merot B. Production of human lactoferrin in

transgenic tobacco plants. Protein Expr Purif 1998;13:127–135.

70. Nandi S, Yalda D, Lu S, Nikolov Z, Misaki R, Fujiyama K and Huang N. Process

development and economic evaluation of recombinant human lactoferrin expressed in

rice grain. Transgenic Res 2005;14:237–249.

71. Li Y, Geng Y, Song H, Zheng G, Huan L and Qiu B. Expression of a human lactoferrin

N-lobe in Nicotiana benthmiana with potato virus X-based agroinfection. Biotechnol

Lett 2004;26:953–957.

72. Cabanes-Macheteau M, Fitchette-Laine AC, Loutelier-Bourhis C, Lange C, Vine ND,

Ma JK, Lerouge P and Faye L. N-Glycosylation of a mouse IgG expressed in

transgenic tobacco plants. Glycobiology 1999;9:365–372.

73. Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, Jordi W,

Lommen A, Faye L, Lerouge P and Bosch D. Galactose-extended glycans of antibodies

produced by transgenic plants. Proc Natl Acad Sci USA 2001;98:2899–2904.

140

74. Vaquero C, Sack M, Chandler J, Drossard J, Schuster F, Monecke M, Schillberg S and

Fischer R. Transient expression of a tumor-specific single-chain fragment and a chi-

meric antibody in tobacco leaves. Proc Natl Acad Sci USA 1999;96:11128–11133.

75. Vaquero C, Sack M, Schuster F, Finnern R, Drossard J, Schumann D, Reimann A and

Fischer R. A carcinoembryonic antigen-specific diabody produced in tobacco. Faseb J

2002;16:408–410.

76. Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J, Williams S, Keen D,

Perrin Y, Christou P and Fischer R. Cereal crops as viable production and storage

systems for pharmaceutical scFv antibodies. Plant Mol Biol 2000;42:583–590.

77. Ko K, Tekoah Y, Rudd PM, Harvey DJ, Dwek RA, Spitsin S, Hanlon CA, Rupprecht

C, Dietzschold B, Golovkin M and Koprowski H. Function and glycosylation of plant-

derived antiviral monoclonal antibody. Proc Natl Acad Sci USA 2003;100:8013–8018.

78. Ma JK, Lehner T, Stabila P, Fux CI and Hiatt A. Assembly of monoclonal antibodies

with IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol

1994;24:131–138.

79. Larrick JW, Yu L, Naftzger C, Jaiswal S and Wycoff K. Production of secretory IgA

antibodies in plants. Biomol Eng 2001;18:87–94.

80. Verch T, Yusibov V and Koprowski H. Expression and assembly of a full-length

monoclonal antibody in plants using a plant virus vector. J Immunol Methods

1998;1:69–75.

81. Briggs K, Zeitlin L, Wang F, Chen L, Fitchen J, Glynn J, Lee VD, Zhang S and Whaley

K. Anti-HSV antibodies produced in rice plants protect mice from vaginal HSV in-

fection. Plant Biol 2000(Stuttg.), Abstract 15.

82. Zeitlin L, Olmsted SS, Moench TR, Co MS, Martinell BJ, Paradkar VM, Russell DR,

Queen C, Cone RA and Whaley KJ. A humanized monoclonal antibody produced in

transgenic plants for immunoprotection of the vagina against genital herpes. Nat

Biotechnol 1998;16:1361–1364.

83. Yuan Q, Hu W, Pestka JJ, He SY and Hart LP. Expression of a functional anti-

zearalenone single-chain Fv antibody in transgenic Arabidopsis plants. Appl Environ

Microbiol 2000;66:3499–3505.

84. De Wilde C, De Neve M, De Rycke R, Bruyns A-M, De Jaeger G, Van Montagu M,

Depicker A and Engler G. Intact antigen-binding MAK33 antibody and Fab fragment

accumulate in intercellular spaces of Arabidopsis thaliana. Plant Sci 1996;114:233–241.

85. De Neve M, De Loose M, Jacobs A, Van Houdt H, Kaluza B, Weidle U, Van Montagu

M and Depicker A. Assembly of an antibody and its derived antibody fragment in

Nicotiana and Arabidopsis. Transgenic Res 1993;2:227–237.

86. Bruyns AM, De Jaeger G, De Neve M, De Wilde C, Van Montagu M and Depicker A.

Bacterial and plant-produced scFv proteins have similar antigen-binding properties.

FEBS Lett 1996;386:5–10.

87. Peeters K, De Wilde C and Depicker A. Highly efficient targeting and accumulation of

a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana.

Eur J Biochem 2001;268:4251–4260.

88. McCormick AA, Kumagai MH, Hanley K, Turpen TH, Hakim I, Grill LK, Tuse D,

Levy S and Levy R. Rapid production of specific vaccines for lymphoma by expression

of the tumor-derived single-chain Fv epitopes in tobacco plants. Proc Natl Acad Sci

USA 1999;96:703–708.

89. Mayfield SP, Franklin SE and Lerner RA. Expression and assembly of a fully active

antibody in algae. Proc Natl Acad Sci USA 2003;100:438–442.

141

90. Ramirez N, Ayala M, Lorenzo D, Palenzuela D, Herrera L, Doreste V, Perez M,

Gavilond JV and Oramas P. Expression of a single-chain Fv antibody fragment specific

for the hepatitis B surface antigen in transgenic tobacco plants. Transgenic Res

2002;11:61–64.

91. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A,

Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM and Dickey

LF. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna

minor. Nat Biotechnol 2006;24:1591–1597.

92. Bouquin T, Thomsen M, Nielsen LK, Green TH, Mundy J and Hanefeld Dziegiel M.

Human anti-rhesus D IgG1 antibody produced in transgenic plants. Transgenic Res

2002;11:115–122.

93. Sharp PM and Li WH. The codon Adaptation Index – a measure of directional synony-

mous codon usage bias, and its potential applications. Nucleic Acids Res 1987;15:

1281–1295.

94. Gustafsson C, Govindarajan S and Minshull J. Codon bias and heterologous protein

expression. Trends Biotechnol 2004;22:346–353.

95. Kurland C and Gallant J. Errors of heterologous protein expression. Curr Opin

Biotechnol 1996;7:489–493.

96. Perlak FJ, Fuchs RL, Dean DA, McPherson SL and Fischhoff DA. Modification of the

coding sequence enhances plant expression of insect control protein genes. Proc Natl

Acad Sci USA 1991;88:3324–3328.

97. Jensen LG, Olsen O, Kops O, Wolf N, Thomsen KK and von Wettstein D. Transgenic

barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during

germination. Proc Natl Acad Sci USA 1996;93:3487–3491.

98. Batard Y, Hehn A, Nedelkina S, Schalk M, Pallett K, Schaller H and Werck-Reichhart

D. Increasing expression of P450 and P450-reductase proteins from monocots in hetero-

logous systems. Arch Biochem Biophys 2000;379:161–169.

99. Hamada A, Yamaguchi K-I, Ohnishi N, Harada M, Nikumaru S and Honda H. High-

level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants

by a combination of signal sequence and codon modification of the phytase gene. Plant

Biotechnol J 2005;3:43–55.

100. Hamilton AJ and Baulcombe DC. A species of small antisense RNA in posttranscrip-

tional gene silencing in plants. Science 1999;286:950–952.

101. Aravin A and Tuschl T. Identification and characterization of small RNAs involved in

RNA silencing. FEBS Lett 2005;579:5830–5840.

102. Voinnet O. Induction and suppression of RNA silencing: insights from viral infections.

Nat Rev Genet 2005;6:206–220.

103. Voinnet O, Rivas S, Mestre P and Baulcombe D. An enhanced transient expression

system in plants based on suppression of gene silencing by the p19 protein of tomato

bushy stunt virus. Plant J 2003;33:949–956.

104. Scholthof HB, Scholthof KB and Jackson AO. Identification of tomato bushy stunt

virus host-specific symptom determinants by expression of individual genes from a

potato virus X vector. Plant Cell 1995;7:1157–1172.

105. Voinnet O, Pinto YM and Baulcombe DC. Suppression of gene silencing: a general

strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci USA

1999;96:14147–14152.

106. Goldstein DA and Thomas JA. Biopharmaceuticals derived from genetically modified

plants. Q J Med 2004;97:705–716.

142

107. Hellwig S, Drossard J, Twyman RM and Fischer R. Plant cell cultures for the pro-

duction of recombinant proteins. Nat Biotechnol 2004;22:1415–1422.

108. Masumura T, Morita S, Miki Y, Kurita A, Morita S, Shirono H, Koga J and Tanaka

K. Production of biologically active human interferon-a in transgenic rice. Plant

Biotechnol 2006;23:91–97.

109. Shirono H, Morita S, Miki Y, Kurita A, Morita S, Koga J, Tanaka K and Masumura

T. Highly efficient production of human interferon-a by transgenic cultured rice cells.

Plant Biotechnol 2006;23:283–289.

110. Raskin I, Ribnicky DM, Komarnytsky S, Ilic N, Poulev A, Borisjuk N, Brinker A,

Moreno DA, Ripoll C, Yakoby N, O’Neal JM, Cornwell T, Pastor I and Fridlender B.

Plants and human health in the twenty-first century. Trends Biotechnol 2002;20:

522–531.

111. Mason HS, Lam DM and Arntzen CJ. Expression of hepatitis B surface antigen in

transgenic plants. Proc Natl Acad Sci USA 1992;89:11745–11749.

112. Frigerio L, Vine ND, Pedrazzini E, Hein MB, Wang F, Ma JK and Vitale A. Assembly,

secretion, and vacuolar delivery of a hybrid immunoglobulin in plants. Plant Physiol

2000;123:1483–1494.

113. Schouten A, Roosien J, van Engelen FA, de Jong GA, Borst-Vrenssen AW,

Zilverentant JF, Bosch D, Stiekema WJ, Gommers FJ, Schots A and Bakker J. The

C-terminal KDEL sequence increases the expression level of a single-chain antibody

designed to be targeted to both the cytosol and the secretory pathway in transgenic

tobacco. Plant Mol Biol 1996;30:781–793.

114. Torres E, Vaquero C, Nicholson L, Sack M, Stoger E, Drossard J, Christou P, Fischer

R and Perrin Y. Rice cell culture as an alternative production system for functional

diagnostic and therapeutic antibodies. Transgenic Res 1999;8:441–449.

115. Fiedler U, Phillips J, Artsaenko O and Conrad U. Optimization of scFv antibody

production in transgenic plants. Immunotechnology 1997;3:205–216.

116. Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, Flynn P,

Register J, Marshall L, Bond D, Kulisek E, Kusnadi AR, Evangelista R, Nikolov ZL,

Wooge C, Mehigh RJ, Hernan R, Kappel WK, Ritland D, Li CP and Howard JA.

Commercial production of avidin from transgenic maize: characterization of trans-

formant, production, processing, extraction and purification. Mol Breeding 1997;

3:291–306.

117. Artsaenko O, Kettig B, Fiedler U, Conrad U and During K. Potato tubers as a bio-

factory for recombinant antibodies. Mol Breeding 1998;4:313–319.

118. Baum TJ, Hiatt A, Parrott WA, Pratt LH and Hussey RS. Expression in tobacco of a

functional monoclonal antibody specific to stylet secretions of the root-knot nematode.

Mol Plant-Microbe Interact 1996;9:382–387.

119. Sandhu JS, Krasnyanski SF, Domier LL, Korban SS, Osadjan MD and Buetow DE.

Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial

virus-F protein induces a systemic immune response. Transgenic Res 2000;9:127–135.

120. Borisjuk NV, Borisjuk LG, Logendra S, Petersen F, Gleba Y and Raskin I. Production

of recombinant proteins in plant root exudates. Nat Biotechnol 1999;17:466–469.

121. Komarnytsky S, Borisjuk NV, Borisjuk LG, Alam MZ and Raskin I. Production of

recombinant proteins in tobacco guttation fluid. Plant Physiol 2000;124:927–934.

122. Staub JM, Garcia B, Graves J, Hajdukiewicz PT, Hunter P, Nehra N, Paradkar V,

Schlittler M, Carroll JA, Spatola L, Ward D, Ye G and Russell DA. High-yield pro-

duction of a human therapeutic protein in tobacco chloroplasts. Nat Biotechnol

2000;18:333–338.

143

123. Stoger E, Sack M, Fischer R and Christou P. Plantibodies: applications, advantages

and bottlenecks. Curr Opin Biotechnol 2002;13:161–166.

124. Gomord V, Wee E and Faye L. Protein retention and localization in the endoplasmic

reticulum and the golgi apparatus. Biochimie 1999;81:607–618.

125. Goldstein DA and Thomas JA. Biopharmaceuticals derived from genetically modified

plants. Qjm 2004;97:705–716.

126. Streatfield SJ. Approaches to achieve high-level heterologous protein production in

plants. Plant Biotechnol J 2006;5:2–15.

127. Gaume A, Komarnytsky S, Borisjuk N and Raskin I. Rhizosecretion of recombinant

proteins from plant hairy roots. Plant Cell Rep 2003;21:1188–1193.

128. Ma JK, Drake PM and Christou P. The production of recombinant pharmaceutical

proteins in plants. Nat Rev Genet 2003;4:794–805.

129. Komarnytsky S, Borisjuk N, Yakoby N, Garvey A and Raskin I. Cosecretion of protease

inhibitor stabilizes antibodies produced by plant roots. Plant Physiol 2006;141:1185–1193.

130. Drake PM, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P and Ma JK.

Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco

roots. Plant Mol Biol 2003;52:233–241.

131. Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D and Higgins TJ. Vicilin with

carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to

high levels in the leaves of transgenic plants. Plant J 1992;2:181–192.

132. Tabe LM, Wardley-Richardson T, Ceriotti A, Aryan A, McNabb W, Moore A and

Higgins TJ. A biotechnological approach to improving the nutritive value of alfalfa. J

Anim Sci 1995;73:2752–2759.

133. Pueyo JJ, Chrispeels MJ and Herman EM. Degradation of transport-competent de-

stabilized phaseolin with a signal for retention in the endoplasmic reticulum occurs in

the vacuole. Planta 1995;196:586–596.

134. Conrad U and Fiedler U. Compartment-specific accumulation of recombinant

immunoglobulins in plant cells: an essential tool for antibody production and immuno-

modulation of physiological functions and pathogen activity. Plant Mol Biol 1998;

38:101–109.

135. Pagny S, Denmat-Ouisse LA, Gomord V and Faye L. Fusion with HDEL protects cell

wall invertase from early degradation when N-glycosylation is inhibited. Plant Cell

Physiol 2003;44:173–182.

136. Triguero A, Cabrera G, Cremata JA, Yuen CT, Wheeler J and Ramirez NI. Plant-

derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-re-

tention signal is N-glycosylated homogeneously throughout the plant with mostly high-

mannose-type N-glycans. Plant Biotechnol J 2005;3(4):449–457.

137. Wandelt CI, Khan MR, Craig S, Schroeder HE, Spencer D and Higgins TJ. Vicilin with

carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to

high levels in the leaves of transgenic plants. Plant J 1992;2:181–192.

138. Gomord V, Denmat LA, Fitchette-Laine AC, Satiat-Jeunemaitre B, Hawes C and Faye

L. The C-terminal HDEL sequence is sufficient for retention of secretory proteins in the

endoplasmic reticulum (ER) but promotes vacuolar targeting of proteins that escape the

ER. Plant J 1997;11:313–325.

139. Park M, Kim SJ, Vitale A and Hwang I. Identification of the protein storage vacuole

and protein targeting to the vacuole in leaf cells of three plant species. Plant Physiol

2004;134:625–639.

140. Park M, Lee D, Lee GJ and Hwang I. AtRMR1 functions as a cargo receptor for

protein trafficking to the protein storage vacuole. J Cell Biol 2005;170:757–767.

144

141. Vitale A and Pedrazzini E. Recombinant pharmaceuticals from plants: the plant endo-

membrane system as bioreactor. Mol Interv 2005;5:216–225.

142. Humphrey BD, Huang N and Klasing KC. Rice expressing lactoferrin and lysozyme

has antibiotic-like properties when fed to chicks. J Nutr 2002;132:1214–1218.

143. Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M and Takaiwa F. A transgenic

rice seed accumulating an anti-hypertensive peptide reduces the blood pressure of

spontaneously hypertensive rats. FEBS Lett 2006;580:3315–3320.

144. Yang D, Guo F, Liu B, Huang N and Watkins SC. Expression and localization of

human lysozyme in the endosperm of transgenic rice. Planta 2003;216(4):597–603.

145. Arcalis E, Marcel S, Altmann F, Kolarich D, Drakakaki G, Fischer R, Christou P and

Stoger E. Unexpected deposition patterns of recombinant proteins in post-endoplasmic

reticulum compartments of wheat endosperm. Plant Physiol 2004;136:3457–3466.

146. Paris N and Neuhaus JM. BP-80 as a vacuolar sorting receptor. Plant Mol Biol

2002;50:903–914.

147. van Rooijen GJ and Moloney MM. Structural requirements of oleosin domains for

subcellular targeting to the oil body. Plant Physiol 1995;109:1353–1361.

148. Seon JH, Szarka S and Molone MM. A unique strategy for recovering recombinant

proteins from molecular farming: affinity capture on engineered oilbodies. J Plant

Biotechnol 2002;4:95–101.

149. Nykiforuk CL, Boothe JG, Murray EW, Keon RG, Goren HJ, Markley NA and

Moloney MM. Transgenic expression and recovery of biologically active recombinant

human insulin from Arabidopsis thaliana seeds. Plant Biotechnol J 2006;4:77–86.

150. Daniell H and Dhingra A. Multigene engineering: dawn of an exciting new era in

biotechnology. Curr Opin Biotechnol 2002;13:136–141.

151. De Cosa B, Moar W, Lee SB, Miller M and Daniell H. Overexpression of the

Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat

Biotechnol 2001;19:71–74.

152. Faye L and Daniell H. Novel pathway for glycoprotein import into chloroplasts. Plant

Biotechnol J 2006;4:275–279.

153. Maliga P. Plant biology: mobile plastid genes. Nature 2003;422:31–32.

154. Millan AF-S, Mingo-Castel A, Miller M and Daniell H. A chloroplast transgenic ap-

proach to hyper-express and purify Human Serum Albumin, a protein highly suscep-

tible to proteolytic degradation. Plant Biotechnol J 2003;1:71–79.

155. Jobling SA, Jarman C, Teh MM, Holmberg N, Blake C and Verhoeyen ME. Immuno-

modulation of enzyme function in plants by single-domain antibody fragments. Nat

Biotechnol 2003;21:77–80.

156. Daniell H, Streatfield SJ and Wycoff K. Medical molecular farming: production of

antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 2001;

6:219–226.

157. Villarejo A, Buren S, Larsson S, Dejardin A, Monne M, Rudhe C, Karlsson J, Jansson

S, Lerouge P, Rolland N, von Heijne G, Grebe M, Bako L and Samuelsson G. Evidence

for a protein transported through the secretory pathway en route to the higher plant

chloroplast. Nat Cell Biol 2005;7:1224–1231.

158. Michaud D, Vrain T, Gomord V and Faye L. Stability of recombinant proteins in

plants. In: Methods in Biotechnology – Recombinant Proteins from Plants Production

and Isolation of Clinically Useful Compounds, AJR P (ed), Totowa, NJ, Humana

Press, 1998, pp. 177–188.

159. Michaud D. Gel electrophoresis of proteolytic enzymes. Anal Chim Acta 1998;

372:173–185.

145

160. Wong EY, Hironaka CM and Fischhoff DA. Arabidopsis thaliana small subunit leader

and transit peptide enhance the expression of Bacillus thuringiensis proteins in

transgenic plants. Plant Mol Biol 1992;20:81–93.

161. Van der Vyver C, Schneideret J, Driscoll S, Turner J, Kunert K and Foyer C.

Oryzacystatin I expression in transformed tobacco produces a conditional growth

phenotype and enhances chilling tolerance. Plant Biotechnol J 2003;1:101–112.

162. Michaud D, Anguenot R and Brunelle F. Method for increasing protein content in

plant cells. (US Patent application) 2003.

163. Rivard D, Anguenot R, Brunelle F, Le VQ, Vezina LP, Trepanier S and Michaud D.

An in-built proteinase inhibitor system for the protection of recombinant proteins

recovered from transgenic plants. Plant Biotechnol J 2006;4:359–368.

164. Aalberse RC, Koshte V and Clemens JG. Immunoglobulin E antibodies that crossreact

with vegetable foods, pollen, and Hymenoptera venom. J Allergy Clin Immunol

1981;68:356–364.

165. Aalberse RC, Koshte V and Clemens JG. Cross-reactions between vegetable foods,

pollen and bee venom due to IgE antibodies to a ubiquitous carbohydrate determinant.

Int Arch Allergy Appl Immunol 1981;66:259–260.

166. Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L and

Lerouge P. Immunoreactivity in mammals of two typical plant glyco-epitopes, core

alpha(1,3)-fucose and core xylose. Glycobiology 2003;13:427–434.

167. Jin C, Bencurova M, Borth N, Ferko B, Jensen-Jarolim E, Altmann F and Hantusch B.

Immunoglobulin G specifically binding plant N-glycans with high affinity could be

generated in rabbits but not in mice. Glycobiology 2006;16:349–357.

168. Nuttall J, Ma JK and Frigerio L. A functional antibody lacking N-linked glycans is

efficiently folded, assembled and secreted by tobacco mesophyll protoplasts. Plant

Biotechnol J 2005;3:497–504.

169. Strasser R, Stadlmann J, Svoboda B, Altmann F, Glossl J and Mach L. Molecular basis

of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking

complex N-glycans. Biochem J 2005;387:385–391.

170. Downing WH, Galpin JD, Clemens S, Lauzon SM, Samuels AL, Pidkowich MS, Clarke

LA and Kermode AR. Synthesis of enzymatically active human alpha-L-iduronidase in

Arabidopsis cgl (complex glycan-deficient) seeds. Plant Biotechnol J 2006;4:169–182.

171. Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, Reski R and

Decker EL. Targeted knockouts of Physcomitrella lacking plant-specific immunogenic

N-glycans. Plant Biotechnol J 2004;2:517–523.

172. Sourrouille C. Inactivation de l’alpha(1,3)-fucosyltransferase et de la beta(1,2)-xylosylt-

ransferase, en vue de la production de proteines recombinantes d’interet therapeutique

chez la luzerne (Universite de Rouen), 2005, p. 164.

173. Sriraman R, Bardor M, Sack M, Vaquero C, Faye L, Fischer R, Finnern R and

Lerouge P. Recombinant anti-hCG antibodies retained in the endoplasmic reticulum

of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues. Plant

Biotechnol J 2004;2(4):279–287.

174. Petruccelli S, Otegui MS, Lareu F, Tran Dinh O, Fitchette A-C, Circosta A, Rumbo M,

Bardor M, Carcamo R, Gomord V and Beachy RN. A KDEL-tagged monoclonal

antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both

partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J

2006;4:511–527.

175. Palacpac NQ, Yoshida S, Sakai H, Kimura Y, Fujiyama K, Yoshida T and

Seki T. Stable expression of human beta1,4-galactosyltransferase in plant cells

146

modifies N-linked glycosylation patterns. Proc Natl Acad Sci USA 1999;96:

4692–4697.

176. Misaki R, Kimura Y, Palacpac NQ, Yoshida S, Fujiyama K and Seki T. Plant cultured

cells expressing human beta1,4-galactosyltransferase secrete glycoproteins with gala-

ctose-extended N-linked glycans. Glycobiology 2003;13:199–205.

177. Fujiyama K, Palacpac NQ, Sakai H, Kimura Y, Shinmyo A, Yoshida T and Seki T. In

vivo conversion of a glycan to human compatible type by transformed tobacco cells.

Biochem Biophys Res Commun 2001;289:553–557.

178. Saint-Jore-Dupas C, Nebenfuhr A, Boulaflous A, Follet-Gueye ML, Plasson C, Hawes

C, Driouich A, Faye L and Gomord V. Plant N-glycan processing enzymes employ

different targeting mechanisms for their spatial arrangement along the secretory path-

way. Plant Cell 2006;18:3182–3200.

179. Bakker H, Rouwendal GJ, Karnoup AS, Florack DE, Stoopen GM, Helsper JP, van

Ree R, van Die I and Bosch D. An antibody produced in tobacco expressing a hybrid

beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes. Proc

Natl Acad Sci USA 2006;103:7577–7582.

180. Seveno M, Bardor M, Paccalet T, Gomord V, Lerouge P and Faye L. Glycoprotein

sialylation in plants? Nat Biotechnol 2004;22:1351–1352, 1352–1353.

181. Zeleny R, Kolarich D, Strasser R and Altmann F. Sialic acid concentrations in plants

are in the range of inadvertent contamination. Planta 2006;224:222–227.

182. Jarvis DL. Developing baculovirus-insect cell expression systems for humanized

recombinant glycoprotein production. Virology 2003;310:1–7.

183. Wee EG, Sherrier DJ, Prime TA and Dupree P. Targeting of active sialyltransferase to

the plant Golgi apparatus. Plant Cell 1998;10:1759–1768.

184. Misaki R, Fujiyama K and Seki T. Expression of human CMP-N-acetylneuraminic acid

synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Bio-

chem Biophys Res Commun 2006;339:1184–1189.

185. Paccalet T, Bardor M, Rihouey C, Delmas F, Chevalier C, D’Aoust M-A, Faye L,

Vezina LP, Gomord V and Lerouge P. Engineering of a sialic acid synthesis pathway in

transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes. Plant

Biotechnol J 2007;5:16–25.

186. Gasdaska JR, Spencer D and Dickey LF. Advantages of therapeutic protein production

in the aquatic plant Lemna. Bioprocess J 2003;3:50–56.

187. Schaefer D, Zryd JP, Knight CD and Cove DJ. Stable transformation of the moss

Physcomitrella patens. Mol Gen Genet 1991;226:418–424.

188. Schaefer DG and Zryd JP. Efficient gene targeting in the moss Physcomitrella patens.

Plant J 1997;11:1195–1206.

189. Schaefer DG, Bisztray G and Zryd J-P. Genetic transformation of the moss Physc-

omitrella patens. In: Plant Protoplasts and Genetic Engineering V, Bajaj YPS (ed),

Berlin, Springer-Verlag, 1994, pp. 349–364.

190. Vietor R, Loutelier BC, Fitchette A, Margerie P, Gonneau M, Faye L and Lerouge P.

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher

plants. Planta 2003;218:269–275.

191. Franklin SE and Mayfield SP. Prospects for molecular farming in the green alga

Chlamydomonas. Curr Opin Plant Biol 2004;7:159–165.

192. Leon-Banares R, Gonzalez-Ballester D, Galvan A and Fernandez E. Transgenic micro-

algae as green cell-factories. Trends Biotechnol 2004;22:45–52.

193. Mayfield SP and Franklin SE. Expression of human antibodies in eukaryotic micro-

algae. Vaccine 2005;23:1828–1832.

147

194. Lienard D, Tran Dinh O, van Oort E, Van Overtvelt L, Bonneau C, Wambre E, Bardor

M, Cosette P, Didier-Laurent A, Dorlhac de Borne F, Delon R, van Ree R, Moingeon

P, Faye L and Gomord V. Suspension-cultured BY-2 tobacco cells produce and mature

immunologically active house dust mite allergens. Plant Biotechnol J 2007;5:93–108.

195. Elbers IJ, Stoopen GM, Bakker H, Stevens LH, Bardor M, Molthoff JW, Jordi WJ,

Bosch D and Lommen A. Influence of growth conditions and developmental stage on

N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in

tobacco leaves. Plant Physiol 2001;126:1314–1322.