Methods 32 (2004) 235–240

www.elsevier.com/locate/ymeth

Over-expression and production of plant allergensby molecular farming strategies

Gerhard Obermeyer,a,* Renate Gehwolf,a Wolfgang Sebesta,a Nichola Hamilton,a

Gabriele Gadermaier,b Fatima Ferreira,b Uli Commandeur,c Rainer Fischer,c

and Friedrich-Wilhelm Bentrupa

a Institute of Plant Physiology, University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austriab Institute of General Biology and Genetics, University of Salzburg, Hellbrunnerstr. 34, 5020 Salzburg, Austria

c Institute of Molecular Biotechnology, RWTH Aachen, Worringer Weg 1, 52074 Aachen, Germany

Accepted 21 August 2003

Abstract

Recombinant allergens have become a valuable tool for diagnosis and may also be used for therapy in the near future. To supply

the required large amounts of functional recombinant proteins on a cost-effective basis, the production of allergens in plants by

molecular farming is an alternative to microbial expression systems. Especially as post-translational modifications of the allergens,

e.g., phosphorylation and glycosylation, may be important for recognition by the human immune system, the plant-based

production of recombinant allergens enables the correct folding, glycosylation, and other modifications of the recombinant allergen.

An introduction to the methods for plant transformation via the tumor-inducing bacterium, Agrobacterium tumefaciens, is given in

this paper.

� 2003 Elsevier Inc. All rights reserved.

Keywords: Agrobacterium tumefaciens; Allergen; Artemisia vulgaris; Art v1; Over-expression; Molecular farming; Plant expression; Plant

transformation

1. Introduction

The sequences of many allergenic proteins have been

identified by molecular biology methods and re-

combinant allergens became a valuable tool for diagnosisand therapy [1]. The recombinant pollen allergens are

produced in bacteria, yeast ormammalian cells; with each

expression system having its respective advantages and

disadvantages, e.g., proteins may not be folded correctly

in bacteria or expression in mammalian cells is very ex-

pensive. Additionally, the polypeptide backbone of an

allergenic plant protein may not be the only epitope rec-

ognized in the allergic response and post-translationalmodifications like glycosylation or phosphorylation may

also be involved in the formation of epitopes [2]. Glyco-

sylation of plant proteins in the dictyosomes is very

* Corresponding author. Fax: +43-662-8044-619.

E-mail address: gerhard.obermeyer@sbg.ac.at (G. Obermeyer).

1046-2023/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.ymeth.2003.08.012

complex and N-linked glycosylation on asparagine resi-

dues as well as O-linked glycosylation on hydroxy proline

may occur. Additionally, plant-specific sugars like xylose

and fucose are incorporated into the glycan chains.

Therefore, glycan chains of recombinant allergens heter-ologously expressed in yeast or mammalian cells may be

very different from the natural plant allergen, and thus not

recognized by IgE antibodies of allergic patients.

To solve this problem, allergenic proteins can be ex-

pressed in a plant expression system. Usually, plants are

transformed by particle bombardment, infected with

recombinant viral vectors or by using a tumor-inducing

bacterium, Agrobacterium tumefaciens [3]. So far, to-bacco plants (Nicotiana benthamiana) have been infected

with a tobacco mosaic virus containing the sequence of

Bet v 1 [4]. However, this approach leads to a transient

expression only and the N. benthamiana plants were not

suitable for large-scale production of the recombinant

allergen due to their low yield per hectare.

236 G. Obermeyer et al. / Methods 32 (2004) 235–240

Therefore, a stable plant expression system for thelarge-scale production of allergens is preferable. The

Agrobacterium-mediated, nuclear transformation of

plants has been established for the production of various

pharmaceuticals and antibodies in plants, and is suitable

for lab-scale as well as large-scale production [5–12].

This paper gives a practical introduction into this tech-

nique to transform plants for the purpose of allergen

production. Special applications may require othertransformation methods, e.g., particle bombardment or

virus-mediated expression (see this issue), that might be

more suitable. But description of all transformation

strategies will be beyond the scope of this paper and the

reader is referred to publications addressing these and

related topics in more detail [3,13–15].

2. Description of methods

2.1. Agrobacterium-based transformation of tobacco

plants

The soil bacterium A. tumefaciens infects wounded

parts of dicot and some monocot plants and causes

undifferentiated, tumorous growth of plant tissue, theformation of so-called crown galls. Upon perception of

wounding signals from the plants, e.g., phenolic sub-

stances, virulence (vir) genes are expressed and a part of

a large (>200 kb), tumor-inducing (Ti) plasmid is

transferred to the plant cells. This transferred DNA

(T-DNA) is integrated into the plant genome and con-

tains genes for synthesis of phytohormones, e.g., cyto-

kines and auxins, promoting a tumor-like proliferationand growth of the infected cells. Additionally, unusual

amino acids (opines) serving as nutrients for the Agro-

bacteria are synthesized by the tumor cells (for review,

see [16–18]). Therefore, this microbe acts as a �natural�genetic engineer transforming a plant for its specific

needs. Usually, a Ti-plasmid contains the oncogenes and

approximately 35 vir genes as well as the T-DNA de-

limited by ca. 25 bp long repeats, the left (LB) and theright border (RB). It was observed that the vir genes

were able to mediate T-DNA transfer also from other,

smaller, so-called �disarmed� Ti plasmids. These vectors

are no longer oncogenic and allow easy manipulation of

the T-DNA, e.g., introduction of multiple cloning site,

marker genes or selectivity marker between the two

borders. The resulting �binary� Ti vectors are able to

replicate in E. coli and in Agrobacterium simplifying thecloning procedure for introduction of foreign genes into

the T-DNA region. All gene manipulation steps can be

performed on small Ti vectors in E. coli and only in a

last step the Ti vector is introduced into Agrobacteria for

subsequent plant transformation. A variety of binary Ti

vectors for Agrobacterium-based plant transformation

are now available [19].

In this paper, we will describe a simple procedure tointroduce a foreign gene encoding a pollen allergen (Art

v 1, Accession No. AF493943) into tobacco plants. In

general, the procedure contains three major steps:

cloning the allergen sequence into the binary Ti vector,

introduction of the Ti vector into Agrobacterium, and

finally, transformation and regeneration of the trans-

genic plants. To introduce the reader to the various

techniques involved in the transformation and culture ofplants, we describe the cloning of Art v 1 into the

pBI121 vector, its introduction into A. tumefaciens

(strain LBA 4404), and the stable as well as the transient

transformation of tobacco plants (Nicotiana tabacum,

�SR1�). An overview of the transformation strategy is

given in Fig. 1. We choose the pBI121 vector and the

A. tumefaciens strain LBA 4404 because both are com-

mercially available (Life Technologies, Rockville, USA,www.lifetech.com) and serve as a convenient test system

to become familiar with the plant transformation tech-

nique. However, when other Ti vectors, allergens or

bacterial strains are used, the protocols have to be

modified and optimized accordingly. We expect that the

reader is familiar with basic molecular biology tech-

niques like plasmid preparation, growth of bacteria,

PCR, etc. For general methods in molecular biology, werefer to Sambrook et al. [20]. A valuable collection of

protocols on plant molecular biology techniques can be

found in the handbook edited by Gelvin and Schil-

peroort [13].

2.1.1. Introduction of foreign DNA into a binary plasmid

The binary vector pBI121 (Fig. 2) was constructed on

the base of pBIN [21] containing a kanamycin resistancegene (NPT II) under the control of the nopaline syn-

thase promoter (NOS-P) and the b-glucuronidase(GUS) gene (ca. 1.87 kb fragment) under the control of

the cauliflower mosaic virus 35 S promoter (35 S-P,

800 bp HindIII–BamHI-fragment) between the left (LB)

and the right border (RB) sequences. The polyadenyla-

tion signal was taken from the terminator of the nopa-

line synthase gene of the Agrobacterium Ti plasmid(NOS-T). The vector backbone contains a low-copy-

number RK2 origin of replication working in E. coli as

well as in Agrobacterium and a kanamycin resistance

gene for selection in bacteria. The unmodified pBI121

vector may be used to perform first transformation ex-

periments to determine GUS activity in plant cells or it

might be modified by exchanging the GUS gene by a

gene of interest, e.g., allergen, using the four possiblerestriction sites. However, due to its large size, the

pBI121 is not the best Ti vector for plant transforma-

tion, it offers a good system to learn the basic principles

of the Agrobacterium-based transformation strategy.

Alternatively, the T-DNA of a Ti vector of the modular

pGREEN II system (Fig. 1; [22]) is shown, which also

contains a kanamycin resistance gene for selection in

Fig. 2. Examples of the T-DNA of two Ti vectors, pBI 121 and

pGREEN II 0049, containing a kanamycin resistance gene for selec-

tion of the transformed plants, a reporter gene (b-glucuronidase or

luciferase, and a multiple cloning side (pGREEN II only) between the

right (RB) and the left border (LB). For selection in bacteria both

vectors contain a kanamycin resistance gene. For further explanations

see text.

Fig. 1. A schematic overview of the Agrobacterium-mediated, stable

transformation of plants.

Table 1

Bacterial growth media

LB (Escherichia coli) 1% (w/v) peptone

0.5% (w/v) NaCl

1% (w/v) yeast extract

�1.5% (w/v) agar

pH 7.0 adjusted with 1N NaOH

YM (Agrobacterium

tumefaciens)

0.04% (w/v) yeast extract

1% (w/v) mannitol

1.7mM NaCl

0.8mMMgSO4

2.2mM K2PO4

�1.5% (w/v) agar

pH 7.0 adjusted with 1N NaOH

For selection of the Agrobacterium strain LBA 4404 add

100lgml�1 streptomycin.

For selection of the Agrobacterium strain LBA 4404 containing the

plasmid pBI 121 or pGREEN II 0049 add 100lgml�1 streptomycin

and 50 lgml�1 kanamycin.

G. Obermeyer et al. / Methods 32 (2004) 235–240 237

plants but has the luciferase reporter gene and most

important, contains a multiple cloning site in a trun-

cated LacZ operon making cloning steps in E. coli

simpler and more convenient.

2.1.1.1. Protocol for E. coli growth, plasmid preparation,

and restriction enzyme digestion.

1. Select a single colony of E. coli containing the pBI121vector which was grown on a plate with LB medium

plus the appropriate antibiotic (in case of pBI121 use

50 lgml�1 kanamycin) and inoculate 5ml LB me-

dium plus 50 lgml�1 kanamycin. Incubate at 37 �Con a rotary shaker at 250 rpm overnight. Introduc-

tion of pBI121 into E. coli was done according tostandard techniques [20] using chemical competent

strains XL-1 blue or DH5-a (see Table 1).

2. Use the overnight culture to isolate the plasmid as de-

scribed by Sambrook et al. [20] or use a plasmid pu-

rification kit (NucleoSpin, Machery and Nagel or

Qiagen). Note: pBI121 is a low-copy-number plas-

mid. Therefore, follow the instructions of the manu-

facturer for these plasmids.3. To insert an allergen sequence cut the plasmid with

BamHI and SstI (or using its isoschizomer SacI) in

single digestion reactions using the appropriate buf-

fers. A double digestion may be performed in Y-Tan-

go (MBI-Fermentas, Vilnius, Lithuania) or all-in-one

buffer but may result in less product. Confirm the di-

gestion on an agarose gel (0.8–1% w/v). An additional

band of ca. 1.8 kb (GUS) is visible upon successfuldigestion.

4. Run a preparative agarose gel and elute the digested

plasmid according to standard techniques. Use the

plasmid immediately for insertion of the allergen

sequence.

2.1.1.2. Protocol for extended PCR and ligation into

pBI 121.1. To insert the allergen cDNA into the BamHI/SstI

sites of the plasmid, the respective restriction sites

have to be added to the allergen sequence of interest.

This can be achieved by PCR and an extension pri-

mer pair containing the 50- and 30-end sequence of

the allergen with additional BamHI- and SstI-specific

sequences, respectively, the restriction sites are added

to both ends of the allergen sequence. Additional tothe restriction, site-specific sequence overhang se-

quences (e.g., GAGGA) have to be added to improve

the restriction reaction.

238 G. Obermeyer et al. / Methods 32 (2004) 235–240

2. Confirm the PCR by separating the products on anagarose gel and elute the product of the appropriate

size from the gel.

3. Digest the PCR product with the respective enzymes

(see above) and purify the digested PCR product by

precipitation with ethanol.

4. The allergen is ligated into the plasmid using a 1:3 ra-

tio of plasmid to allergen DNA (other ratios may be

tested to improve the ligation reaction). Ligation iscarried out overnight at 16 �C using T4 DNA ligase

(3–5 units in a reaction volume of 110 ll).5. Incorporate the ligated plasmid into a chemically

competent E. coli strain by heat shock transformation

[20]. Select positive clones on kanamycin containing

agar plates and confirm the successful ligation and/

or plasmid incorporation by performing a PCR using

primer sets hybridizing to sequences of the insertedallergen and to the plasmid, respectively. Prepare

glycerol stocks from positive E. coli clones for long-

term storage at )80 �C.

2.1.1.3. Protocol for A. tumefaciens growth.

1. Inoculate a plate (solidifiedYMmedium+100 lgml�1

streptomycin) with A. tumefaciens, strain LBA 4404,

from a )80 �C frozen stock.2. Incubate at 28 �C for 2–3 days until single colonies

are visible.

3. Inoculate 10ml liquid YM medium with a single col-

ony and incubate at 28 �C for 3 days on a rotary sha-

ker (200 rpm).

4. Measure the absorption at 600 nm (OD600) to moni-

tor the growth of the bacterial culture. Usually, an

OD600 of 0.5–0.6 is obtained after 30 h under theseconditions.

2.1.2. Transformation of Agrobacterium

Several methods have been developed to mobilize

plasmids into Agrobacterium cells, e.g., tri-parental

mating, a freeze–thaw method [23], and electropora-

tion. As the strain LBA 4404 is already electrocom-

petent, we describe here the electro-transformation ofAgrobacterium.

2.1.2.1. Preparation of electrocompetent Agrobacterium

[29,24].

1. Pellet an overnight culture of Agrobacterium by cen-

trifugation (3000 rpm, 4 �C, 15min) and discard the

supernatant. Resuspend the bacteria pellet with 1/10

volume of sterile, ice-cold 10% (v/v) glycerol by gentlevortexing. Fill up to initial volume (10/10) with ice-

cold 10% (v/v) glycerol.

2. Repeat the centrifugation and washing steps with

10% (v/v) glycerol twice.

3. Finally resuspend in a 1/100 vol with 10% (v/v) ice-

cold glycerol, aliquot, and freeze in liquid N2. Store

at )80 �C for up to 3 months.

2.1.2.2. Electro-transformation of Agrobacterium.1. Thaw a fresh aliquot of electrocompetent Agrobacte-

ria on ice. Add plasmid samples in water or TE buffer

(<5 ll) in a pre-cooled electroporation cuvette

(0.1 cm distance between the electrode plates). Then,

add 20 ll of the electrocompetent bacteria and mix

gently.

2. Set the parameters of the electroporation device to a

field strength of 20 kV cm�1 and a time constant of5ms and apply one pulse. Many manufacturers pro-

vide good electroporation protocols for a variety of

bacteria in their instruction manual, e.g., BioRad Mi-

cropulser: select �Agr� (2.2 kV) or Gibco-BRL cell

porator: voltage 400V, 333 lF, low resistance, but

electroporation parameters should be optimized to

improve the transformation frequency.

3. Add electroporated Agrobacteria immediately to 1mlYM medium at room temperature and incubate for

3 h at 28 �C on a shaker at 250 rpm.

4. Plate on YM plates in the presence of 50 lgml�1

kanamycin at 28 �C for 2 days. Check for successful

transformation.

5. Grow transformed Agrobacterium clones in liquid

YM medium and prepare glycerol stocks.

2.1.3. Transformation of tobacco plants

For the production of large amounts of recombinant

allergens, a stable transformation of tobacco plants is

preferable to a transient expression. However, transient

expression of the foreign gene can be done quite fast and

it takes only about 1 week until the expression products

can be monitored instead of 6–8 months in a stable

transformation system. In some cases, it is important totest the constructed Ti vectors especially when the

physiological function of the introduced gene is not

known and its expression might be lethal for the plant.

An elegant and convenient transient expression method

for plants is the Agro-infiltration [25,26] in which plant

leaves are infiltrated with an Agrobacterium suspension

harbouring the constructed Ti vector. Alternatively, the

Ti vector itself can be introduced into plant cells usingparticle bombardment. But this method requires addi-

tional equipment (gene gun, etc.) and time-consuming

optimization of the bombardment procedure for the

respective plant tissue and vector (pressure, distance,

post-bombardment treatments, etc.).

Stable transformation of plants can be obtained by

the so-called �leaf-disc method� where parts of a leaf are

co-cultured with Agrobacteria that will infect some ofthe leaf cells. The infected, and subsequently trans-

formed, cells are able to grow on antibiotic-containing

media and form a callus from which shoots are

growing depending on the concentrations of the added

phytohormones. The shoots are then transferred to

another, hormone-free medium allowing the generation

of roots. The small plantlets may then be planted into

Table 2

Preparation of tobacco growth media Linsmaier–Skoog (LS) medium

(based on Murashige–Skoog (MS) salt solutions)

10�Macroelements per 1L

188mM KNO3 19.0 g

206mM NH4NO3 16.5 g

30mM CaCl2 4.4 g

15mM MgSO4 3.7 g

12.5mM KH2PO4 1.7 g

100�Fe-EDTA solution per 1L

10mM Na2-EDTA 3.73 g

10mM FeSO4 � 7H2O 2.73 g

Filter sterilize the solution!

B5 vitamin solution per 100ml

Nicotinic acid 100mg

Thiamine HCl 1000mg

Pyridoxine HCl 100mg

Myo-inositol 10,000mg

100�Microelements per 1L

10mM MnSO4 � 4H2O 2.25 g

3mM ZnSO4 0.85 g

10mM H3BO3 0.62 g

10mM KI 0.082 g

0.1mM Na2MoO4 � 2H2O 0.025 g

0.01mM CuSO4 � 5H2O 0.0025 g

0.01mM CoCl2 � 6H2O 0.0025 gL�1

Stock solutions (filter sterilized)

a-Naphthaleneacetic acid (a-NAA, 100mgml�1 in ethanol)

Benzyladenine (BA, 1mgml�1 in ethanol)

A ready-to-use mixture of MS salts is commercially available, e.g.,

Sigma, Life Technologies.

For 500ml LS-1 mix 50ml of macroelement sol. with 5ml of mi-

croelement sol. and add 15 g of sucrose. Add distilled water to a vol-

ume of 490ml and adjust pH with 1N NaOH between 5.7 and 5.8.

When solidified medium is needed add also 4 g of agar. Autoclave at

120 �C, 1 bar for 20min. Let cool to about 50 �C (hand warm) and add

5ml vitamin sol. and 5ml Fe-EDTA sol. before use.

For LS-2 prepare LS medium, autoclave and let cool to below

50 �C. Add the sterile-filtered hormones: 0.5 ll a-NAA and 500ll BA.

For LS-1-kan and LS-2-kan add kanamycin to a final concentra-

tion of 50lgml�1 to the appropriate medium.

G. Obermeyer et al. / Methods 32 (2004) 235–240 239

pots on normal soil or on a special substrate until theyare ready for analysis or purification of the expressed

gene product. It is important to harvest seeds from

plants showing the highest expression rates and to

analyse the F2 generation for antibiotic resistance and

expression levels. This transformation method requires

knowledge of aseptic techniques of cell and plant cul-

tures and it is of advantage to establish these tech-

niques before transformation experiments areperformed (see books on plant culture techniques

[14,27]). All steps of plant cultivation have to be per-

formed in a laminar flow hood.

2.1.3.1. ‘Leaf-disc’ transformation protocol.

1. Inoculate a YM (+ appropriate antibiotic) agar plate

with a frozen stock of A. tumefaciens containing the

appropriate Ti vector and incubate at 28 �C for 2–3days. Add one clone to 2ml of liquid YM medium

from this plate.

2. Grow to OD600 of 0.6–0.8.

3. Take a young leaf from tobacco plants (about 5–7

leaf stage), sterilize the surface of the leaf by dipping

into 70 (v/v)% ethanol, washing for 2min in 35 (v/

v)% commercial bleach, and finally, wash thor-

oughly with sterile water (2–5�). When sterile grownplants are used, surface sterilization is not necessary.

4. Prepare leaf sections by cutting small pieces

(5� 5mm) out of the leaf or by using a sharp cork

borer with a diameter of 5–10mm.

5. Transfer 3–5 leaf discs always with the upper side

down into small petri dishes containing 4–6ml LS

medium (no antibiotics and no hormones).

6. Add 50–100 ll of the overnight Agrobacterium cul-ture. Determine the optimal amount of bacteria to

be added experimentally by using dilutions of the

overnight cultures from OD¼ 0.6 to OD¼ 1.4.

7. Seal the petri dishes with Parafilm and incubate

them in a plant growth chamber at 24 �C for 2–3

days in the dark.

8. Wash the leaves in LS medium (Table 2) with car-

benicillin (500mgL�1) or another appropriate anti-biotic to kill the Agrobacteria. Take care not to

wet the lower leaf side. Place leaf discs on solidified

LS medium (LS-2-kan) containing kanamycin to se-

lect for kanamycin resistance and hormones (a-NAA and BA) to stimulate shoot growth.

9. Seal the plates with Parafilm and place in a lighted

growth chamber (16 h light/8 h dark) at 24 �C.10. Examine the leaf discs once or twice a week for con-

taminations. After 1–2 weeks, callus growth can be

noticed at the rims of the leaf discs and within 3–4

weeks tiny, green shoots can be observed whereas

the rest of the disc turns bleached.

11. Transfer 10–15mm long shoots to solidified LS me-

dium (LS-1-kan) in Magenta boxes to allow the

transformed shoots to regenerate roots.

12. At a height of at least 4 cm transfer plants to soil in

pots. Cover them with plastic bags with vent holes to

maintain high humidity conditions and gradually

adapt the plants to the standard greenhouse condi-

tions.

2.1.3.2. Agro-infiltration protocol.

1. Use a culture of Agrobacterium (LBA 4404 with pBI121) grown in YM medium supplemented with anti-

biotics to inoculate a YM medium. For vir gene in-

duction: YM+10mM Mes (pH 5.6) in supplement

with antibiotics and 20 lM acetosyringone. Grow

the culture overnight (OD600 0.6–0.8) at 28 �C and

pellet the bacteria for 20min at 15 �C, 4000g. Resus-

pend the bacterial pellet with infiltration medium

(INF-Med) to a final volume of 50ml and incubatefor 1–2 h at room temperature (see Table 3).

Table 3

Infiltration medium

(INF-Med, 500ml)

50ml macroelements

5ml microelements

10 g sucrose

200lM acetosyringone

pH 5.8 adjusted with 1N NaOH

Table 4

Reagents for GUS assay

Incubation medium

(INC-Med)

0.1M Na2HPO4/NaH2PO4, pH 7.0

1M Na2 EDTA

1mM K3Fe(CN)61mM K4Fe(CN)62% (v/v) Triton X-100

�0.5mgml�1 x-Gluc (5-bromo-4-

chloro-3-indolyl-b-glucuronic acid)

240 G. Obermeyer et al. / Methods 32 (2004) 235–240

2. Submerge 3–4 tobacco leaves into 50–150ml of Agro-

bacterium suspension in a beaker and weight them,

e.g., with a mortar, so that the leaves are fully cov-

ered with the bacterial suspension.

3. Place the beaker in an excicator and apply a low pres-

sure of 60–80mbar for ca. 20min. Turn off the vac-

uum pump and release the vacuum fast but notsuddenly. Infiltrated leaves have a transparent ap-

pearance.

4. Put the infiltrated leaves with the upper side up in a

incubation chamber and close with saran wrap. High

humidity should be maintained in the chamber. Place

the chamber in a plant growth cabinet and incubate

for 3–5 days at 24 �C.5. Use a reporter gene assay, e.g., GUS assay for pBI

121 or luciferase assay for pGREEN II 0049, to mon-

itor the successful transformation, or detect the ex-

pressed allergen with a specific antibody in an

immunoblot.

2.2. GUS assay [28]

1. Wash leafs or leaf discs in 100% ethanol. Incubate inice-cold acetone for 1 h. Wash twice with incubation

medium without x-Gluc (INC-med).

2. Vacuum-infiltrate the leafs with INC-med plus

0.5mgml�1 x-Gluc and incubate at 37 �C overnight.

3. Wash twice with INC-med without x-Gluc and ob-

serve under a light microscope (see Table 4).

3. Concluding remarks

The Agrobacterium-mediated transformation of

plants represents a powerful technique for the produc-

tion of pharmaceutical proteins on a lab scale as well as

on a field scale. Although the transformation is labori-

ous and time consuming, a stable transformation of a

variety of plants including crop plants can be achieved

and the expressed proteins, e.g., allergens, hypo-aller-gens, antibodies, are functional. The plant expression

system is especially preferable for the production of

complex proteins and proteins that become functional

after plant-specific post-translational modifications.

Plants produce a large amount of biomass and there-

fore, almost unlimited quantities of recombinant pro-

teins for use as diagnostic and therapeutic tools can be

obtained on a low cost-basis making plants the leadingexpression system in the near future.

Acknowledgments

The work in the laboratories of G.O. and F.F. was

financed by grants from the FWF (S8804, S8802).

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