ORIGINAL PAPER

Expression and subcellular targeting of human insulin-likegrowth factor binding protein-3 in transgenic tobacco plants

Stanley C. K. Cheung Æ Samuel S. M. Sun ÆJuliana C. N. Chan Æ Peter C. Y. Tong

Received: 13 October 2008 / Accepted: 16 May 2009 / Published online: 6 June 2009

� Springer Science+Business Media B.V. 2009

Abstract Human insulin-like growth factor binding

protein-3 (hIGFBP-3) is a multifunctional protein

which has high affinity for insulin-like growth factor-

I (IGF-I). It combines with IGF-I to form a tertiary

complex in circulation, thus regulating the activity of

IGF-I. Furthermore, recombinant hIGFBP-3 (rhI-

GFBP-3) has been found to negatively regulate cell

proliferation and induce apoptosis. In this study, we

have established an efficient plant bioreactor platform

for mass production of rhIGFBP-3. Different expres-

sion constructs, driven by the seed-specific phaseolin

promoter, were designed and transformed into tobacco

plant via Agrobacterium. To enhance protein expres-

sion level, the signal peptide (SP) and the C-terminal

tetrapeptide AFVY of phaseolin were used to direct

rhIGFBP-3 to protein storage vacuole (PSV) in tobacco

seed for stable accumulation. Western blot analysis

showed that rhIGFBP-3 was successfully synthesized

in transgenic tobacco seeds, with the highest protein

expression of 800 lg/g dry weight. The localization of

rhIGFBP-3 in PSV was also evident by confocal

immunofluorescence microscopy. Our results indi-

cated that protein sorting sequences could benefit the

expression level of rhIGFBP-3 and it is feasible to use

plant as ‘‘bio-factory’’ to produce therapeutic recom-

binant proteins in large quantity.

Keywords IGFBP-3 � Transgenic tobacco �Protein targeting � Phaseolin � Codon modification �Recombinant human protein

Introduction

The mitogenic activities of insulin-like growth factors

(IGFs) are modulated by a family of six insulin-like

growth factor binding proteins (IGFBPs). Among the

six homologous multifunctional proteins, IGFBP-3 is

the most abundant IGFBP in human serum and

produced by non-parenchymal hepatic cells. It consists

of 264 amino acids (Wood et al. 1988), with the

molecular weight of 40–45 kDa (Martin and Baxter

1986). It binds IGF-I with high affinity by forming a

150 kDa complex with an acid liable subunit (ALS),

thus determining the bioavailability of IGF-I to tissues.

In addition to its function as a high-affinity binding

protein for IGF-I, IGFBP-3 has been shown to be a

growth-inhibitory, apoptosis-inducing molecule that

can act via IGF-dependent and IGF-independent

mechanisms (Mohan and Baylink 2002; Ali et al.

Electronic supplementary material The online version ofthis article (doi:10.1007/s11248-009-9286-8) containssupplementary material, which is available to authorized users.

S. C. K. Cheung � J. C. N. Chan � P. C. Y. Tong (&)

Department of Medicine and Therapeutics, Prince

of Wales Hospital, The Chinese University of Hong Kong,

Shatin, Hong Kong SAR, China

e-mail: ptong@cuhk.edu.hk

S. S. M. Sun

Department of Biology, The Chinese University

of Hong Kong, Shatin, Hong Kong SAR, China

123

Transgenic Res (2009) 18:943–951

DOI 10.1007/s11248-009-9286-8

2003). In the absence of IGF-I, IGFBP-3 is able to

interact with a number of growth-inhibitory proteins

and agents, such as p53, retinoic acid, tumor necrosis

factor-a and transforming growth factor-b (Butt et al.

1999) and hence is a potential anti-tumor agent.

The synthesis of recombinant human proteins from

microbials has several limitations including high

equipment and production costs, and potential con-

tamination with pathogens. With the advance in

recombinant DNA technology, transgenic plant has

emerged as an attractive expression system for large-

scale production of industrial enzymes and pharma-

ceutical proteins (Goddijn and Pen 1995; Daniell

et al. 2001). Unlike many organisms, plant can be

engineered to accept and express genetic information

from a wide range of prokaryotic and eukaryotic

sources (Walden and Wingender 1995). Plant can be

grown easily and inexpensively in large quantities

with the available agronomic infrastructures. The

recombinant proteins produced are correctly folded,

as the post-translational modifications systems of

plant and animal are very similar. Besides, safe

recombinant proteins can be obtained, since contam-

ination with human or animal pathogens does not

occur in plants (Fischer and Emans 2000). Recombi-

nant proteins can also be generated in plant seeds,

which can be stored for long time at ambient

temperature (Mett et al. 2008). In this study, we

demonstrated the use of tobacco seeds as bioreactor

platform to produce rhIGFBP-3. In an attempt to

enhance protein expression of rhIGFBP-3 in tobacco,

the codon of hIGFBP-3 was modified to plant-

optimized sequence. Two protein sorting signals,

including the signal peptide (SP) and the C-terminal

tetrapeptide AFVY sequences from phaseolin were

also added to the expression constructs in order to

direct rhIGFBP-3 to protein storage vacuole (PSV)

for stable accumulation. We hypothesized that by

adopting these strategies, rhIGFBP-3 could be pro-

duced in transgenic tobacco and its expression level

would be greatly enhanced.

Materials and methods

Plant materials and growth conditions

Wild type tobacco (Nicotiana tabacum cv. Xanthi)

seeds were surface sterilized in 50% Clorox (5.25%

sodium hypochlorite) and 0.04% Tween-20 for

10 min. The seeds were rinsed with sterilized distilled

water for several times to remove the detergent and

allowed to germinate on Murashige and Skoog (MS)

medium (42.4 g/l MS basal medium (Sigma, MS-

9274), pH5.7) at 25�C with a 16-h photoperiod.

Tobacco leaves about one-month old were used for

Agrobacterium-mediated transformation.

Chimeric genes construction

The plasmid IGFBP-3 (pIGFBP-3) containing hI-

GFBP-3 coding sequence was modified to optimize

its expression in plants (MWG Biotechnology Com-

pany), with 14.8% change of codon (Supplemen-

tary Fig. 1). The plant codon-optimized hIGFBP-3

sequence was excised from pIGFBP-3 using AccI and

subcloned into vector pTZ/Phas (Yu 2001), resulting in

construct B, pTZ/Phas/IGFBP-3, which contained

phaseolin promoter (Phaspro) and terminator (Phaster)

flanking the hIGFBP-3 cDNA. It is used for cytosolic

expression of rhIGFBP-3 in tobacco seeds. To produce

rhIGFBP-3 in apoplast, phaseolin SP was included in

the protein targeting constructs. The modified hI-

GFBP-3 coding sequence was first amplified to intro-

duce a 50 NdeI and 30 AccI restriction sites by

polymerase chain reaction (PCR). PCR was performed

using primers LeftBP-3,50-CAT ATG CCA CCG GAG

CTA GCT CTG GAG GTT T-30 and ACCBP-3,50-GAA GTA TAC TCA CTT GCT CTG C-30. A 25 ll

reaction mix containing 100 ng of DNA template, 19

Pfu buffer (Strategene), 2 mM MgCl2, 0.2 mM

dNTPs, 1 lM 50 primer, 1 lM 30 primer, 1.25 units

Pfu DNA polymerase (Strategene) was prepared. The

PCR conditions were as follows: 94�C for 5 min, then

25 cycles of 94�C for 30 s, 53�C for 1 min and 72�C

for 1 min, followed by 1 cycle of 72�C for 7 min. The

PCR products were digested with NdeI and AccI, and

then subcloned into pTZ/Phas/SP/MP42 (Lau 2003) to

form construct SB, pTZ/Phas/SP::IGFBP-3, with

phaseolin promoter and SP, modified hIGFBP-3 cod-

ing sequence and phaseolin terminator sequence. The

targeting construct with AFVY was synthesized as the

procedure described above except for the primers used.

Primers LeftBP-3 and RightBP-3,50-GTA TAC AAA

TGC CTT GCT CTG CAG GCT GTA GCA-30, were

used to introduce a 50 NdeI site and 30 AFVY targeting

sequence (AccI site is the ‘‘VY’’) to the hIGFBP-3

fragment, leading to construct SBA, pTZ/Phas/

944 Transgenic Res (2009) 18:943–951

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SP::IGFBP-3::AFVY. All chimeric constructs were

then cut with HindIII to release the gene expression

cassettes and ligated into the Agrobacterium binary

vector pBI 121 (Clonetech), which contains the

neomycin phosphotransferase II (NPTII) antibiotic

selectable marker and b-glucuronidase (GUS) protein

color reporter genes (Supplementary Fig. 2). The

identities of the cloned fragments were verified by

DNA sequencing.

Plant transformation and selection

All chimeric genes in pBI 121 expression vectors were

transformed into Agrobacterium tumefaciens

LBA4404/pAL4404 by electroporation (Sambrook

et al. 1989). For tobacco transformation, wild type

tobacco leaves were cut into small pieces and

submerged in Agrobacterium culture using the standard

protocol (Horsch et al. 1985). After co-cultivation, the

putative transformants were selected on MS medium

containing 500 mg/l carbenicillin (Sigma) and 100 mg/l

kanamycin (Sigma). Regenerated tobacco plants were

finally transferred to soil and grown in greenhouse for

maturity.

Southern blot analysis

Leaf genomic DNA of T0 transgenic tobacco plant was

extracted by the Cetyltrimethylammonium bromide

(CTAB) method (Doyle et al. 1990). Fifteen micro-

gram of genomic DNA was digested overnight with

BamHI, separated on 0.8% agarose gel and transferred

to positively charged nylon membrane (Roche) using

the VacuGeneXL Vacuum blotting System (Pharmacia

Biotech). Hybridization and detection were carried out

according to the method described in the DIG Nucleic

Acid Detection Kit (Roche). Double strand DIG-

labeled DNA probes (IGFBP-3) were prepared by

amplifying the whole IGFBP-3 cDNA using DIG DNA

labeling Kit (Roche) and heated to denature at 99�C

before use.

Western blot analysis

Seed total soluble protein was extracted from mature

seed (20 mg) of T0 transgenic tobacco plants by

grinding in 200 ll of extraction buffer (7.75% SDS,

0.125 M Tris-HCl, pH7.0, 10% b-mercaptoethanol

(b-mer) and one tablet per 50 ml of protease inhibitor

cocktail (Roche)). The quantity of seed total soluble

protein was determined by the bicinochoninic acid

(BCA) method and bovine serum albumin (BSA) was

used as standard. For western blot, total soluble

protein from 1 mg of seed was resolved in 10% SDS

polyacrylamide gels and electrophoretically trans-

ferred onto PVDF membrane (Bio-Rad) using Towbin

buffer (48 mM Tris, 39 mM Glycine and 20% meth-

anol). After blocking in blocking solution (ICN), the

blot was incubated in primary polyclonal antibody

(anti-human IGFBP-3 from Santa Cruz) at 1:200

dilution and then in secondary AP-conjugated anti-

rabbit antibody (Bio-Rad) at 1:3,000 dilution. Finally

the blot was subjected to non-radioactive detection

with chemiluminescent StarlightTM Substrate (ICN) as

described in the manual of AuroraTM western blot

chemiluminescent detection system (ICN).

Confocal immunofluorescence study

Mature seeds from T0 transgenic tobacco and wild-

type plants were first fixed in FAA (50% ethanol, 10%

formaldehyde, 5% acetic acid) for 24 h at room

temperature separately. Samples were then dehy-

drated with automated Enclosed Tissue Processor

(ETP) as described in the user manual and embedded

with paraffin wax. The embedded samples were

sectioned (6 lm), followed by paraffin removal using

xylene and then rehydration. The labeling and anal-

ysis by confocal immunofluorescence have been

described previously (Jiang et al. 2000). The pre-

treated sections were blocked in 5% BSA blocking

solution. Afterwards, the sections were incubated with

rabbit polyclonal anti-a-TIP antibody (provided by

Prof. LW Jiang, CUHK) at 4 lg/ml for 5 h, then with

the same concentration of mouse monoclonal anti-

IGFBP-3 (Oncogene Research Products) overnight.

After the primary antibodies incubation, the sections

were incubated with rhodamine-conjugated anti-

rabbit antibody and FITC-conjugated anti-mouse

antibody (both diluted to 1:100, Jackson ImmunoRe-

search Laboratory Inc.). The sections were covered

with cover slip and mounted with mowiol for

fluorescence images taking using a Bio-Rad Radiance

2100 system with the LaserShape2000 software (Bio-

Rad). A FITC/rhodamine software program was used

to collect images under conditions where no crossover

between FITC and rhodamine emissions occurred and

Transgenic Res (2009) 18:943–951 945

123

the two images were collected sequentially from the

same optical section. The FITC images were pseudo-

colored in green whereas the rhodamine images were

pseudocolored in red.

Results

Transgenic integration of modified hIGFBP-3

in tobacco genome

The three chimeric constructs were transferred into

tobacco plants through A. tumefaciens transforma-

tion. Genomic DNA from transgenic tobacco leaves

was isolated, digested with BamHI and used for

Southern blot analysis. As BamHI cuts only once on

the pBI 121 vector but not the transgenes, the copy

number of the transgenes integration could be

estimated. While most of the transformants contained

1–2 copies of the transgenes (Supplementary Fig. 3),

no observable signal was detected in wild type plant.

These results confirmed the presence and integration

of the transgenes in tobacco genome.

Expression of rhIGFBP-3 in transgenic

tobacco seeds

Protein extracts from transgenic tobacco seeds were

analysed by western blot detection. Transformants

harboring the targeting constructs SB (Fig. 1b, SB1, 4

and 9) and SBA (Fig. 1b, SBA1, 2 and 3), showed

‘‘positive signals’’ on chemiluminescent detection.

No protein expression was found in transformant B

(Fig. 1a, B1 and 3), which was supposed to have

rhIGFBP-3 expressed in cytosol.

The expression levels of rhIGFBP-3 in transfor-

mants SB and SBA were compared. Total soluble

protein was extracted with 200 ll SDS-Tris buffer

from 20 mg SB and SBA seeds. Commercial rhI-

GFBP-3 protein was used as standard to estimate the

amount of plant-produced rhIGFBP-3 in these trans-

formants. As shown in Fig. 2, the expression level of

rhIGFBP-3 was about three times higher in SBA than

that in SB. When comparing the western blot signals

from SBA and commercial rhIGFBP-3, the maximum

amount of rhIGFBP-3 in SBA was estimated to be

800 lg/g dry weight.

Fig. 1 Expression of rhIGFBP-3 in transgenic tobacco seeds.

Total protein was extracted from 1 mg of tobacco seeds,

resolved in 10% Tricine SDS–PAGE and blotted on PVDF

membrane. Western blot analysis using anti-human IGFBP-3

polyclonal antibody (Santa Cruz) was carried out. Commercial

rhIGFBP-3 protein (125 ng; Gropep) was used as the positive

control. a Lane 1: positive control (?ve)—commercial

rhIGFBP-3 protein; lane 2: wild type tobacco (wt); lanes 3and 4: pBI/Phas/IGFBP-3 transformants B1 and 3. b Lanes 1and 6: positive control (?ve)—commercial rhIGFBP-3 protein;

lanes 2–4: pBI/Phas/SP::IGFBP-3 transformants SB1, 4 and 9;

lane 5: wild type tobacco (wt); lanes 7–9: pBI/Phas/SP::IG-

FBP-3::AFVY transformants SBA1, 2 and 3; lane 10: Precision

plus protein dual color standards (Bio-Rad)

946 Transgenic Res (2009) 18:943–951

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Confocal immunofluorescence study

The effect of protein sorting sequence AFVY on

subcellular localization of the plant-produced rhI-

GFBP-3 was investigated by confocal immunofluo-

rescence microscopy. Transgenic tobacco seeds from

T0 SB and SBA plants were used. To detect whether

AFVY could direct rhIGFBP-3 to PSV, double

labeling using both anti-IGFBP-3 and anti-a-TIP

was performed. a-TIP is a membrane protein that is

present on PSV tonoplast and could served as a

marker for this sub-cellular compartment (Jauh et al.

1999). The green signal represented the location of

rhIGFBP-3 while the red one represented the mem-

brane of PSV in the seed endosperm. For construct

SBA with AFVY, signal from anti-IGFBP-3 was

superimposed with the signal from anti-a-TIP, as

illustrated by the yellow color in Fig. 3a and c. This

finding suggested that rhIGFBP-3 colocalized with

the membrane of PSV in the endosperm of transgenic

tobacco seeds. In contrast, for construct SB without

AFVY, rhIGFBP-3 was found diffusely in PSV

(Fig. 3e, g).

Discussion

In this study, different chimeric gene constructs were

designed to drive the expression of plant-optimized

hIGFBP-3 cDNA and introduced into tobacco plants

via Agrobacterium-mediated transformation. All

transgenic plants (B, SB and SBA) showed positive

Fig. 2 Comparison between the expression levels of SBA1

and SB1 in transgenic tobacco seeds. Total protein was

extracted with 200 ll SDS-Tris buffer from 20 mg tobacco

mature seeds of the transformants SBA1 and SB1. Different

amounts of total protein were resolved in 10% Tricine SDS–

PAGE and blotted on PVDF membrane. Western blot analysis

using anti-human IGFBP-3 polyclonal antibody (Santa Cruz)

was carried out. Commercial rhIGFBP-3 protein (Gropep) was

used as standard to estimate the amount of rhIGFBP-3 in these

transformants. Lanes 1–3: pBI/Phas/SP::IGFBP-3::AFVY

transformants SBA1 2, 10 and 15 ll; lanes 4–6: pBI/Phas/

SP::IGFBP-3 transformants SB1 2, 10 and 15 ll; lane 7:

Precision Plus Protein Dual Color Standards (Bio-Rad); lanes8–10: commercial rhIGFBP-3 protein 0.1, 0.5 and 1 lg

Fig. 3 Confocal immunofluorescence labeling of rhIGFBP-3

with (SBA) or without (SB) AFVY in the endosperm of

transgenic tobacco seeds. Recombinant hIGFBP-3 was

detected by FITC-conjugated anti-human IGFBP-3 antibody

and was stained green; while the membrane of PSV was

detected by rhodamine-conjugated anti-a-TIP antibody (a, c, e,

g) and was appeared in red. All images were obtained using the

same photomultiplier gain and offset settings. Panels b, d, f and

h are corresponding differential interference contrast images.

Panels c, d and g, h are corresponding enlarged images (39) of

Panels a, b and e, f, respectively. Bars, 15 lm

Transgenic Res (2009) 18:943–951 947

123

results confirming the integration of the transgenes

into tobacco genome by Southern blot analysis.

Protein expression was only found in transformants

harboring the protein targeting constructs, SB and

SBA, as illustrated in western blot analysis. Confocal

immunofluorescence study indicated that SBA pro-

tein was accumulated in the membrane of PSV

present in seed endosperm, while SB protein was

found diffusely in PSV.

Codon modification of hIGFBP-3 cDNA

The codon of hIGFBP-3 was plant-optimized in an

attempt to increase its expression in tobacco. Earlier

studies have shown that codon biases play an

important role in gene expression levels. The optimal

codons would enhance translation accuracy and

efficiency of heterogeneous expression (Marais and

Duret 2001). One example on enhanced gene expres-

sion in plants by changing the rare codons to more

typical ones is the green fluorescent protein (GFP)

from Aequorea victoria. By increasing the G?C

content of the gene and removing the cryptic introns

and potential polyadenylation sites, full-size GFP was

found in transgenic tobacco while only small and

truncated fragments of wild-type GFP were observed

in transgenic plants harboring the unmodified gene

(Rouwendal et al. 1997). Based on the assumption

that the more frequently used codons have a greater

proportion of tRNAs and hence better translation,

these frequently used codons are thus more preferred

in that organism. As hIGFBP-3 cDNA is originated

from human, its difference in codon usage from

plants might result in poor expression. The codon of

hIGFBP-3 cDNA was modified based on the pre-

ferred codons in two plant seed storage proteins,

lysine-rich protein (LRP) from winged bean and

methionine-rich 2S albumin (PN2S) from Paradise

nut. They were chosen as the basis for modification

because their high expression and stable accumula-

tion, 3–10 and 3–15% of total extractable seed

proteins for LRP and PN2S, respectively, in trans-

genic Arabidopsis was observed in previous studies

(Cheng 1999; Chen 2000).

Phaseolin protein sorting signals

Owing to post-synthesis and/or post-secretion instabil-

ity and degradation, the yields of most pharmaceutical

proteins produced in plants are usually low, at levels

between 0.01 and 0.1% total soluble protein (Doran

2006). With the addition of appropriate protein sorting

determinants, the expression levels of recombinant

proteins can be accumulated up to 100-fold (Bencha-

bane et al. 2008). Targeting recombinant proteins to

specific compartments is thus the most efficient way to

enhance protein accumulation. Signal peptide has been

employed to deliver the target protein to the secretory

pathway, including the endoplasmic reticulum (ER),

the Golgi complex and hydrolytic vacuoles, or to

secretion from the cell. It has been found that secretory

proteins could be accumulated to high level than those

expressed in cytosol (Schillberg et al. 1999). In the

present study, phaseolin SP and its tetrapepide AFVY

were used to drive the expression of rhIGFBP-3 in

transgenic tobacco seeds. As shown in Fig. 1, western

blot results demonstrated that rhIGFBP-3 was suc-

cessfully expressed in the presence of phaseolin SP

(transformants SB and SBA), while no protein expres-

sion was found in the transformant carrying hIGFBP-3

cDNA alone (transformant B), though transcript was

detected in the northern blot (data not shown). Without

SP, rhIGFBP-3 is presumed to enter the default

pathway and delivered to the cytoplasm, where it

may be susceptible to degradation by the proteolytic

enzymes. On the other hand, rhIGFBP-3 is directed by

SP to the secretory pathway via endoplasmic reticulum

(ER) (Barrieu and Chrispeels 1999). It is then trans-

ported downstream the pathway by a ‘bulk flow’

process and finally delivered to different destinations

by additional protein sorting signals.

Having slightly acidic or neutral environment,

protein storage vacuole (PSV) is believed to be an

ideal place for protein storage (Robinson et al.

2005). Protein accumulation levels of recombinant

proteins in PSV can be amounted to 20,000-fold

when compared to those expressed in cytosol

(Benchabane et al. 2008). The hydrophobic tetra-

peptide AFVY (Ala, Phe, Val and Tyr) of phaseolin

was the C-terminal protein sorting signal for the

delivery of rhIGFBP-3 from ER to PSV of trans-

genic tobacco seeds. According to Frigerio et al.

(1998), removal of AFVY allowed correct trimer

formation but caused phaseolin to be secreted into

apoplast instead of accumulated in PSV. The fact

that the AFVY tetrapeptide alone is sufficient for

sorting protein to the vacuole was further demon-

strated by fusing this tetrapeptide to a secreted

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version of GFP under the control of CaMV 35S

promoter (Frigerio et al. 2001). As shown in Fig. 1b,

rhIGFBP-3 could be detected in both transformants

harboring the constructs with (SBA) or without (SB)

AFVY. It is hypothesized that SBA construct would

target rhIGFBP-3 to PSV while SB might direct the

protein to the apoplast. Confocal immunofluores-

cence study proved that AFVY would target

rhIGFBP-3 to the membrane of PSV present in

seed endosperm, as illustrated in Fig. 3a and c.

While for transformant SB, it was surprising to find

that rhIGFBP-3 was present diffusely in PSV

(Fig. 3e, g). Similarly unexpected results were also

found in other recombinant proteins expressed in

plant seeds, for instance, an antigenic glycoprotein

from human cytomegalovirus carrying a SP only

was found in PSV of tobacco seed endosperm

(Wright et al. 2001). The same phenomenon

appeared in transgenic cereals as well. The recom-

binant phytase, which was expected to be secreted

to the apoplast, was found accumulated in the

prolamin vacuoles of transgenic wheat and rice

endosperm instead (Arcalis et al. 2004; Drakakaki

et al. 2006). These data suggested that protein

sorting may be cell- or tissue-specific, especially in

storage tissues such as seed endosperm (Abranches

et al. 2008). The storage function of the seed tissue

may be able to override the default pathway of

being secreted to the apoplast in leaf cells. On the

other hand, with the tetrapeptide AFVY, the protein

can clearly be directed to the membrane of PSV.

Also, detergent like SDS or TritonX-100 was

required when extracting rhIGFBP-3 with AFVY

(data not shown), suggesting that rhIGFBP-3 with

AFVY is membrane-associated. A study using

transgenic tobacco leaves confirmed that the asso-

ciation of AFVY with phaseolin has high affinity

with membranes, whereas mutated phaseolin

deprived of AFVY does not undergo membrane

association (Castelli and Vitale 2005). When com-

paring the expression level of rhIGFBP-3, trans-

formant SBA was 3 times higher than that of SB

(Fig. 2). These results indicated that protein trapped

in the membrane of PSV leads to high content in the

seeds, thus providing a suitable location for protein

accumulation. As mentioned before, the rhIGFBP-3

expression in SBA1 amounted to 800 lg/g dry

weight.

Molecular farming and limitations

Molecular farming is the production of pharmaceu-

tically important and commercially valuable proteins

in plants at economic price, with an aim to address

the growing medical needs worldwide (Franken et al.

1997). Recent research and development in the plant-

made pharmaceuticals has been focused on agricul-

tural crops, including maize, rice and potato. About

eighteen plant-made veterinary and pharmaceutical

proteins are reported to be in clinical trials and

several of them are approaching market stage,

including maize-produced gastric lipase for cystic

fibrosis, carrot-produced human glucocerebrosidase

for Gaucher’s disease and safflower-produced insulin

for diabetes (Spok et al. 2008). Tobacco has been

used commonly to produce pharmaceuticals such as

antibodies to treat carries prophylaxis and cancer

vaccine (Spok et al. 2008). Although it is a well-

known expression host in biopharmaceutical industry,

its high content of nicotine and other toxic alkaloids

is a disadvantage (Fischer et al. 2004). During

purification, the toxic substances must be removed

completely, which contributes to higher production

cost. With this proof-of-concept study, however, the

expression technology could be extended to other

suitable plants, such as rice for rhIGFBP-3 protein

production. Rice grains not only contain no noxious

chemicals, but also have low allergenicity in human

(Stoger et al. 2000). Owing to its edible nature,

transgenic rice grains may be developed as seed pills

for direct oral delivery. In this connection, the costly

purification processes can be avoided. Yang et al.

(2006) reported that oral administration of rice-

produced anti-hypertensive peptide (RPLKPW) could

reduce systolic blood pressure in rats. Takagi et al.

(2005) also demonstrated that the rice-produced T

cell epitope peptides could induce oral tolerance

against pollen allergen-specific responses in trans-

genic mice through oral delivery. Given the problem

of gastrointestinal degradation is addressed, the usage

of ‘‘seed pills’’ should be an important implication

waiting to be developed in future. To our knowledge,

this is the first report of producing rhIGFBP-3 in

transgenic plants and with high yield.

Acknowledgments We would like to express our sincere

thanks to Prof. L. W. Jiang, Dept. of Biology, CUHK for

providing the anti-a-Tip antibody for confocal immunofluo-

Transgenic Res (2009) 18:943–951 949

123

rescence study. Financial support for this research was provided

by CUHK-Direct Grant (2041134), RGC grant (CUHK 4580/

05 M) and AoE grant (AoE/B-07/99) from the University Grants

Committee of the Hong Kong Special Administrative Region,

China.

References

Abranches R, Arcalis E, Marcel S, Altmann F, Ribeiro-Pedro

M, Rodriguez J, Stoger E (2008) Functional specialization

of Medicago truncatula leaves and seeds does not affect

the subcellular localization of a recombinant protein.

Planta 227:649–658. doi:10.1007/s00425-007-0647-3

Ali O, Cohen P, Lee KW (2003) Epidemiology and biology of

insulin-like growth factor binding protein-3 (IGFBP-3) as

an anti-cancer molecule. Horm Metab Res 35:726–733.

doi:10.1055/s-2004-814146

Arcalis E, Marcel S, Altmann F, Kolarich D, Drakakaki G,

Fisher R, Christou P, Stoger E (2004) Unexpected depo-

sition patterns of recombinant proteins in post-endoplas-

mic reticulum compartments of wheat endosperm. Plant

Physiol 136:3457–3466. doi:10.1104/pp.104.050153

Barrieu F, Chrispeels MJ (1999) Delivery of a secreted soluble

protein to the vacuole via a membrane anchor. Plant Physiol

120:961–968. doi:10.1104/pp.120.4.961

Benchabane M, Goulet C, Rivard D, Faye L, Gomord V,

Michaud D (2008) Preventing unintended proteolysis in

plant protein biofactories. Plant Biotechnol J 6:633–648.

doi:10.1111/j.1467-7652.2008.00344.x

Butt A, Firth SM, Baxter RC (1999) The IGF axis and pro-

grammed cell death. Immunol Cell Biol 77:256–262. doi:

10.1046/j.1440-1711.1999.00822.x

Castelli S, Vitale A (2005) The phaseolin vacuolar sorting

signal promotes transient, strong membrane association

and aggregation of the bean storage protein in transgenic

tobacco. J Exp Bot 56:1379–1387. doi:10.1093/jxb/eri139

Chen L (2000) Transgenic manipulation of aspartate family

amino acid biosynthetic pathway in higher plants for

improved plant nutrition. Doctoral Thesis, The Chinese

University of Hong Kong, Hong Kong

Cheng MK (1999) Transgenic expression of a chimeric gene

encoding a lysine-rich protein in Arabidopsis. Master

Thesis, The Chinese University of Hong Kong, Hong Kong

Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular

farming: production of antibodies, biopharmaceuticals

and edible vaccines in plants. Trends Plant Sci 6:219–226.

doi:10.1016/S1360-1385(01)01922-7

Doran MP (2006) Foreign protein degradation and instability in

plants and plant tissue cultures. Trends Biotechnol

24:426–432. doi:10.1016/j.tibtech.2006.06.012

Doyle JD, Doyle JL, Bailey LH (1990) Isolation of plant DNA

from fresh tissue. Focus 12:13–15

Drakakaki G, Marcel S, Arcalis E, Altmann F, Gonzalez-Melendi

P, Fisher R, Christou P, Stoger E (2006) The intracellular fate

of a recombinant protein is tissue dependent. Plant Physiol

141:578–586. doi:10.1104/pp.106.076661

Fischer R, Emans N (2000) Molecular farming of pharma-

ceutical proteins. Transgenic Res 9:279–299. doi:10.1023/

A:1008975123362

Fischer R, Stoger E, Schillberg S, Christou P, Twyman RM

(2004) Plant-based production of biopharmaceuticals.

Curr Opin Plant Biol 7:152–158. doi:10.1016/j.pbi.

2004.01.007

Franken E, Teuschel U, Hain R (1997) Recombinant proteins

from transgenic plants. Curr Opin Biotechnol 8:411–416.

doi:10.1016/S0958-1669(97)80061-4

Frigerio L, De Virgilio M, Prada A, Faoro F, Vitale A (1998)

Sorting of phaseolin to the vacuole is saturable and requires

a short C-terminal peptide. Plant Cell 10:1031–1042

Frigerio L, Foresti O, Felipe DH, Neuhaus JM, Vitale A (2001)

The C-terminal tetrapeptide of phaseolin is sufficient to

target green fluorescent protein to the vacuole. J Plant

Physiol 158:499–503. doi:10.1078/0176-1617-00362

Goddijn OJ, Pen J (1995) Plants as bioreactors. Trends Bio-

technol 13:379–387. doi:10.1016/S0167-7799(00)88985-4

Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley

RT (1985) A simple and general method for transferring genes

into plants. Science 227:1229–1231. doi:10.1126/science.227.

4691.1229

Jauh GY, Phillips TE, Rogers JC (1999) Tonoplast intrinsic

protein isoforms as markers for vacuolar functions. Plant

Cell 11:1867–1882

Jiang L, Phillips TE, Rogers SW, Roger JC (2000) Biogenesis

of the protein storage vacuole crystalloid. J Cell Biol

150:755–770. doi:10.1083/jcb.150.4.755

Lau OS (2003) Transgenic expression of the Malaria surface

antigens, MSP142 and MSP119, in plant seeds. Master

Thesis, The Chinese University of Hong Kong, Hong Kong

Marais G, Duret L (2001) Synonymous codon usage, accuracy

of translation, and gene length in Caenorhabditis elegans.

J Mol Evol 52:275–280

Martin JL, Baxter RC (1986) Insulin-like growth factor binding

protein from human plasma. Purification and character-

ization. J Biol Chem 261:8754–8760

Mett V, Farrance CE, Green BJ, Yusibov V (2008) Plants as bio-

factories. Biologicals 36:354–358. doi:10.1016/j.biologicals.

2008.09.001

Mohan S, Baylink DJ (2002) IGF-binding proteins are multi-

functional and act via IGF-dependent and–independent

mechanisms. J Endocrinol 175:19–31. doi:10.1677/joe.0.

1750019

Robinson DG, Oliviusson P, Hinz G (2005) Protein sorting to

the storage vacuoles of plants: a critical appraisal. Traffic

6:615–625. doi:10.1111/j.1600-0854.2005.00303.x

Rouwendal GJ, Mendes O, Wolbert EJ, Douwe de Boer A

(1997) Enhanced expression in tobacco of the gene

encoding green fluorescent protein by modification of its

codon usage. Plant Mol Biol 33:989–999. doi:10.1023/

A:1005740823703

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning:

a laboratory manual, 2nd edn. Cold Spring Harbor Lab-

oratory Press, Cold Spring Harbor

Schillberg S, Zimmermann S, Voss A, Fischer R (1999)

Apoplastic and cytosolic expression of full-size antibodies

and antibody fragments in Nicotiana tobacum. Transgenic

Res 8:255–263. doi:10.1023/A:1008937011213

Spok A, Twyman RM, Fischer R, Ma JK, Sparrow PA (2008)

Evolution of a regulatory framework for pharmaceuticals

derived from genetically modified plants. Trends Bio-

technol 26:506–517. doi:10.1016/j.tibtech.2008.05.007

950 Transgenic Res (2009) 18:943–951

123

Stoger E, Vaquero C, Torres E, Sack M, Nicholson L, Drossard J,

Williams S, Keen D, Perrin Y, Christou P, Fischer R (2000)

Cereal crops as viable production and storage systems for

pharmaceutical ssFv antibodies. Plant Mol Biol 42:583–590.

doi:10.1023/A:1006301519427

Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y, Takamura K, Ish-

imitsu R, Kawauchi H, Kiyono H, Takaiwa F (2005) A rice-

based edible vaccine expressing multiple T cell epitopes

induces oral tolerance for inhibition of Th2-mediated IgE

responses. Proc Natl Acad Sci USA 102:17525–17530. doi:

10.1073/pnas.0503428102

Walden R, Wingender R (1995) Gene-transfer and plant

regeneration techniques. Trends Biotechnol 13:324–331.

doi:10.1016/S0167-7799(00)88976-3

Wood WI, Cachianes G, Henzel WJ, Winslow GA, Spencer SA,

Hellmiss R, Martin JL, Baxter RC (1988) Cloning and

expression of the growth hormone-dependent insulin-like

growth factor-binding protein. Mol Endocrinol 2:1176–

1185. doi:10.1210/mend-2-12-1176

Wright KE, Prior F, Sardana R, Altosaar I, Dudani AK, Ganz

PR, Tackaberry ES (2001) Sorting of glycoprotein B from

human cytomegalovirus to protein storage vesicles in

seeds of transgenic tobacco. Transgenic Res 10:177–181.

doi:10.1023/A:1008912305913

Yang L, Tada Y, Yamamoto MP, Zhao H, Yoshikawa M, Tak-

aiwa F (2006) A transgenic rice seed accumulating an anti-

hypertensive peptide reduces the blood pressure of spon-

taneously hypertensive rats. FEBS Lett 580:3315–3320.

doi:10.1016/j.febslet.2006.04.092

Yu WS (2001) Plants as bioreactors: expression of toxoplasma

gondii surface antigen P30 in transgenic tobacco plants.

Master Thesis, The Chinese University of Hong Kong,

Hong Kong

Transgenic Res (2009) 18:943–951 951

123