Journal of Biotechnology 125 (2006) 159–172
Accumulation of hEGF and hEGF–fusion proteins in
chloroplast-transformed tobacco plants is higher
in the dark than in the light
Sonia Wirth, Maria Eugenia Segretin, Alejandro Mentaberry,
Fernando Bravo-Almonacid ∗
Instituto de Investigaciones en Ingenierı́a Genética y Biologı́a Molecular, INGEBI-CONICET, and FCEN-UBA,
Vuelta de Obligado 2490, 1428 Buenos Aires, Argentina
Received 7 October 2005; received in revised form 27 January 2006; accepted 17 February 2006
Abstract
Chloroplast transformation has many potential advantages for the production of recombinant proteins in plants. However, it
has been reported that heterologous protein accumulation in chloroplasts could be hindered by post-transcriptional mechanisms
not yet characterized. Here, we describe the development and characterization of transplastomic tobacco plants transformed with
four different transformation vectors for the expression of human epidermal growth factor (hEGF). We showed that, although
the corresponding transcript was present in all of the analyzed plants, hEGF could only be detected when fused to the first 186
amino acids of bacterial ␤-glucuronidase (GUS). In addition, we observed that the expression levels of recombinant protein
increased when plants were placed in the dark or when leaves were incubated in the presence of electron transport inhibitors,
such as methyl viologen (MV) and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These results suggest that the mechanism
responsible for hEGF instability in chloroplasts is regulated by light.
© 2006 Elsevier B.V. All rights reserved.
Keywords: hEGF; Chloroplast transformation; Transplastomic tobacco; Molecular farming; Fusion protein
1. Introduction
Chloroplast transformation has many advantages for
the production of recombinant proteins as compared
∗ Corresponding author. Tel.: +54 11 4783 2871;
fax: +54 11 4786 8578.
E-mail address: [email protected] (F. Bravo-Almonacid).
0168-1656/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2006.02.012
with other expression systems in plants. Transgene
integration is accomplished by homologous recombination into a defined region of the chloroplast genome.
As a consequence, the positional effects that can concern transgene expression in nuclear transformation
are not observed. On the other hand, photosynthetic
cells contain hundreds of polyploid chloroplasts and
therefore, the integrated transgene can be present in
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thousands of copies per cell. As a result, and since
gene silencing has not been reported in plastids, high
expression levels of the heterologous protein can be
potentially achieved in transplastomic plants (Bock and
Khan, 2004; Maliga, 2004).
A further advantage of chloroplast transformation
is the possibility of inserting multiple genes in a
single transformation round as a polycistronic array.
This strategy was used to express the B. thuringiensis
cry2Aa2 toxin operon in tobacco chloroplasts, leading to an outstanding accumulation of the recombinant
protein (45% of total proteins; De Cosa et al., 2001).
Finally, in most plant species plastid DNA is exclusively maternal-inherited resulting in significant transgene containment since it is not found in the pollen
(Zhang et al., 2003).
Examples as the expression of human serum albumin (Fernández-San Millán et al., 2003), cholera toxin
subunit B (Daniell et al., 2001) and tetanus toxin
fragment C (Tregoning et al., 2003) demonstrate that
chloroplasts could be a suitable system for the production of biopharmaceuticals. However, in some cases
such as in those of human somatotropin (Staub et al.,
2000), 5-enolpyruvylshikimate-3-phosphate synthase
(Ye et al., 2001) and gamma-interferon (Leelavathi
and Reddy, 2003), high levels of expression were only
obtained when the candidate products were synthesized as fusion proteins. As no differences in mRNA
accumulation were observed in comparison with nonfused proteins, it was suggested that the fusion could
be acting by protecting the recombinant protein against
degradation. Moreover, two recent reports showed that
though high levels of transcripts were detected, neither
the human hemoglobin nor the maize ␤-zein accumulated in transgenic tobacco chloroplasts (Magee et al.,
2004; Bellucci et al., 2005).
A better understanding of the post-transcriptional
mechanisms involved in chloroplast genetic regulation,
would allow the design of strategies to increase heterologous protein accumulation. In this work, we obtained
transplastomic tobacco plants transformed with four
different transformation vectors for the expression of
mature human epidermal growth factor (hEGF), a
6.2 kDa non-glycosylated polypeptide involved mainly
in the maintenance and repairing of epithelial and epidermal tissues (Berlanga Acosta and Mella Lizama,
1998). Albeit we detected the specific mRNAs in all
of the transplastomic plants analyzed, we found that
hEGF accumulates only when fused to an N-terminal
fragment of Escherichia coli ␤-glucuronidase (GUS).
We also found that recombinant protein accumulation increased when the transplastomic plants were
placed in the dark or when leaves were incubated in the
presence of electron transport chain inhibitors. These
results strongly suggest a connection between the stability of recombinant hEGF and the state of chloroplast
photosynthetic activity.
2. Materials and methods
2.1. Transformation vectors
Four transformation vectors were designed to
express hEGF and hEGF–fusion proteins in tobacco
chloroplasts. For the construction of plasmid pBSWsdEGF the hegf sequence that codes for the mature
hEGF polypeptide, was digested from pETEGF (Wirth
et al., 2004) with enzymes Nde I and Xba I and
cloned downstream of the aadA sequence, that confers
spectinomycin resistance. In both the hegf and the
aadA sequences, a ribosome binding site containing
the Shine–Dalgarno-like element GGAGG (SD) was
inserted upstream of the initial ATG codon. This
di-cistron was cloned under the transcriptional control
of the rrn promoter (Prrn) and the rps16 termination
sequence (Trps16), which were obtained by PCR
amplification from isolated Nicotiana tabacum plastid
DNA, using primers 5 -TTTACTAGTTGGATTTGCTC-3 and 5 -TAGACACCGCGGATTCG-3 complementary to rrn promoter sequence and 5 -AATCTAGACTAATCAACCGA-3 and 5 -ACGGGATCCAATGGAA-GC-3 complementary to rps16 termination sequence. For targeting this transcriptional unit by
homologous recombination into the intergenic region
between rrn16 and trnI genes in the inverted repeat of
the tobacco plastome, the full construction was cloned
between a left flanking region (LFR) that includes
1169 bp of the 3 -region of rrn16 gene defined by positions 103235 (Sac II restriction site) and 104404 (Avr II
restriction site) of the N. tabacum plastome (GenBank
accession no. NC 001879); and a right flanking region
(RFR) that includes the 1015 bp containing the full trnI
sequence and the 5 -region of trnA defined by positions
104404 (Avr II restriction site) and 105419 (Sac I
restriction site) of the N. tabacum plastome (Fig. 1E).
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
161
Fig. 1. Chloroplast transformation vectors. (A) pBSW-sdEGF: the hegf sequence was cloned as part of a transcriptional unit with the aadA
sequence under the control of the Prrn promoter and the Trps16 termination sequence. In both, the hegf and aadA sequences a ribosome
binding (SD) was inserted upstream of the initial ATG codon. (B) pBSW-utrEGF was obtained by replacing the SD sequence upstream of hegf
in pBSW-sdEGF by the 5 -untranslated sequence and the promoter of the psbA gene (5 psbA) at Kpn I and Nde I sites. (C) pBSW-utrHISE
was obtained by replacing the hegf sequence in Nde I and Xba I sites of pBSW-utrEGF by the HISE sequence that encodes, in a continuous
frame, a tract of six histidines (6xHIS), the IDGR peptide recognized by factor Xa and the hegf coding sequence. The arrowhead indicates
the cleavage site for factor Xa. (D) pBSW-utrGEKE was obtained by replacing the hegf sequence in Nde I and Xba I sites of pBSW-utrEGF
by the GEKE sequence that encodes a continuous frame containing the first 186 amino acids of the E. coli ␤-glucuronidase (N-t GUS),
the peptide DDDDK, cleavage site for the enzyme enterokinase (ENK) and the hegf sequence. The arrowhead indicates the cleavage site
for the enterokinase. For simplicity, only the initial ATG of GUS and the amino acids close to the fusion region are shown in the detailed
sequences. The four constructions were cloned between a left flanking region (LFR, 1169 bp) that includes the sequence of the 3 -region
of rrn16 gene and a right flanking region (RFR, 1015 bp) containing the full trnI and 5 -region of trnA. (E) Chloroplast genomic sequence
at the insertion site (arrowhead) showing the LFR and RFR. rrn23: sequence coding the 23S rRNA. trnI/A: probe used in Southern blots
assays.
Plasmid pBSW-utrEGF is a derivative of pBSWsdEGF in which the ribosome binding site upstream of
hegf was replaced by the promoter and 5 -untranslated
region of psbA gene (5 psbA), at the Kpn I and
Nde I sites (Fig. 1B). The 5 psbA sequence (Eibl et
al., 1999) was obtained by PCR amplification from
tobacco plastid DNA, using oligonucleotides UTR5
(5 -TCGGTACCGAGCTCCGTATTTTTCC-3 ; Kpn I
site underlined) and UTR3 (5 -CTAAAATTGCAGTCATATGAAAATC-3 ; Nde I site underlined).
Plasmids pBSW-utrHISE and pBSW-utrGEKE
were obtained by replacing the hegf sequence between
the Nde I and Xba I sites of plasmid pBSW-utrEGF,
by the HISE and GEKE sequences, respectively. The
HISE sequence encodes, in a continuous frame, a
tract of six histidines, the IDGR peptide recognized
by factor Xa (HISXa) and the hegf coding sequence
(Fig. 1C).
Similarly, the GEKE sequence encodes a continuous
frame containing the first 186 amino acids of the E.
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coli ␤-glucuronidase, the DDDDK (cleavage site for
the enzyme enterokinase) and hegf sequence (Fig. 1D).
The plasmid pBSW-utrGUS-used as a control was
obtained by replacing the hegf sequence by the complete GUS coding sequence in pBSW-utrEGF (not
shown).
2.2. Chloroplast transformation
Chloroplast transformation was carried out as
previously described (Daniell, 1997), using the PDS
1000/He biolistic particle delivery system (Bio-Rad).
Fully expanded leaves of in vitro cultured N. tabacum
cv Petit Havana plants were bombarded with 50 mg
of 0.6 ␮m gold particles (Bio-Rad, USA) coated
with 10 ␮g of plasmid DNA using 1100 psi rupture
discs (Bio-Rad). Transformants were regenerated
in selective RMOP medium containing 500 mg l−1
spectinomycin dihydrochloride, and insertion of the
different transgenes was assessed by PCR amplification. To obtain homoplasmic plants, leaves from
PCR-positive shoots were cut into pieces and taken
through two to three additional regeneration cycles in
selective medium.
2.3. Southern blot analysis
Total DNA was extracted from leaves as described
by Dellaporta et al. (1983). The DNA (4 ␮g) was
digested with Nco I (New England Biolabs, USA),
electrophoresed in 0.8% agarose gels and blotted
onto Hybond N+ Nylon membranes (Amersham
Biosciences, USA). Specific DNA sequences were
detected by hybridization with 32 P-labeled hEGF or
trnI/A DNA probes generated by random priming with
a Prime-a-Gene kit (Promega, USA). Pre-hybridization
and hybridization were carried out at 65 ◦ C in Church’s
hybridization solution (Church and Gilbert, 1984) for 2
and 16 h, respectively. Membranes were washed twice
with gentle shaking for 30 min in 0.2× SSC, 0.1%
SDS at 65 ◦ C. For re-hybridization, membranes were
washed at 95 ◦ C with 0.5% SDS to remove probes.
2.4. Northern blot analysis
Total RNA was extracted from fully expanded
young leaves using TRiZOL Reagent (Invitrogen, USA). Four micrograms of formaldehyde-
denatured RNA were electrophoresed in a 1.5%
agarose/formaldehyde gel and blotted onto Hybond N+
Nylon membranes (Amersham Biosciences). Specific
mRNA sequences were detected by hybridization with
32 P-labeled hEGF or 18S rRNA DNA probes generated
by random priming with a Prime-a-Gene kit (Promega).
The blot was pre-hybridized, hybridized and washed as
described for Southern blot.
2.5. Gene expression in E. coli
Total proteins from E. coli DH5␣ cultures transformed with plasmids pBSW-sdEGF, pBSW-utrEGF,
pBSW-utrHISE, pBSW-utrGEKE and pBSW-utrGUS
were extracted in protein extraction buffer (50 mM
Tris–HCl, pH 6.8, 10 mM EDTA–Na2 , 1 mM PMSF,
0.5 ␮g ml−1 leupeptine, 2 ␮g ml−1 aprotinine, 0.1 mM
benzamidine and 2 ␮g ml−1 trypsine inhibitor), and
quantified by BCA protein assay (Pierce, USA).
Analysis of hEGF and fusion recombinant proteins
was performed by Western blot. Total protein from E.
coli extracts were separated by 16% Tris–tricine SDSPAGE in non-reducing conditions (pBSW-sdEGF,
pBSW-utrEGF and pBSW-utrHISE extracts) or by
12% Tris–glycine SDS-PAGE in reducing conditions
(pBSW-utrGUS and pBSW-utrGEKE extracts) and
blotted onto nitrocellulose membranes (Amersham
Biosciences). Blots were probed with 2 ␮g ml−1 of
monoclonal anti-hEGF antibody (R&D Systems Inc.,
USA) and detected by a chromogenic reaction using
5-bromo-4-chloro-3-indolyl phosphate and nitroblue
tetrazolium (Sigma Chemical Co., USA) as substrates
or ECL peroxidase detection system (Amersham
Biosciences).
2.6. Recombinant protein expression in
transplastomic plants
Total protein from transformed and nontransformed soil-grown plants was extracted from fully
expanded leaves using extraction buffer containing
a protease inhibitor cocktail (Sigma Co.) and 0.01%
Triton X-100, and quantified by the BCA protein assay
(Pierce). Expression of hEGF and fusion proteins
were analyzed by Western blot as described for E.
coli extracts. Quantification of hEGF was performed
by an ELISA assay using commercial anti-hEGF
antibodies (R&D Systems Inc.), and recombinant
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
hEGF as standard (Sigma Chemical Co.) as previously
described (Wirth et al., 2004).
163
taken after 2, 4 and 8 h of incubation and GEKE fusion
protein expression was analyzed by Western blot.
2.7. In vitro stability assays
3. Results
Chloroplasts were isolated from fully expanded
young leaves from greenhouse-grown N. tabacum cv
Xanthi D8 plants, essentially as described by Tewari
(1986) and Triboush et al. (1998). Briefly, 5 g of
leaves from plants previously placed in darkness for
48 h to reduce starch content were mechanically disrupted by chopping tissue in presence of 25 ml of
STM buffer (0.5 M sucrose, 5 mM MgCl2 and 50 mM
Tris–HCl, pH 8). This homogenate was cleared by
filtering through Miracloth (Calbiochem, USA) followed by centrifugation at 1000 × g for 10 min. The
pellet was then washed once with 4 ml of STM buffer
and resuspended in same buffer plus 2.5% (v/v) Triton X-100 for differential rupture of chloroplasts.
Lysed chloroplasts were separated from intact nuclei
by centrifugation at 5000 × g for 30 min. In vitro stability assays were performed by incubation of commercial recombinant hEGF (Sigma Chemical Co.) or
extracts from E. coli cells transformed with pBSWutrGEKE, with chloroplasts extracts (100 ␮g ml−1
chlorophyll) at 26 ◦ C under continuous illumination
(300 ␮mol quanta m−2 s−1 ) or in the dark. Samples
were taken at the beginning of the assay and after 1,
2 or 16 h and remaining hEGF or GEKE fusion protein were analyzed by ELISA or Western blot assays,
respectively. In assays containing ATP, this component was incorporated at a final concentration of 5 mM
before the addition of hEGF or GEKE fusion protein.
2.8. Protein expression analysis using electron
transfer inhibitors
Treatment with photosynthetic electron transfer
inhibitors was performed following a modification of the protocol described by Palatnik et al.
(1999). Leaf discs (1 cm in diameter) from pBSWutrGEKE-transformed homoplasmic plants grown
in vitro were incubated at 26 ◦ C in darkness or
under continuous light (300 ␮mol quanta m−2 s−1 ) in
a 0.05% Tween-20 solution or in same solution but
with the addition of 100 ␮M of methyl viologen
(MV) or 100 ␮M DCMU [3-(3,4-dichlorophenyl)-1,1dimethylurea; Sigma Chemical Co.]. Samples were
3.1. Chloroplast transformation
Four transformation vectors were designed for the
expression of hEGF and hEGF–fusion proteins in
tobacco chloroplasts. In plasmid pBSW-sdEGF the
hegf sequence comprises a single transcription unit
with the aadA sequence under the control of the Prrn
promoter and the Trps16 termination sequence. To
direct the recruitment of the plastidic small ribosomal
subunit, the Shine–Dalgarno-like element GGAGG
was placed 10 and 11 nucleotides upstream of the initial
ATG of aadA and hegf coding sequences, respectively,
according to the consensus observed for many plastidic
genes (Hirose and Sugiura, 2004). The entire transcriptional unit was then cloned between a left flanking
region and a right flanking region of 1169 and 1015
nucleotides in length, respectively, to direct the insertion by homologous recombination into the intergenic
region between rrn16 and trnI genes in the tobacco
plastome (Fig. 1A and E).
As it was demonstrated that the presence of some
plastidic 5 -untranslated sequences can increase the
transcript levels and translation efficiency of the
genes cloned downstream (Eibl et al., 1999), the
SD sequence upstream of hegf in plasmid pBSWsdEGF was replaced by the 5 -untranslated sequence
and the promoter of psbA gene (5 psbA), to obtain
the transformation vector pBSW-utrEGF (Fig. 1B).
Also, as recombinant protein expression in chloroplasts can be stabilized by fusion to different peptides
(Ye et al., 2001; Leelavathi and Reddy, 2003; Staub
et al., 2000), two other transformation vectors were
designed to test this strategy for hEGF production in
transplastomic plants. Thus, plasmids pBSW-utrHISE
and pBSW-utrGEKE were obtained by replacing the
hegf sequence in plasmid pBSW-utrEGF by sequences
HISE or GEKE for the expression of hEGF as a fusion
protein (Fig. 1C and D). The sequence HISE encodes
hEGF as a C-terminal fusion to a tract of six histidines
and the peptide recognized by factor Xa (Fig. 1C). Similarly, sequence GEKE, encodes hEGF as a C-terminal
fusion to the firsts 186 amino acids of GUS protein
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and the peptide recognized by the enzyme enterokinase (Fig. 1D).
The expression of hEGF and hEGF–fusion proteins in E. coli was used as a first broad criterion
to verify the functionality of the transformation vectors. Since the prokaryotic protein synthesis machinery
can recognize some of the plastidic transcriptional and
translational elements, expression in E. coli can be
used as a quick method for testing the genetic constructions, despite it cannot be used as a precise way
to predict its behavior in chloroplasts (Magee et al.,
2004). The Western blot analysis of extracts from E.
coli cells transformed with each of the four chloroplast transformation vectors showed that although no
recombinant hEGF was detected in pBSW-sdEGFtransformed bacteria; plasmids pBSW-utrEGF, pBSWutrHISE and pBSW-utrGEKE were able to direct the
synthesis of hEGF or the respective fusion proteins
(Fig. 2).
Fig. 2. Western blot analysis of transformed E. coli cells. (A)
One hundred and fifty micrograms of total protein from pBSWutrHISE-transformed (HISE) or pBSW-utrEGF-transformed (UTR)
cells and from non-transformed cells (Ctrl) were electrophoresed in
Tris–tricine SDS-PAGE and recombinant protein revealed by Western blot analysis with a monoclonal anti-hEGF antibody. rhEGF:
40 ng of commercial recombinant hEGF. (B) Fifty micrograms of
total proteins from pBSW-utrGEKE-transformed (GEKE) or pBSWutrGUS-transformed (GUS) cells and from non-transformed cells
(Ctrl) were electrophoresed in Tris–glycine SDS-PAGE and Western
blot analysis was carried out as described in (A). The arrow indicates the position of the GEKE fusion protein (27 kDa). M: molecular
weight marker.
Transplastomic tobacco plants were obtained by
bombardment of N. tabacum cv Petit Havana leaves
with each of the four transformation vectors described.
Regenerated plants were initially analyzed by PCR
to detect presence of the transgenes (not shown) and
PCR-positive plants were subjected to two to three
additional regeneration rounds in spectinomycin containing media before grown for further analysis.
3.2. Southern blot characterization of
transplastomic plants
Stable integration of the transgene and homoplasmy
of the transformed plants was confirmed by Southern
blot analysis. Total leaf DNA was cut with Nco I restriction enzyme that recognizes two positions flanking
the insertion site outside the left and right recombination regions (Fig. 1). Therefore, wild type plastomes
release a 6.4 kbp DNA fragment while transformed
plastomes release a higher size fragment, depending
on the length of the construction inserted (pBSWsdEGF, 7.8 kbp; pBSW-utrEGF, 7.9 kbp; pBSWutrHISE, 8.0 kbp; pBSW-utrGEKE, 8.5 kbp). The analysis of three independent lines transformed with each
of the transformation vectors is showed in Fig. 3. In all
of them, the EGF probe that hybridizes with the hegf
sequence revealed a DNA fragment of the expected size
not present in non-transformed plants, confirming the
insertion of the transgene in the plastome (Fig. 3A).
The same membrane was stripped and re-probed with
the trnI/A probe to detect the wild type plastomes.
Absence of the 6.4 kbp wild type DNA fragment in
plants transformed with pBSW-sdEGF, pBSW-utrEGF
and pBSW-utrGEKE indicates that, within the limit
of detection, these lines are essentially homoplasmic.
In plants transformed with pBSW-utrHISE a slight
wild type band was still detected indicating that further regeneration rounds should be addressed to attain
homoplasmy (Fig. 3B).
3.3. Northern blot analysis
Transcription of the hegf sequence and the HISE
and GEKE fusions was confirmed by Northern blot
analysis of total leaf RNA extracted from the different
transplastomic lines. Specific transcripts were detected
by hybridization with the EGF probe. In pBSW-sdEGF
transformed lines, di-cistronic transcripts including
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
165
Fig. 3. Southern blot analysis of representative transplastomic lines. (A) Total cellular DNA (4 ␮g per lane) was digested with Nco I, electrophoresed and blotted onto a Nylon membrane. Presence of hegf sequence was detected by hybridization with a specific probe (EGF probe).
(B) Nylon membranes from (A) were stripped and re-hybridized with the trnI/A probe (Fig. 1) that revealed a 6.4 kbp fragment in wild type
plastomes and higher size DNA fragments in the transformed plastomes, according to the length of inserted constructs (pBSW-sdEGF, 7.8 kbp;
pBSW-utrEGF, 7.9 kbp; pBSW-utrHISE, 8.0 kbp; pBSW-utrGEKE, 8.5 kbp). Each number or code indicates a different plant line. C: DNA from
a non-transformed control plant. Position of DNA marker (lambda BstE II digested DNA, New England Biolabs, USA) is indicated at the left
and right side of the respective panels.
hegf and aadA sequences and a higher size transcript arising by read-through transcription from the
Prrn promoter of the rRNA operon, were observed
(Fig. 4A, rows ii and iii, respectively). In the case
of pBSW-utrEGF, a third smaller band, corresponding to the monocistronic hEGF transcript produced
from the psbA promoter present in 5 psbA sequence
was observed (Fig. 4B, row i). Monocistronic and dicistronic transcripts exhibiting higher sizes due to histidine tag and GUS fusion were also detected in pBSWutrHISE- and pBSW-utrGEKE-transformed lines. Differences in band intensities between lines pBSWutrGEKE 2A, 2F and 2G are due to differences in
loading (not shown).
Presence of aadA sequence in the di-cistronic and
the polycistronic transcript obtained by read-through
transcription from the Prrn promoter of the rRNA
operon was also confirmed by hybridization with a specific probe (not shown).
3.4. Expression of hEGF and fusion proteins in
transplastomic plants
Analysis of recombinant protein expression in
transplastomic plants revealed that N-terminal GUS
fusion could play a role in stabilizing hEGF. No recombinant hEGF was detected in plants transformed with
pBSW-sdEGF, pBSW-utrEGF or pBSW-utrHISE, even
when using an ELISA assay with detection limit as
low as 0.1 ng g−1 of fresh leaf. As a recent report
has shown that accumulation of recombinant rotavirus
VP6 protein decreased as the leaves matured (BirchMachin et al., 2004), we also tested the presence of
hEGF in cotyledons and young leaves, although it
remained still undetectable in these plants. On the other
hand, in plants transformed with pBSW-utrGEKE a
band of the expected size for the GEKE fusion protein
(27 kDa) could be observed in Western blot, indicating that this fusion but not the histidine tag could
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Fig. 4. Northern blot analysis of representative transplastomic lines. Total RNA (4 ␮g per lane) was electrophoresed in denaturing conditions,
blotted onto a Nylon membrane and hybridized with a hEGF specific probe. Polycistronic transcripts, synthesized from the Prrn promoter
incorporated with the constructions and read-trough transcripts synthesized from the Prrn promoter of the rrn operon, are denoted as (ii) and (iii),
respectively. Monocistronic transcripts synthesized from the psbA promoter located into the 5 psbA sequence are denoted as (i). (A) Analysis
of plants transformed with pBSW-sdEGF, (B) analysis of plants transformed with pBSW-utrEGF and (C) analysis of plants transformed with
pBSW-utrHISE or pBSW-utrGEKE. RNA marker: 0.24–9.5 RNA ladder (Invitrogen, USA). The bottom panel shows a transcription map with
the expected mRNAs (i), (ii) and (iii).
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
Fig. 5. Expression of GEKE fusion protein in transplastomic plants.
Leaf extracts containing 150 ␮g of total proteins from the pBSWutrGEKE-transformed plants (2A, 2F and 2G) and from nontransformed control plants (C) were subjected to Western blot analysis using a monoclonal anti-hEGF antibody. (+) Extract of pBSWutrGEKE-transformed E. coli cells (50 ␮g of total proteins) and (M)
pre-stained protein ladder.
167
now incubating them in the dark. The fact that more
than 86% of the initial hEGF remained stable after
a 2-h incubation in the dark, suggested a connection
between the light and a presumed degradation activity (Fig. 6A). Remarkably, when these reactions were
visualized in a non-reducing acrylamide gel, the monoclonal anti-hEGF antibody recognized the expected
monomeric and dimeric forms hEGF, but also another
band of higher molecular weight, not present in the
reaction performed in control buffer. This band could
reflect either aggregation of unfolded hEGF molecules,
or the binding of hEGF to a non-characterized plastidic
component (Fig. 6C).
help to stabilize the hEGF expression in choloplasts
(Fig. 5).
3.6. The effect of light on hEGF and GEKE fusion
protein expression in transplastomic plants
3.5. Stability of hEGF and GEKE fusion protein in
tobacco chloroplast extracts
Accumulation of hEGF and GEKE fusion protein
increased in transplastomic plants incubated in the
dark. To evaluate the effect of light on the stability of hEGF and HISE or GEKE fusion proteins in
vivo, we compared the expression levels in transplastomic plants previously grown in soil, under a 16-h
light:8-h dark cycle, when placed for 20 h in continuous
light (300 ␮mol quanta m−2 s−1 ) or 24 h in darkness.
In plants transformed with pBSW-utrEGF or pBSWutrHISE, levels of recombinant protein remained below
the detection limit of the ELISA assay (0.1 ng g−1 of
fresh leaf) independently of the light conditions in
that they were placed. However, when plants transformed with pBSW-sdEGF were placed for 24 h in the
dark we were able to detect a small increase in hEGF
expression, near the detection limit of ELISA assay.
Considering that the efficiency of translation initiation from the psbA 5 -untranslated sequence decreases
in the dark (Trebitsh et al., 2000), the discrepancies
between constructs having or not this sequence could be
attributed to a counterbalance between this effect and
a putative increased protein stability in the dark. The
most dramatic effect was observed in pBSW-utrGEKEtransformed plants in which accumulation of GEKE
fusion protein was considerably increased after incubation in the dark (Fig. 7A).
The increase in hEGF and GEKE fusion protein
expression levels in the dark is not due to an increase in
mRNA stability. As the increase in recombinant protein
levels in plants placed in the dark could be the result of
an enhanced stability of their transcripts, we compared
Both hEGF and GEKE fusion protein were unstable
when incubated in a tobacco chloroplast extract. As a
first approach to elucidate whether the discrepancies
observed in hEGF and GEKE expression in transplastomic plants were due to different translation rates or to
a differential susceptibility to proteolysis, we assayed
the stability of both recombinant proteins when incubated in vitro with a wild type chloroplast extract. As
shown in Fig. 6A, commercial recombinant hEGF is
fully stable when incubated by 2 h at room temperature
in control buffer. However, less than 20% of the initial
recombinant hEGF was detected after a 2-h incubation
in a chloroplast extract under continuous light conditions. Since one of the best-characterized proteases
of the chloroplast stroma is the Clp ATP-dependent
protease (Adam and Clarke, 2002), we performed the
same assay but supplemented with ATP to test the putative participation of this enzyme in hEGF stability.
As observed in Fig. 6A, ATP did not stimulate hEGF
degradation, on the contrary, it seems to promote some
degree of protection. Similar results were observed
when the assays were performed using recombinant
GEKE protein extracted from E. coli cells transformed
with pBSW-utrGEKE (Fig. 6B).
Stability of recombinant hEGF in the chloroplast
extracts depends on light incubation conditions. To
test a putative effect of light in the hEGF stability we
performed in vitro assays herein before described but
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S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
Fig. 6. Stability of hEGF and GEKE fusion protein in chloroplast extracts. (A) Thirty nanograms of commercial hEGF were incubated at
26 ◦ C with 150 ␮l of control buffer (open triangles), chloroplast extracts in continuous light (open circles) or in darkness (closed circles) and
chloroplast extracts in the light or in darkness supplemented with 5 mM ATP (open and closed squares, respectively). Remaining hEGF after 1
and 2 h of incubation is represented as the percentage of initial concentration. The indicated values are the mean of at least three independent
assays and error bars correspond to the standard deviation. (B) Extracts of pBSW-utrGEKE-transformed E. coli cells containing 50 ␮g of total
protein were incubated at 26 ◦ C with control buffer (control), chloroplast extracts (Cp extract) or chloroplast extract supplemented with 5 mM
ATP (Cp extract + ATP) under continuous illumination. Twenty-microliter aliquots were sampled at the beginning of the assay (0) and at 1, 2
or 16 h (ON). The remaining GEKE fusion protein was detected by Western blot analysis using a monoclonal anti-hEGF antibody. (C) Two
hundred nanograms of commercial hEGF were incubated for 2 h with chloroplast extraction buffer (C), chloroplasts extracts under continuous
light (L) and dark (D), or chloroplast extract under continuous light supplemented with 5 mM ATP (ATP), and analyzed by Western blot using
anti-hEGF antibody. The arrow indicates the high molecular weight aggregates of hEGF produced after incubation in chloroplast extracts. Cp:
control chloroplast extract.
the specific mRNA levels in transplastomic plants after
20 h of incubation in continuous light or 24 h in the
dark. As shown in Fig. 7B, the levels of transcripts for
hEGF or GEKE fusion, were not increased in plants
placed in the dark, moreover, they seemed to be less
stable than in light conditions.
The stability of GEKE fusion protein in transplastomic plants is related to chloroplast photosynthetic
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
169
Fig. 7. Light effect on GEKE fusion protein and transcripts stability in transplastomic plants. (A) Western blot analysis of pBSW-utrGEKEtransformed (lines 2A and 2G) and non-transformed (wt) plants placed in light for 20 h (white bar) or in darkness for 24 h (black bar). Western blot
assays were performed with 30 ␮g of total leaf extracts. EGF: 100 ng of commercial recombinant hEGF. The lower panel shows the Coomasie
blue staining of the rubisco large subunit of the same acrylamide gel. (B) Upper panel: Northern blot analysis of total RNA obtained from
pBSW-utrEGF-, pBSW-utrGEKE- and pBSW-sdEGF-transformed plants placed in continuous light for 16 h (white bars) or in darkness for
24 h (black bars). Numbers correspond to the different plant lines transformed with the indicated transformation vectors. Lower panel: Loading
control with a specific 18S rRNA probe (C) Leaf discs from plant 2A were incubated for 2, 4 and 8 h, under light or dark conditions with or
without addition of 100 ␮M MV or 100 ␮M DCMU. Samples were analyzed as described in (A). The lower panel shows the Coomasie blue
staining of the rubisco small subunit of the same acrylamide gel.
activity. To evaluate whether the differences in GEKE
expression levels in transplastomic plants placed in
the light or darkness were mediated by the status of
the photosynthetic electron transport chain, leaf discs
from in vitro grown plants 2A and 2G transformed
with pBSW-utrGEKE were incubated in the presence
of the electron transport inhibitors MV or DCMU and
GEKE protein was visualized by Western blot at different times. Fig. 7C shows the results from plant 2G
in that both inhibitors led to a considerable increase in
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S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
the accumulation of GEKE fusion protein in the light,
supporting the idea of a connection between the stability of hEGF and GEKE and photosynthesis. The fact
that in this example GEKE seemed to increase at 8 h
in light or decrease at 4 h in MV and then increase
again at 8 h should be interpreted with caution, since
this assay was performed using individual leaf discs
from the same plant, but that were incubated separately, therefore comparisons between different time
points could not be made. Nevertheless, the same tendency to higher GEKE levels in the dark or in the light
in presence of the photosynthetic chain inhibitors was
observed in independent repeats of the same assay performed on discs of plants 2G and 2A (not shown).
4. Discussion
In a first attempt to express hEGF in tobacco
chloroplasts, we obtained transplastomic plants transformed with plasmids pBSW-sdEGF and pBSWutrEGF. In pBSW-sdEGF the hegf sequence was assembled as a di-cistronic transcriptional unit with the aadA
sequence, under the control of the Prrn promoter,
both sequences containing its own Shine–Dalgarnolike ribosome binding site (Fig. 1A). On the other hand,
in pBSW-utrEGF, the Shine–Dalgarno-like sequence
upstream of hegf was replaced by the promoter and
5 -untranslated sequence of psbA gene (Fig. 1B). Both
plasmids directed the insertion of the constructions into
the same intergenic region in the plastome, between
the rrn16 and trnI sequences. Although we verified
the correct integration of the transgene into the plastome (Fig. 3) and determined that accumulation of
hEGF transcripts was high in all of the analyzed plants
(Fig. 4A and B), we were not able to detect the recombinant protein in any of the transgenic lines.
In order to exclude an incorrect assembling of the
transcriptional units in our transformation vectors we
tested hEGF expression in E. coli, although it was
demonstrated that expression in bacteria could not
always be directly related to expression in plastids
(Magee et al., 2004). While no hEGF was detected in
bacteria transformed with pBSW-sdEGF, we were able
to detect the recombinant hEGF in those transformed
with pBSW-utrEGF (Fig. 2).
As it was previously shown that N-terminal fusions
could increase the accumulation of some recombinant
proteins in chloroplasts (Ye et al., 2001; Leelavathi
and Reddy, 2003; Staub et al., 2000) we designed
two additional transformation vectors derived from
pBSW-utrEGF, in that the hegf sequence was fused
to an N-terminal histidine tag (pBSW-utrHISE) or
to the first 186 amino acids of the GUS protein
(pBSW-utrGEKE). Northern blot analysis of transplastomic plants showed that hEGF mRNA was correctly
transcribed (Fig. 4C). Nevertheless, recombinant protein accumulation could only be detected in pBSWutrGEKE-transformed plants, indicating that fusion to
the GUS sequence – but not to the histidine tag – could
partially stabilize protein accumulation (Fig. 5). These
results highly agree with the earlier observations of
Leelavathi and Reddy (2003) for protein expression in
tobacco chloroplasts, in that the fusion of the complete
GUS protein to gamma-interferon conferred a greater
stability than fusion to a six-histine tag, leading to a
higher accumulation of the recombinant protein (6% of
total soluble proteins in the GUS fusion against 0.1%
of total soluble proteins in the histidine–tag fusion).
The fact that despite the absence of the recombinant
protein we detected the specific mRNAs in all of the
analyzed plants, suggests the contribution of a posttranscriptional mechanism involved in the instability
of hEGF. These observations are also in agreement
with previous reports concerning expression of human
hemoglobin (Magee et al., 2004) and maize ␤-zein
(Bellucci et al., 2005) in tobacco chloroplasts in which
despite the presence of functional mRNA transcripts,
no recombinant protein could be detected. Similarly,
although levels of rotavirus VP6 transcripts remained
high when expressed in tobacco chloroplasts, protein
accumulation declined as young leaves and cotyledons
matured (Birch-Machin et al., 2004). Moreover, the
observation that ␤-zein fused to a chloroplast transit
peptide in nuclear transformed plants did not accumulate in chloroplasts, supports the assumption of a posttranslational mechanism (Bellucci et al., 2005). Even
though the precise mechanisms involved in recombinant protein instability in chloroplasts were not elucidated in any of these cases, it was suggested that lack
of protein accumulation could be due to chloroplast
proteolytic activity.
In order to test this hypothesis we designed a series
of in vitro assays using chloroplast extracts to evaluate
the effect of a putative proteolytic activity on hEGF
and GEKE fusion protein. Incubations performed with
S. Wirth et al. / Journal of Biotechnology 125 (2006) 159–172
both proteins showed that they were rapidly degraded
in these extracts. Strikingly, the activity involved in
this process depended on the presence of light (Fig. 6).
Confirming this observation, GEKE fusion protein significantly accumulated when transgenic plants were
incubated in the dark or in presence of the electron
transport chain inhibitors MV and DCMU (Fig. 7).
These results strongly suggest that instability of the
GEKE fusion protein depends on a degrading mechanism induced by light.
Considering that photosynthesis is the main
function performed by chloroplasts, it is not surprising that light has a preponderant role in adapting
protein turnover to variable light conditions. A
well-known mechanism to achieve this implies the
activation/deactivation of plastidic enzymes by thioredoxins (Trxs), through light-dependent reduction of
their disulfide bonds (Schurmann and Jacquot, 2000).
Since correct folding of mature hEGF requires three
disulfide bonds, the recombinant protein could be an
appropriate target for plastid Trxs. Supporting this
view, Trxs have been recently shown to potentially
interact with multiple proteins containing disulfide
bonds (Balmer et al., 2003). This assumption is also
sustained by the fact that secreted hEGF was stabilized
when expressed in a Brevibacillus choshinensis strain
that overproduces CatA, a periplasmic thiol disulfideoxidorreductase belonging to the Trx super-family
(Tanaka et al., 2003). Moreover, previous studies
performed by us showed that hEGF expression in
tobacco plants is highly increased when targeted to
the cell apoplast (Wirth et al., 2004). It could be then
hypothesized that acquisition of correct hEGF folding
in the endoplasmic reticulum (ER) depends on the
interaction with plant disulfide isomerases (PDIs), a
family of CatA homologues that act on target proteins
by shuffling disulfide bonds and promote formation of
thermodynamically stable structures.
In chloroplasts, the PDI-like RB60 protein is a major
component of an mRNA binding complex regulating the light-dependent translation of psbA transcripts
(Trebitsh et al., 2000; Kim and Mayfield, 2002). However, the fact that RB60 is the only identified PDI in
Chlamydomonas reinhardtii that is targeted to both the
chloroplasts and the ER (Levitan et al., 2005), suggests
that this enzyme could be involved in protein folding. In
the same direction, it has been shown that the reduced
form of mammal PDI can unfold cholera toxin (Tsai
171
et al., 2001). Therefore, one possibility to explain the
hEGF and hEGF–fusions instability in the chloroplast
is that these proteins could be assisting correct folding
in the ER and preventing the same process in the reducing environment of illuminated plastids. If this were the
case, abnormal folding could subsequently target these
proteins for their degradation by the chloroplast proteolytic systems. Nevertheless, presence of alternative
mechanisms, such as the direct action of putative redoxdependent proteases or oxidative damage of hEGF,
cannot be entirely discarded on the basis of the present
evidence.
Even so other two disulfide containing proteins have
been expressed in chloroplasts, one of them, the human
serum albumin, forms inclusion bodies that could protect it from degradation (Fernández-San Millán et al.,
2003); and the other one, human somatotropin, has
been expressed as a fusion protein to ubiquitine. (Staub
et al., 2000).
To conclude, although restriction of hEGF accumulation by a light-dependent activity seems clear from
the experiments showed here, the molecular mechanisms leading to this process remain highly hypothetical and more than a model could be postulated to
explain these observations. To further elucidate this
issue, each of the possible mechanisms discussed here
will be specifically addressed in future research.
Acknowledgements
This work was supported by Grant BID 802/OCAR PICT 00 No. 08-08702 from the Agencia Nacional
de Promoción Cientı́fica y Tecnológica (ANPCyT).
MES and SW are fellows of CONICET, FBA and
AM are Research Scientists of CONICET (Argentina).
We are grateful to Dr. Jorge Muschietti and Marı́a
M. Rivero (INGEBI-CONICET) for critical reading
of the manuscript, to Dr. Néstor Carrillo (Universidad
Nacional de Rosario) for the gift of DCMU and Dr. Santiago Mora (Instituto de Investigaciones Bioquı́micas,
Fundación Leloir) for the MV.
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