enhanced proteolysis [11]. However, a recent re- port demonstrated that scFv fragments derived
from phage display reached up to 0.3 and 1 of total soluble protein in the cytoplasm of Petunia
leaves and petals, respectively, indicating that phage display selection can be used for enrichment
of more stable scaffolds which tolerate the absence of disulfide bonds [12].
Accumulation of
cytoplasmic recombinant
proteins in prokaryotes has been enhanced by the addition of a stabilizing fusion protein, and anti-
body stability in plants has been improved by the fusion of peptide sequences to the antibody C-ter-
minal. Schouten et al. [13,14] demonstrated that addition of a C-terminal KDEL sequence signifi-
cantly increased scFv protein levels in the plant cytoplasm, indicating that fusion to short polypep-
tides may protect the scFv fragment from prote- olytic degradation. In Escherichia coli, linkage to
N-terminal fusion partners significantly increased cytoplasmic accumulation levels of functionally
active protein. Small cytoplasmic proteins, such as thioredoxin
TRX, glutathione
S-transferase GST or the maltose binding protein [15 – 17] were
successfully used and improved the solubility and stability of heterologous proteins.
The success of protein stabilization with fusion partner proteins in the E. coli system prompted us
to evaluate the suitability of fusion partners to stabilize scFv fragments in the plant cytoplasm.
Here, we used the TMV-specific scFv24, which has a high affinity towards epitopes present on intact
virions and confers virus resistance to transgenic plants [9]. scFv24 has been successfully expressed
to high level in the apoplast of tobacco plants but expression only reaches low levels in the cytoplasm
[9]. Our rationale was to improve cytoplasmic protein levels by linking scFv24 to small cytoplas-
mic proteins. scFv24 was fused to GST from Schistosoma japonicum, TMV coat protein CP,
thioredoxin from tobacco TRXt or thioredoxin from E. coli TRXe and these scFv-fusion
proteins were expressed in the ER or the cyto- plasm of tobacco leaves. The results demonstrated
that all four scFv24 fusion proteins were func- tional but their protein levels were directly related
to the fusion partner. The highest levels of func- tional fusion protein were observed for the ER
targeted GST-scFv24 fusion protein, whereas in the cytoplasm only the CP fusion gave a signifi-
cantly increased level of functional fusion protein. Cytosolic levels of CP-scFv24 could be further
increased by the addition of a C-terminal KDEL sequence.
2. Materials and methods
2
.
1
. Plasmids, bacteria, plants The following plasmids, bacterial strains and
plants were used throughout this study. Plasmids: pUC18 [18], pSS [19]; bacterial strains: E. coli
SCS110 Stratagene,
Heidelberg, Germany,
Agrobacterium tumefaciens GV3101: pMP90RK, GmR KmR RifR, [20]; plants: Nicotiana tabacum
cv. L. Petite Havana SR1, Nicotiana benthamiana.
2
.
2
. Vector design and construction The gene fusion partners glutathione S-trans-
ferase GST from S. japonicum, coat protein CP from TMV, thioredoxin from tobacco TRXt and
thioredoxin from E. coli TRXe were amplified by PCR or RT-PCR. cDNAs were amplified from the
pGEX-5x-3 vector Pharmacia, Freiburg, Ger- many containing GST, a cDNA clone from TMV
kindly provided by Dr. Dennis Lewandowski, CREC, FA, USA, total RNA isolated from to-
bacco [9] and the pTrxFus vector Invitrogen, Leek,
The Netherlands
containing bacterial
thioredoxin as a template. The forward primers introduced a NcoI restriction site 5 end and the
backward primers a C-terminal Gly
4
Ser
2
linker sequence and an AatII restriction site 3 end. The
following forward and backward primers were used for PCR amplification: For GST: GST-for
5-ACT GCG CCA TGG CGT CCC CTA TAC TAG GT-3, GST-back 5-CCG TCA GAC GTC
AGA ACC TCC ACC TCC ACT TCC GCC GCC TCC ATC CGA TTT TGG AGG ATG
GTC GCC ACC ACC-3; for TMV CP: CP-for 5-ACT GCG CCA TGG CTT ACA GTA TCA
CT-3, CP-back 5-CCG TCA GAC GTC AGA ACC TCC ACC TCC ACT TCC GCC GCC TCC
AGT TGC AGG ACC AGA GGT CCA AAC CAA ACC-3; for tobacco TRX: TRXt-for 5-
GCG GAA TTC CAT GGC AGA GGA AGG ACA AGT C-3, TRXt-back 5-GCG TCT AGA
CGT CAG AAC CTC CAC CTC CAC TTC CGC CGC CTC CGG CTG TTG AGG TAC
TAC TGA T-3; and for E. coli TRX: TRXe-for
5-ACT GCG CCA TGG GGA GCG ATA AAA TTA TT-3, TRXe-back 5-CCG TCA GAC GTG
AGA ACC TCC ACC TCC ACT TCC GCC GCC TCC GGC CAG GTT AGC GTC GAG
GAA CTC TTT CAA-3. The 5-NcoI and 3- AatII restricted PCR fragments were subcloned
into a pUC18 derivative containing the TMV spe- cific scFv24 [9] flanked by the 5 untranslated
region omega-sequence, V and 3 untranslated region Pw sequence from TMV [21,22]. A C-ter-
minal His6- H or a KDEL-sequence K were added to scFv24 of all four constructs by PCR
using the backward primers: His6-back 5-CTA CCC CTC GAG TTT AGT GAT GGT GAT
GGT GAT GAG CGG CCG CGT CGA CTG CAG AGA CAG TGA CCA GAG TC-3 and
KDEL-back 5-CCC TCA CTC GAG TTT AGA GCT CAT CTT TCT CAG ATC CAC GAG
CGG CCG CAG AAC CTC CAC CTC CGT CGA CTG CAG AGA CAG TGA CCA G-3.
The subsequent ligation of the EcoRI-AscI frag- ments into the plant expression vector pSS, con-
taining an double enhanced 35S promoter [19], resulted in the final expression constructs GST-
scFv24H, GST-scFv24K,
CP-scFv24H, CP-
scFv24K, TRXt-scFv24H,
TRXt-scFv24K, TRXe-scFv24H and TRXe-scFv24K, which were
used for analyzing scFv-fusion protein accumula- tion in the cytoplasm Fig. 1A.
For ER targeting and retention, the plant codon optimized leader sequence derived from the light
chain of the murine monoclonal antibody mAb24 [19] was integrated between the 5 V untranslated
region and scFv24 of the four different cytoplas- mic constructs containing the KDEL sequence,
giving the four plant expression vectors L-GST- scFv24K, L-CP-scFv24K, L-TRXt-scFv24K and
L-TRXe-scFv24K Fig. 1C.
Two expression vectors lacking a leader se- quence and an N-terminal fusion partner but con-
taining scFv24 with a C-terminal His6 or KDEL sequence were used as controls for cytoplasmic
accumulation scFv24H, scFv24K, Fig. 1B.
Fig. 1. Constructs for expression of scFv24 fusion proteins in the cytoplasm and ER of plant cells. scFv24 cDNA, comprising variable light chain V
L
and heavy chain V
H
domains connected by a 14 amino acid 212 linker linker 2, were fused to GST, CP, TRXt or TRXe using the Gly
4
Ser
2
linker linker 1 and subcloned into the plant expression vector pSS [19]. A Cytoplasmic targeting vectors containing a C-terminal His6 or KDEL sequence. B Cytoplasmic targeting control vectors lacking a fusion
partner. C ER retention vectors. 35SS = enhanced CaMV-35S promoter; V=5 untranslated region of TMV; LP=codon optimized original mouse leader peptide sequence from mAb24; FP = fusion partner GST, CP, TRXt or TRXe; His6 = histidine
6-tag; KDEL = ER retention signal; Pw = TMV 3 untranslated region.
2
.
3
. Transformation of Agrobacterium tumefaciens and tobacco plants
Plant expression constructs were transferred into A. tumefaciens GV3101 by N
2
transformation [23].
Transient transformation
of Nicotiana
tabacum cv. Petite Havana SR1 and N. benthami- ana was performed as described [24].
2
.
4
. Generation of anti-mAb
24
polyclonal antibodies
Anti-mAb24 polyclonal antibodies were raised in rabbits Charles River Wiga, Hannover, USA
and affinity purified, as described [9].
2
.
5
. Protein extraction and analysis To extract total soluble proteins, tobacco leaves
were frozen and ground in liquid nitrogen and scFv-fusion protein level was analysed by ELISA
and Western blot [25], ELISA III. A Fab frag- ment of the mAb24 was used as the ELISA stan-
dard. For surface plasmon resonance analysis, tobacco leaves were extracted using HBS-buffer
containing dextran matrix 150 mM NaCl, 3.4 mM EDTA, 0.05 vv Surfactant P20 BioSen-
sor, Upppsala, Sweden, 10 mM HEPES, pH 7.4, 1 mgml dextran matrix BioSensor and cen-
trifuged at high speed 40 000 × g, 15 min, 4°C to remove insoluble precipitate. Protein concentra-
tions were determined with the BioRad protein assay using bovine serum albumin BSA as the
standard.
2
.
6
. Affinity purification For affinity purification of scFv-fusion proteins
from plant extracts prepared as described above, TMV virions were coupled to an activated CNBr
sepharose matrix. 300 mg of CNBr activated sep- harose 4B matrix Pharmacia, Freiburg, Germany
was resuspended in 1 ml PBS pH 7.4 1.37 M NaCl, 27 mM KCl, 81 mM Na
2
HPO
4
, 15 mM KH
2
PO
4
and incubated for 1 h at RT on a rotator. The matrix was pelleted 5000 × g, 5 min,
RT, resuspended in 1 ml PBS pH 7.4 containing 10 mg TMV virions and incubated for 2 h at RT
on a rotator. The TMV coupled matrix was cen- trifuged 5000 × g, 5 min, RT, resuspended in 1
ml PBS pH 7.4 containing 1 wv BSA and 1 wv powdered milk and rotated over night at 8°C
to block nonspecific binding sites. The TMV cou- pled matrix was washed three times with PBS pH
7.4 and resuspended in 1 ml PBS pH 7.4. 30 ml TMV-matrix was added to 1.5 ml plant extract
prepared as described above and incubated for 1 h at RT on a rotator. Then the TMV-matrix was
washed three times with PBS and the TMV-matrix bound proteins were solubilised in sample buffer
and analysed by SDS-PAGE [26].
2
.
7
. Surface plasmon resonance Biomolecular interaction analyses were carried
out in HBS-buffer 150 mM NaCl, 3.4 mM EDTA, 0.05 vv Surfactant P20, 10 mM
HEPES, pH 7.4 using the BIAcore
®
2000 BioSensor, Uppsala, Sweden. TMV was immobi-
lized on a CM5-rg sensorchip BioSensor using the Amine Coupling Kit BioSensor, Uppsala,
Sweden [27]. The surface of the sensorchip was activated with 70 ml EDCNHS buffer 100 mM
N - ethyl - N - dimethylaminopropyl - carbodimide- hydrochloride, 400 mM N-hydroxy-succinimide
using a flow rate of 10 mlmin. For immobilization of the virus, 200 mg of TMV in 100 ml 10 mM
formic acid pH 3.0 were applied flow rate: 5 m
lmin. Subsequently, the sensorchip was deacti- vated with 70 ml 1M ethanolamine hydrochloride
pH 8.5 flow rate: 10 mlmin and conditioned with 10 ml 100 mM HCl flow rate: 5 mlmin. Between
sample injections the surface was regenerated with 10 ml 30 mM HCl flow rate: 30 mlmin.
3. Results
