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
3
.
1
. Analysis of fusion protein accumulation in the ER
Four different cytoplasmic expressed proteins were selected as fusion partners to analyse their
effects on the function and stability of the TMV- specific scFv24. Glutathione S-transferase GST
from S. japonicum [16], the tobacco mosaic virus coat protein CP [28], thioredoxin h2 from to-
bacco TRXt [29] and thioredoxin from E. coli TRXe [30] were cloned 5 upstream of the
scFv24. Accumulation of functional fusion protein in the ER was tested first, because the protein
levels of recombinant antibody fragments are sig-
Fig. 2. Protein levels of ER retained scFv-fusion proteins. N. tabacum cv. Petite Havana SR1 leaves were transiently trans-
formed with recombinant agrobacteria containing the con- structs L-TRXt-scFv24K, L-TRXe-scFv24K, L-CP-scFv24K,
L-GST-scFv24K and incubated for 3 days. Total soluble protein was isolated and levels of functional scFv24-fusion
proteins, were quantified in a TMV-specific ELISA and indi- cated as ngg of leaf material. Each column represents the
mean value of four leaves. Standard deviations are indicated.
m gg of leaf material and the thioredoxin fusion
proteins L-TRXt-scFv24K and L-TRXe-scFv24K showed the lowest accumulation average 190 ng
or 40 ngg of leaf material, respectively. To verify the integrity of scFv24 fusion proteins,
western blot analysis was carried out using affinity purified
L-GST-scFv24K and
L-CP-scFv24K, which showed the highest protein levels in tran-
sient expression assays. In both cases, intact fusion proteins were detected with the expected size of
56.5 kDa for L-GST-scFv24K and 49.5 kDa for L-CP-scFv24K Fig. 3. However, a faint prote-
olytic 27 kDa product of the GST-scFv24 fusion protein was detected using an anti-GST antisera.
It is likely that cleavage occurred in the linker region 1 because the detected faint band corre-
sponds to the predicted size of GST 27 kDa.
In addition to ELISA the capacity of the GST- fusion protein scFv24 domain to bind antigen was
confirmed by surface plasmon resonance based biomolecular interaction analysis [31]. The dextran
matrix of a CM5-rg sensorchip was coated with intact TMV virions, using standard amine cou-
pling chemistry. After surface stabilization, a protein extract from a non transformed tobacco
leaf was injected followed by anti-GST antisera, to monitor nonspecific binding. As shown in Fig. 4,
no binding was observed when these controls were applied. A protein extract from a tobacco leaf
Fig. 3. Western blot analysis of ER retained fusion proteins. Affinity purified L-GST-scFv24K and L-CP-scFv24K were
separated by 12 SDS-PAGE and proteins were transferred to a nitrocellulose membrane. Blots were probed with anti-
GST rabbit antisera and CP-specific mAb29 primary anti- body followed by alkaline phosphatase conjugated goat-anti
rabbit and goat-anti mouse secondary antibody and NBT BCIP staining. Lane 1: Prestained protein marker; lane 2:
affinity purified L-GST-scFv24K; lane 3: affinity purified L-CP-scFv24K.
Fig. 4. Surface plasmon resonance analysis of the L-GST- scFv24K fusion protein. TMV virions were immobilized on
the surface of a CM5rg sensorchip and extracts from a non-transformed and a transformed tobacco leaf were
analysed by surface plasmon resonance using a BIAcore 2000
®
. The sensogram shows injection of: 1 non-trans- formed tobacco leaf extract; 2 25 mgml anti-GST antisera;
3 total soluble protein extract from a L-GST-scFv24K producing tobacco leaf; 4 25 mgml anti-GST antisera. All
injections were made in HBS-buffer with a flow rate of 5 m
lmin.
nificantly higher in the ER than cytoplasmic accu- mulation [1,13], which facilitates analysis of func-
tionality and integrity of the recombinant fusion proteins.
The fusion protein levels in the ER were analysed by transient expression in N. tabacum cv.
Petite Havana SR1 leaves and functional scFv24 was detected by a TMV-specific ELISA. The
highest levels of functional scFv24 were detected for the L-GST-scFv24K fusion protein, with a
maximum level of 3 mgg of leaf material average 2.3 mgg of leaf material, Fig. 2. The protein level
of the coat protein-scFv fusion protein, L-CP- scFv24K, was lower maximum 1 mg; average 0.6
Fig. 5. Levels of cytoplasmic expressed fusion proteins. N. benthamiana leaves were transiently transformed with recombinant agrobacteria and incubated for 3 days. Total soluble protein was isolated and levels of functional scFv24, expressed as part of the
fusion proteins, were quantitated in a TMV-specific ELISA and indicated as ngg of leaf material. Each column represents the mean value of four leaves. Standard deviations are indicated with bars. A Protein levels of constructs GST-scFv24H,
CP-scFv24H, TRXt-scFv24H, TRXe-scFv24H and scFv24H containing a C-terminal His6 sequence. B Protein levels of constructs GST-scFv24K, CP-scFv24K, TRXt-scFv24K, TRXe-scFv24K and scFv24K containing a C-terminal KDEL sequence.
producing L-GST-scFv24K was injected and bind- ing was observed 1470 response units, as the
TMV-specific scFv24 domain of the fusion protein was captured by the TMV virions. The presence of
the fusion partner GST was subsequently confi- rmed by additional binding of an anti-GST antis-
era 800 response units, Fig. 4.
3
.
2
. Analysis of fusion protein accumulation in the cytoplasm
For cytoplasmic
accumulation of
fusion proteins N. benthamiana leaves were used, because
protein level was higher than using N. tabacum cv. Petite Havana SR1 leaves data not shown. Anal-
ysis using the four constructs containing the C-ter- minal His6 sequence demonstrated that only
CP-scFv24H was detectable in a TMV-specific ELISA, with an average protein level of 0.9 ng
functional active scFv24g of leaf material. Protein levels
of GST-scFv24H,
TRXt-scFv24H and
TRXe-scFv24H were below the ELISA detection limit 0.5 ng as was the control construct
scFv24H lacking an N-terminal fusion partner Fig. 5A.
We evaluated the influence of a C-terminal KDEL sequence on the accumulation of scFv24
fusion proteins. Addition of the C-terminal KDEL sequence increased the level of fusion proteins
Fig. 5B. The average protein level of the KDEL tagged CP-scFv24K was 3-fold higher than CP-
scFv24H 2.9 ng per gram leaf material. However, levels of GST-scFv24K, TRXt-scFv24K, TRXe-
scFv24K and the control construct scFv24K were below the ELISA detection threshold. A control
ELISA performed without the antigen TMV gave no signal, indicating that values of CP-scFv24H
and CP-scFv24K did not correspond to binding of CP-fusions to anti TMV polyclonal sera data not
shown.
4. Discussion
