Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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LARGE SCALE PRODUCTION OF HUMAN OR ANIMAL PROTEINS
US ING PUTT BIORBACTORs
BACKGROUND OF T8E INVENTION
(a) Field of the Invention
The invention relates to a vector for large
scale production of human or animal proteins using
plant cells, a method of large scale production of
human or animal proteins using plant cells, to a method
of large scale production of human protein C using
plant cells and to plant bioreactors for the expression
of human or animal proteins using plant cells.
tb) Description of Prior Art
Several foreign proteins have been expressed in
plants for diverse purposes: pest resistance, viral
and fungal resistance, environmental stress tolerance,
herbicide resistance or tolerance, food quality and
processing, experimental studies, and for the expres
sion of specialty chemicals.
Among the proteins which were expressed in plant
cells, there is the following:
A human neuropeptide which was linked to a frag-
ment of the plant 2S albumin gene. The peptide
accumulated in the seeds of Arabidopsis and of
oilseed rape at levels up to 200 nmol and 50
nmol respectively per gram of seeds;
~ Chicken ovalbumin in alfalfa with levels of
ovalbumin up to 0.01 of total soluble proteins:
~ The prepro-human serum albumin gene was trans
fected in potatoes. The prosequence was not
cleaved before secretion from the plant cells
but the human signal sequence was recognized by
the plant endoplasmic reticulum; and
~ Antibodies were produced in tobacco plants.
Using plant transgenics for the mass production
of foreign proteins has several advantages, such as the
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low cost of growing the plants, the possibility of
growing the transgenic plants on a very large scale,
the transformation procedures which are well estab-
lished, and the possibility of using specific plant
parts as a sink for engineered proteins. However, in
the case of human or animal proteins requiring post-
translational modifications, the lack of knowledge
concerning the post-translational modifications in
plants represents a potential problem.
Agrobacterium-mediated gene transfer is the most
widely used technique for plant transformation. Agro-
bacterium tumefaciens is a saprophytic soil bacterium
which is also a pathogen of many dicotyledonous plants
causing the formation of crown galls. Pathogenic Agro-
bacterium has a megaplasmid of approximately 200 kilo
base pairs called the Ti plasmid or tumor-inducing
plasmid. The T-DNA (or transferred DNA) within the
megaplasmid is delimited by two 25 base-pair (bp)
sequences called the right and left borders. Virulence
genes are located outside the T-DNA. These contain the
information for the excision of the T-DNA at the T-DNA
borders, and its transfer to the plant cells.
Agrobacterium-mediated gene transfer takes advantage of
this system to transfer foreign DNA.
The binary vector strategy exploited here uses
E. coli-Agrobacterium shuttle vector. This vector con-
tains the T-DNA borders flanking the foreign DNA. This
vector is introduced into Agrobacterium which moves the
T-DNA in trans to the plant cells. The binary vector
strategy uses disarmed Ti plasmids.
There is a demand for many human or animal pro-
teins which have therapeutical applications. These
proteins are sometimes difficult to produce in large
quantities.
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Since the use of human protein C (HPC) concen-
trate as a therapy appears promising (Dreyfus, M. et
al., 1991, N. Engl. J. Med., 325:1565-1568), several
isolation and production systems have been studied.
The purification of HPC from human plasma con-
stitutes a challenge since HPC is present in the plasma
at concentrations of approximately 4 ~g/ml and con-
taminants from similar vitamin K-dependent plasma pro-
teins may be difficult to remove. In addition, there
is always the possibility of infectious agent contami-
nation. Nevertheless, Velander et al. (1991, In pro-
tein C and related anticoagulants, Bruley, D.F. and
W.N. Drohan (eds), Portfolio, The Woodlands, Texas,
p.ll-27) designed a protocol to purify HPC from plasma.
The starting material is either cryopoor plasma or
reconstituted Cohn IV-1 paste which is filtered and
adsorbed on an anion-exchange chromatography column.
The eluate containing HPC is treated with solvent and
detergent to inactivate viruses and it is adsorbed on a
protein C immunoaffinity column. The eluate is again
adsorbed on anion-exchange chromatography and HPC
finally undergoes diafiltration before formulation.
The amount of purified HPC is small and may not be use-
ful for industrial applications.
Synthesis of biologically active recombinant
protein C by bacteria or yeast is precluded because
those organisms are unable to perform some of the
critical post-translational modifications. Production
of vitamin K-dependent plasma proteins by most
mammalian cells resulted in partially processed pro-
teins and low transcription levels (Anson et al., 1985,
Nature, 315:683-685; Busby et al., 1985, Nature,
316:271-273; Grinnell et al., 1987, BiolTechno.Iogy,
5:1189-1192; de la Salle et al., 1985, Nature, 316:268-
270). However, improved cell lines have been described
*rB
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transgenic animals. Velander et al. (1991, Ann. N.Y.
Acad. Sci., 665:391-403) demonstrated that encineered
mice could produce biologically active HPC in their
milk at concentrations of up to 3 ~cg/ml. Velander et
al. (1992, Proc. Natl. Acad. Sci. USA, 89:12003-12007) ,
also reported that transgenic swine were capable of
high-level expression of HPC. A concentration of 1 g
per litre of milk was detected from the best animal.
The use of animals for the production of human protein
t U ~. i 5 i i W . a c o i 1 ci it i c a a c ~. v ~. i W :: ~:. ~ y ~ W . v.:
~ ~. s. ,., ,,:.; ,.; .;
with the purification of the human transgenic protein
away from related animal proteins.
International Application published under No. WO
97/04122 on February 6, 1997 discloses an expression
o vector comprising DNA coding for a plant promoter, a
transcription terminator and a protein to be expressed,
such as hormones and growth factors.
International Application-published under No. WO
91/02066 on February 21, 1991 discloses a DNA construct
zo for the expression of human serum albumin (HSA) in
plants.
It would be highly desirable to be provided with
a vector for the large scale production of human or
animal proteins using plant cells.
zs It would be highly desirable to be provided with
L.; ..~-~~r.t-~,r ~r"-~ tho l ~,-r_ro crV~~l c r,rnr7osnt; nn of lpoman nr
...._..__~_,.~_ _,.~ ___ __- __ ,.._ __
animal proteins using plant cells.
It would be highly desirable to be provided with
a method of large scale production of human or animal
3o proteins using plant cells.
It would be highly desirable to be provided with
a method of large scale production of human protein C
using plant cells.
~i,r~rJDED sHEET
CA 02300383 2000-02-09
r. _ »a . . ,
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SUMMARY OF THE INVENTION
One aim of the present invention is to provide a
vector for the large scale production of human or ani
s mal proteins using plant cells.
Another aim of the present invention is to pro-
vide a method of large scale production of human or
animal proteins using plant cells.
Another aim of the present invention is to pro-
1U VlliC a 1J1V1tCGt.tV1 ivW .iic iui~c .~s.~..y:. ~i:.:~.:: .......~
human or animal proteins using plant cells.
F,ivi~fVGED SHEET .
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Another aim of the present invention is to pro-
vide a bioreactor for the large scale production of
human or animal proteins using plant cells.
Another aim of the present invention is to pro
s vide a method of large scale production of human pro
tein C using plant cells.
In accordance with one embodiment of the present
invention, there is provided an expression vector for
the large scale production of a human or animal
protein, which comprises a DNA construct consisting of
operatively linked DNA coding for a plant promoter, a
transcription terminator and said human or animal pro-
tein to be expressed. More specifically, the expres-
sion vector is referred to as pCP2.
In accordance with another embodiment of the
present invention, there is provided an expression vec-
tor for the large scale production of a human or animal
protein, which comprises a DNA construct consisting of
operatively linked DNA coding for a plant promoter, a
mRNA stabilizer, a transcription terminator, and said
human or animal protein to be expressed. More specifi-
cally, the expression vector is referred to as pLG3.
In accordance with the present invention there
is provided a plant bioreactor for the large scale pro
duction of a human or animal protein, which comprises
dicotyledonous plants transformed with a DNA construct
consisting of operatively linked DNA coding for a plant
promoter, a transcription terminator and said human or
animal protein to be expressed flanked by T-DNA borders
and a suitable selectable marker for plant transforma-
tion.
In accordance with the present invention there
is provided a plant bioreactor for the large scale pro-
duction of a human or animal protein, which comprises
dicotyledonous plants transformed with a DNA construct
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consisting of operatively linked DNA coding for a plant
promoter, a mRNA stabilizer, a transcription terminator
and said human or animal protein to be expressed
flanked by T-DNA borders and a suitable selectable
marker for plant transformation.
In accordance with the present invention there
is provided a method of large scale production of human
or animal proteins, which comprises the steps of:
a) inserting a suitable recombinant expression vec
for in plant cells using Agrobacterium transfor
mation, said expression vector comprising
operatively linked DNA coding for a plant pro
moter, a transcription terminator and said human
or animal protein to be expressed flanked by T
DNA borders and a suitable selectable marker for
plant transformation; and
b) recovering said expressed human or animal pro-
tein of step a) from said culture medium.
In accordance with the present invention there
is provided a method of large scale production of human
or animal proteins, which comprises the steps of:
a) inserting a suitable recombinant expression vec-
for in plant cells using Agrobacterium transfor-
mation, said expression vector comprising
operatively linked DNA coding for a plant pro
moter, a mRNA stabilizer, a transcription termi
nator and said human or animal protein to be
expressed flanked by T-DNA borders and a suit
able selectable marker for plant transformation;
and
b) recovering said expressed human or animal pro-
tein of step a) from said culture medium.
In accordance with the present invention there
is provided a method of large scale production of human
protein C, which comprises the steps of:
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a) inserting a suitable recombinant expression vec-
tor in plant cells using Agrobacterium transfor-
mation, said expression vector comprising
operatively linked DNA coding for a plant pro-
s moter, a transcription terminator and said human
protein C to be expressed flanked by T-DNA bor-
ders and a suitable selectable marker for plant
transformation; and
b) recovering said expressed human protein C of
step a) from said culture medium.
In accordance with the present invention there
is provided a method of large scale production of human
protein C, which comprises the steps of:
a) inserting a suitable recombinant expression vec
for in plant cells using Agrobacterium transfor
mation, said expression vector comprising
operatively linked DNA coding for a plant pro
moter, a mRNA stabilizer, a transcription termi
nator, and said human protein C to be expressed
flanked by T-DNA borders and a suitable select-
able marker for plant transformation; and
b) recovering said expressed human protein C of
step a) from said culture medium.
For the purpose of the present invention the
following terms are defined below.
The term "human or animal proteins" is intended
to mean any human or animal protein which include with-
out limitation, human protein C (HPC), factor VIII,
growth hormone, erythropoietin, interleukin 1 to 7,
colony stimulating factors, relaxins, polypeptide hor-
mones, cytokines, growth factors and coagulation fac-
tors.
The term "plants" is intended to mean any
dicotyledonous plants, which include without limitation
tobacco, tomato, potato, crucifers.
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The term "operatively linked" is intended to
mean that the elements are physically joined on the
same piece of DNA to produce a unit with a specific
purpose.
The term "T-DNA borders" is intended to mean the
25 base pair Agrobacterium-derived sequences that
delimit the fragment of DNA that will be transferred to
the plant cell with the help of Agrobacterium proteins.
The expression "a suitable selectable marker for
plant transformation" is intended to mean any gene cod
ing for a function that will allow the identification
of transformed plant cells, such as kanamycin resis
tance.
BRILF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the construction of plasmid
pCP2;
Fig. 2 illustrates the construction of plasmid
pLG3;
Fig. 3 is a graph of first screen for HPC pro-
duction of samples 1 to 27;
Fig. 4 is a graph of first screen for HPC pro-
duction of samples 28 to 54;
Fig. 5 is a graph of first screen for HPC pro-
duction of samples 55 to 81;
Fig. 6 is a graph of first screen for HPC pro-
duction of samples 82 to 104;
Fig. 7 illustrates a Western immunoblot of
reduced samples from ion-exchange #7 using a rabbit
anti-HPC serum.
DETAILED DESCRIPTION OF '~1HE INVENTION
The present invention was designed to express
human or animal proteins, namely human protein C (HPC),
in plants. This novel approach has several advantages
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over mammalian transgenics: the low cost of growing
the plants, the ability to produce the protein on a
very large scale, and the elimination of contamination
by related animal proteins during purification of the
expressed human or animal protein.
The preferred bioreactors in accordance with the
present invention include most dicotyledonous plants,
particularly the solonacae, which include (but are not
limited to) tobacco, potato and tomato, and the cruci-
fern as transformed with the vector of the present
invention.
The preferred plants in accordance with the pre-
sent invention include without limitation tobacco,
tomato, potato and crucifers.
Many other human or animal proteins, beside
human protein C, may be prepared in accordance with the
present invention. The preferred human or animal pro-
teins to be expressed in accordance with the present
invention include, but are not limited to, anti-coagu-
lation proteins, such as human protein C, factor VIII,
or tissue plasminogen activator, and other proteins of
pharmaceutical or veterinary interest.
Human protein C (HPC) is a vitamin K-dependent
plasma glycoprotein which is a key element of the anti
coagulation cascade. It is synthesized by the liver
cells as a single peptide but is modified into a het-
erodimer linked by a disulfide bond before its secre-
tion to the bloodstream. Some individuals are par-
tially or completely HPC deficient, a situation that
increases the likelihood of an early thrombotic event
which may be lethal. Purified HPC injection has been
used as an experimental treatment for homozygous defi-
cient patients who are not producing HPC, but is also a
promising drug for several other complications such as
septic shock, thrombolytic therapy, and hip replace-
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ment. The annual demand in the U.S.A. for HPC repre-
sents about 96 kg. At the moment, HPC is purified from
human plasma. Several researchers are experimenting
with the synthesis of HPC in the milk of transgenic
animals. In accordance with the present invention, the
production of HPC is intended to serve as an example of
the human or animal proteins which can be produced at a
very large scale using plants as bioreactors.
The method of the present invention involved
engineering tobacco plants using Agrobacterium-mediated
gene transfer associated with the binary vector
strategy of Hoekema et a1. (1983, Nature, 303:179-180).
Agrobacterium-mediated gene transfer takes advantage of
the gene transfer system provided by the bacterium.
The binary vector strategy consists of using A. tvmefa-
ciens with a Ti plasmid, inserting an accessory plasmid
called the binary vector into the bacterium, and allow-
ing T-DNA transfer. The accessory plasmid contains T-
DNA border sequences with the desired genes and regu-
lating elements located between them.
Engineering tobacco plants to produce human or
animal proteins, or for example HPC, was achieved via
the use of either of the two plasmids with different
elements for the regulation of the expression of the
introduced cDNA. The first plasmid included the con-
stitutive caulif lower mosaic virus ( CaMV ) 3 5S promoter
to drive gene expression, the second included a dimer
of the 35S constitutive promoter with an alfalfa mosaic
virus (AMV) leader sequence to enhance stability of the
transcript. Duplicating the promoter has previously
been found to enhance transcription, while the leader
sequence enhanced translation.
Expression of the T-DNA was verified by ana
lyzing HPC and neomycin phosphotransferase II (NPTII)
synthesis by enzyme linked immunosorbent assay (ELISA)
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and inheritance of the T-DNA insert was observed by
germinating R1 seeds on antibiotic-containing medium or
by ELISA to HPC on Rl seedlings.
Purification protocols were created and pre
y liminary experiments are described. Biological activ
ity of various protein fractions was measured.
Tobacco plants engineered with the human protein
C (HPC) cDNA and plant promoters expressed HPC. This
was demonstrated by ELISA and Western assays using a
combination of antibodies to human protein C. No
similar protein was found in non-transformed plants.
The protein had the expected molecular weight of the
uncleaved form of HPC.
Changes in coagulation times were observed in
several experiments when tobacco extracts were tested
for clotting activity.
Plant transformation and selection
Several cell types or tissues can be used but
cells must be totipotent, that is, able to regenerate
mature plants. Plant cells or tissues are co-culti
vated with Agrobacterium for a few days to allow T-DNA
transfer. After co-cultivation, plant cells and tis
sues are grown on media with antibiotic which sup
presses bacterial growth. Engineered plant cells sur-
vive because an antibiotic resistance marker gene is
transferred with the foreign DNA. This system allows
elimination of non-transformed plant cells. Plantlets
are regenerated for analysis using plant tissue culture
media with various plant growth regulator levels.
HPC structure and post-translational modifications
HPC is a complex vitamin K-dependent plasma gly
coprotein. The HPC mRNA codes for a single peptide
including a signal peptide and a propeptide sequence.
After cleavage of the single chain by removal of the KR
dipeptide (Lys-Arg), HPC is secreted to the bloodstream
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as a two-chain glycoprotein with a molecular weight of
62,000 Da. The light chain (21,000 Da) and the heavy
chain (41,000 Da) remain attached by a disulfide bond.
However, before secretion, HPC must undergo several
post-translational modifications.
Determination of SPC's biological activity
A cDNA clone coding for HPC was inserted down
stream of the CaMV 35S promoter and of a dimer of the
CaMV 35S promoter. Tobacco plants were transformed
using Agrobacterium and a binary vector strategy.
Kanamycin resistant plants were regenerated. T-DNA
integration was tested to insure that plants were
stably transformed. Rl seedlings were also analyzed.
A second round of transformation was performed in order
to increase the level of HPC expression. Partial pro-
tein purification (using ion-exchange chromatography),
dialysis and ultrafiltration were followed by various
analyses (SDS-PAGE, Western immunoblot) in order to
assess protein purity and activity. Clotting assays
were performed in order to determine whether the plant-
produced HPC was biologically active.
1. Recombinant D~tA manipulations
The binary vector pBI121 and a culture of Agro-
bacterium tumefaciens strain LBA4404 were purchased
from Clontech. The plasmid pBI524 and pLPC were pro-
vided by Dr. Bill Crosby from Agriculture Canada and by
Dr. Jeff Turner (Department of Animal Science, McGill
University). Restriction and modifying enzymes were
purchased from New England Biolabs and the DNA marker
(IKB DNA ladder) from BRL. Standard recombinant DNA
manipulations were used during the construction of the
binary vectors and all plasmid manipulations were
performed using E. coli strain DHSa if not specified.
Finally, the concentration of agarose for gel
electrophoresis was 0.8~ unless mentioned, and gels
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were run in 0.7X TBE buffer (89mM Tris, 89mM boric
acid, 2mM EDTA, pH 8.3). Specific DNA fragments to be
recovered after enzymatic digestion were electropho-
resed in TAE buffer (40mM Tris-acetate, 2mM Na2EDTA, pH
8.5) and the GenecleanT"t II kit (Bio 101) was used to
purify and extract the DNA from the agarose gel.
1.1 Non-radioactive hybridisation
Plasmid DNA was digested with Xbal and EcoRI,
electrophoresed in l~ agarose gels (TBE), and trans
ferred onto HybondTM N+ nylon membrane (Amersham) by
alkaline transfer with 0.4 M NaOH according to the mem
brane manufacturer's instructions. The Bcll fragment
containing the HPC cDNA from pLPC was digested, elec
trophoresed in an agarose gel in TAE, and isolated by
GenecleanTM II. The Bcll fragment was used as the
probe. Probe labeling, hybridization, and detection
was carried out using non-radioactive DIG'S"" DNA Labeling
and Detection Kit (Boehringer Mannheim) according to
the manufacturer's instructions.
1.2 Genomic DNA isolation
Plant genomic DNA from transgenic tobacco was
isolated using the CTAB (hexadecyltrimethylammonium
bromide) method. Five grams of leaf material (previ
ously frozen at -70°C) were homogenized in 15 ml of
prewarmed CTAB buffer (100 mM Tris-Cl pH 8.0, 1.4 M
NaCl, 20 mM EDTA, 0.2$ 13-mercaptoethanol, 2$ CTAB) in a
WaringT"' blender and incubated for 30 minutes at 60°C.
The solution was mixed every 5 minutes. An equal vol-
ume of chloroform-isoamyl alcohol (24:1) was added to
the tube, mixed well and centrifuged 3000 g for 15
minutes at 4°C. The aqueous phase was collected and
chloroform-extracted once more. The aqueous phase was
transferred to a new tube and precooled (-20°C) isopro-
panol was added 2/3 volume of the aqueous solution.
The mixture was inverted a few times and incubated at -
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20°C for at least 60 minutes. DNA was precipitated by
centrifugation 10,000 g for 10 minutes at 4°C. Super-
natant was removed and the DNA pellet washed with 10 mM
ammonium acetate in 76$ ethanol. DNA was vacuum-dried
and resuspended in TE buffer (10 mM Tris-C1 and 1 mM
EDTA, pH 8.0).
1.3 Southern hybridization
Genomic DNA (20 fig) was digested with Sau3AI and
DNA fragments were separated by agarose gel electro
phoresis (l~ agarose) in TBE buffer. DNA was trans
ferred to HybondT'" N+ nylon membrane (Amersham) using
alkaline transfer following the manufacturer's instruc
tions. The DNA probe was the cDNA of HPC (Bc~I frag
ment of pLPC) labeled with oc32P-dCTP (ICN) using the T7
QuickprimeT"~ kit of Pharmacia. The nylon membrane was
incubated in 30 ml of prehybridization buffer (250 mM
NaHP04 pH 7.2, 2.5 mM EDTA, 7~ sodium dodecyl sulfate
(SDS), 1$ blocking reagent (Boehringer Mannheim), 50~
deionized formamide) for 24 hours at 42°C. The prehy-
bridization buffer was replaced by 15 ml of hybridiza-
tion buffer (prehybridization buffer with 10~ dextran
sulfate) and incubated with the probe and the membrane
overnight at 42°C. The membrane was washed three times
15 minutes at 42°C with 2X SSC (20X SSC was 3 M NaCl
and 0.3 M Na3citrate, pH 7.0) and 0.1$ SDS, followed by
0.5X SSC and 0.1$ SDS, and then O.1X SSC and 0.1~ SDS.
The final wash was with O.1X SSC and 0.1~ SDS for 30
minutes at 52°C. The X-ray film (KodakTM X-OMAT AR) was
exposed for approximately 7 days.
1.4 Transfer of plasmids into Agrobacterium
Three plasmids (pBI121, pCP2, and pLG3) were
purified from E. coli using an alkaline lysis DNA
minipreparation. Vector DNA was transferred to A.
tumefaciens strain LBA4404 using a freeze and thaw
method.
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2. Plant transformation
2.1 Plant material propagation
Seeds of Nicotiana tabacum cv. Xanthi were ster
ilized for 15 minutes in a 10~ bleach solution with a
drop of detergent (TweenT~-20). Seeds were washed at
least five times with sterile distilled water and
allowed to germinate and grow on artificial medium
composed of basal MS salts, B5 vitamins, 3~ sucrose,
and 0.6~ agar (Anachemia) at pH 5.7-5.8. Seed were
grown under a 16 hour photoperiod with a light inten-
sity of 50 ~E and a temperature of 24°C.
2.2 Preparation of Agrobacterium inoculum
A. tumefaciens was grown in Luria Broth (LB) (l~
tryptone, 0.5~ yeast extract, 85 mM NaCl, pH 7.0)
medium supplemented with 50 ~g/ml kanamycin and
~g/ml of streptomycin for 18 hours or until the
optical density at 595 nm reached 0.5 to 1Ø Cells
20 were spun down at 3,000 g for 5 minutes and the pellet
was resuspended to its initial volume with MS-104
medium (MS basal salts, B5 vitamins, 3$ sucrose,
1.0 ~g/ml benzylaminopurine (BAP), 0.1 ~g/ml naph
taleneacetic acid (NAA), pH 5.7-5.8, and 0.8$ agar)
25 without agar.
2.3 Leaf disc transformation
Leaf squares of about 64 mm2 were dissected us
ing a sharp scalpel, immersed in the inoculum for 15-30
minutes and plated onto MS-104 medium for 2 days under
a 16 hour photoperiod, under low light intensity (20
~E) at 24°C for cocultivation. Leaf discs were washed
alternately three times with sterile distilled water
for 1 minute and sterile distilled water supplemented
with 500 ~g/ml of carbenicillin for 5 minutes. Leaf
discs were transferred to MS-104 medium with 500 ug/ml
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of carbenicillin for another 2 days under the same
environmental conditions.
2.4 Selection and regeneration
Leaf discs were washed as above, plated on MS-
104 medium with 500 ~tg/ml of carbenicillin and
100 ~g/ml of kanamycin, and grown using the above envi-
ronmental conditions until calluses appeared. The
light intensity was increased to 50 ~E and the explants
were allowed to grow until development of well-formed
shoots. Shoots were excised and transferred onto MS-
rooting medium (MS-104 but with 0.6~ agar and no plant
growth regulators) with 500 ~rg/ml of carbenicillin and
100 ~g/ml of kanamycin. Surviving plantlets with well-
formed roots were removed from the artificial medium,
dipped alternately in a 0.06 50WP BenlateTM solution
and in a rooting powder (Stim-root #1) containing
indole-3 butyric acid, and transplanted into
pasteurized PromixT"~ soil mixture. Plantlets were cov-
ered with a transparent cover which was gradually
lifted during the following 7 days.
3. T-DNA expression analysis
3.1 NPTII immunoassay
A double antibody sandwich enzyme linked immu-
nosorbent assay (DAS-ELISA) was used to analyze T-DNA
expression. The DAS-ELISA for NPTII detection was
based on the Nagel et a1. (1992, Plant Mol. Biol. Rep.,
10:263-272) procedure. Approximately 100 mg of leaf
material was homogenized in 300 ~l of PBS-TP (137 mM
NaCl, 43 mM Na2HP04, 27 mM KC1, 14 mM KH2P04, 0.05
Tween'~-20, 2$ polyvinylpyrrolidone (PVP), pH 7.4).
Debris was removed by centrifugation (10,000 g for 2
minutes) and the concentration of soluble proteins was
determined for every sample using the Bradford method
(Bradford, N.M. 1976, Anal. Bio. Chem., 72:248-254).
Samples were diluted to 400 ~g/ml in PBS-TP.
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Microtiter plates (Falcon) were coated with 200 ~1 of
rabbit anti-NPTII (5 Prime -~ 3 Prime Inc.) diluted
1:500 in carbonate buffer (35 mM NaHC03, 15 mM Na2C03,
pH 9.6). The antibody was incubated for 2 hours at 37°
C. Wells were washed five times with PBS-T (PBS-TP
without PVP) by alternately filling the wells with a
multichannel pipette and emptying the plates in the
sink. Wells were blocked with a solution of PBS-T
containing 2$ BSA for 30 minutes at room temperature
(RT). Wells were washed five more times. Leaf samples
(200 ~l) were added and incubated for 2 hours at RT.
Wells were washed five times and 200 ~1 of biotinylated
NPTII antibody (5 Prime -~ 3 Prime Inc.), diluted 1:500
in PBS-TPO (PBS-TP with 0.2$ BSA), was added and incu-
bated at RT for 1 hour. Wells were washed five times
and 200 ~1 of P-nitrophenyl phosphate (PNP) diluted to
1 mg/ml in substrate buffer (9.7$ diethanolamine, pH
9.8) was incubated for approximately 40 minutes.
Absorbance was measured by a microtiter plate reader
(Bio-RadT" 450) with a 405 nm filter.
3.2 HPC immuaoassay
Plant samples were homogenized as for the NPTII
ELISA. A polyclonal rabbit anti-HPC (Sigma) was
diluted 1:2000 in carbonate buffer and used to coat the
wells of microtiter plates (Falcon) for 2 hours at 37°
C. Wells were washed five times with PBS-T and blocked
with a solution of PBS-T and 2$ BSA for 30 minutes at
RT. Wells were washed five more times and 200 ~1 of
leaf extract was added and incubated overnight at 4°C.
Wells were washed five times and 200 ~1 of a polyclonal
goat anti-HPC (Biopool), diluted 1:2000 in PBS-TPO, was
added and incubated at RT for 2 hours. Wells were
washed five times and 200 ~1 of a swine anti-goat IgG
(Cedarlane), diluted 1:3000 in PBS-TPO, was added and
incubated at RT for 1 hour. Wells were washed five
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more times and 200 ~1 of 1 mg/ml PNP dissolved in sub-
strate buffer was added. Color development was allowed
to proceed in the dark for at least 1 hour and color
intensity was measured using a microtiter plate reader
with a 405 nm filter. A standard curve was also made
using purified HPC (American Diagnostica) diluted in
PBS-T.
3.3 Germination of R1 seeds on kanamycin-containing
medium
Seeds produced by RO plants were collected and
germinated on artificial medium ( see described in sec-
tion 2.1) containing 100 ~.g/ml of kanamycin to assay
for antibiotic resistance among the R1 generation and
segregation of the transferred gene.
3.4 Double transformation
Rl seeds from S-2B transformed tobacco plant
were grown in vitro and transformed using the pCP2 vec
for inserted in Agrobacterium tumefaciens (as described
in section 2. above).
A total of three S-2B controls (transformed
once) and 104 potentially double transformant plants
were analyzed for their HPC production. DAS-ELISA was
used to determine HPC concentration (using PBS-T as a
blank) while the Bradford method was used to measure
the soluble protein concentration (the latter analysis
was made with the Bio-RadT~ protein assay kit using
ddH20 as a blank).
HPC production [~] - DAS-ELISA (HPC) x 100
Bradford (soluble proteins)
4. Rngineering tobacco for the expression of pro-
tein C
The tobacco genome was modified in order to syn-
thesize HPC. This involved the construction of two
plasmids which contained a T-DNA and the HPC cDNA, the
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transfer of HPC cDNA into tobacco and the analysis of
T-DNA expression among regenerated plants.
4.1 Binary vectors
Two binary vectors for the expression of plant
HPC were constructed in order to avoid using a single
construct which could have errors acquired during DNA
manipulations. Both pCP2 (Fig. 1) and pLG3 (Fig. 2)
constructs contained the right and left T-DNA border
sequences and the selectable marker gene NPTII, which
provides resistance to kanamycin and allows quick
selection of engineered plants.
Plasmid pCP2 is a derivative of pBI 121. Its T
DNA is delimited by two T-DNA border sequences
(triangles, Fig. 1) which flank the selectable marker
gene neomycin phosphotransferase II (NPTII) preceded by
the nopaline synthase promoter (P) and terminated with
the nopaline synthase terminator (T). In addition, the
cDNA of HPC ( cDNA HPC ) was c loned in between the CaMV
35S promoter (35S) and a nopaline synthase terminator
(T). Approximate sizes of the elements between the
border sequences are indicated and key restriction
sites are indicated above the diagram.
Plasmid pLG3 is a derivative of pBI 121. Its T
DNA is delimited by two T-DNA border sequences
(triangles, Fig. 2)) which flank the selectable marker
gene neomycin phosphotransferase II (NPTII) preceded by
the nopaiine synthase promoter (P) and terminated with
the nopaline synthase terminator (T). In addition, the
cDNA of HPC (cDNA HPC) was cloned downstream of a
double CaMV 35S promoter (35S) and an AMV leader
sequence and upstream of a nopaline synthase terminator
(T). Approximate sizes of the elements between the
border sequences are indicated and key restriction
sites are indicated above the diagram.
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The NPTII gene is regulated by the nopaline
synthase promoter and the NOS-T.
pCP2 construct
A 1420 by BcII fragment, which contained the
cDNA of HPC, was cut out from pLPC and cloned into the
BamHI site of vector pBI524. pBI524 is a derivative of
pUC9 with, (5' to 3'), a dimer of the CaMV 35S pro-
moter, an alfalfa mosaic virus (AMV) leader sequence, a
polylinker (NcoI, XbaI, BamHI), and a NOS-T. The new
construct was called pCPl. The cDNA sequence was pre-
ferred to the genomic sequence because it is easier to
manipulate smaller DNA sequences, and it is not known
if plant cells will correctly splice out human introns.
In order to verify the orientation of the cloned
HPC cDNA, a BglII restriction digestion was performed
on plasmid DNA isolated from recovered E. coli colo-
nies. BglII was expected to cleave at the 3' end of the
double CaMV 35S promoter and 210 by away from the 5'
end of HPC cDNA thus generating two DNA fragments of
approximately 300 by and 4800 by if the cDNA was well
oriented, that is the ATG codon from the HPC cDNA was
immediately downstream of the AMV leader sequence. Two
bands of the correct size were observed.
An XbaI-EcoRI cassette was isolated from pCPl
and ligated in place of the XbaI-EcoRI cassette from
pBIl2l. Therefore, the GUS gene with the NOS-T was
replaced by the cDNA of HPC with its accompanying NOS-T
forming the plasmid pCP2. The HPC cDNA is under the
control of the constitutive CaMV 35S promoter from
pBI121 which is known to highly express foreign pro-
teins.
pLG3 construct
Vector pBI524 contained an undesirable ATG which
is part of the NcoI restriction site. The ATG was
deleted by cleaving pBI524 with NcoI, removing single
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stranded sticky ends with mung bean nuclease, and
ligating the modified vector which was named pLGl. The
removal of the Ncol restriction site was verified with
a double digestion with Ncol and ScaI. Two bands were
observed when pBI524 was digested with the two restric-
tion enzymes while only one band appeared for pLGl
indicating that the NcoI site was missing.
The HPC cDNA BclI fragment was cloned into the
BamHI site of pLGl to create pLG2. Then, a HindIII
EcoRI cassette from pLG2 was cloned in place of the
HindIII-EcoRI from pBIl2l. Therefore, the CaMV 35S
promoter along with the GUS gene and a NOS-T were
replaced by a dimer of the CaMV 35S promoter with an
AMV leader sequence, the cDNA of HPC, and a NOS-T.
Doubling the CaMV 35S promoter is known to markedly
increase transcription, while the leader sequence
enhances translation of the expressed protein.
Transfer of Plasmids pCP2 and pLG3 into A. tu~uefaciens
Plasmids pCP2 and pLG3 were transferred into A.
tumefaciens LBA4404 using a freeze/thaw method. In
order to verify whether the plasmids were successfully
transferred, a non-radioactive Southern hybridization
was attempted. The cDNA of HPC was observed to hybrid-
ize to plasmid DNA isolated from A. tumefaciens and E.
coli transfected with pCP2 while there was no hybridi-
zation with the control plasmid pBI121.
4.2 $ngineering tobacco and selection of transfor-
wants
Leaf discs were inoculated with four Agrobacte-
rium inocula:
A) Seventy-five leaf discs were inoculated with
LBA4404 without any binary vector. Half of the leaf
discs were grown on kanamycin-containing medium in
order to verify the efficacy of kanamycin selection
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while the other half were grown on medium without anti-
biotic in order to recover negative control plants
(untransformed tobacco).
B) One hundred leaf discs were inoculated with LBA4404
with the binary vector pBI121 as a control to monitor
the transformation procedure's efficiency.
C) Two hundred leaf discs were inoculated with LBA4404
with the binary vector pCP2 for the expression of HPC.
D) Two hundred leaf discs were inoculated with LBA4404
with the binary vector pLG3 for the expression of HPC.
Kanamycin repressed regeneration of leaf discs
inoculated with Agrobacterium without binary vector
when cultivated on kanamycin-containing medium. Thus,
the antibiotic selection was efficient in removing
untransformed plants. Thirty-six plants were kanamycin
resistant and survived transplantation to the
greenhouse following inoculation with the binary vector
pBI121. This indicates that the T-DNA transfer took
place. A total of 230 kanamycin-resistant plants were
regenerated: 118 engineered with pCP2 and 112
engineered with pLG3.
4.3 Analysis of HPC and NPTII expression
Screening Rp plants for HPC expression
A DAS-ELISA procedure was preferred to other ELISA
methods because this type of assay is less susceptible
to non-specif is binding of antibodies. Two polyclonal
antibodies for HPC were selected for the sandwich com-
plex with the HPC antigen because polyclonal antibodies
recognize several epitopes of HPC. The ELISA for HPC
was used to screen ail recovered plants engineered with
the pCP2 and pLG3 binary vectors, and to find which RO
plants were potentially highly expressing HPC. A
dilution of 1:2,000 was used for coating the rabbit
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anti-HPC antibody and 1:1,000 for the goat anti-HPC
antibody. The dilution of swine anti-goat IgG was
1:3,000 as recommended by the manufacturer. Finally,
the concentration of soluble protein in sap extracts
was adjusted to 1 mg/ml. Negative control plants had
ELISA values, after the background was removed, (PBS-TP
buffer in place of sap extract) ranging from -0.003 to
0.125 with~an average of 0.061 (n - 38). Results for
engineered plants varied from 0.025 to 0.513. All
plants with ELISA values above 0.375 were selected for
the optimization of the ELISA conditions and for the
accurate quantification of HPC expression.
Optimisation of HPC DA8-BLISA
Prior to the final HPC quantification, different
ELISA conditions were tested in order to optimize the
assay. Dilutions of the rabbit anti-HPC antibody and
of the swine anti-goat IGg were kept as before while a
1:2,000 dilution of the goat anti-HPC antibody was used
since reducing the quantity of antibody was found to
only slow down color development. Eight concentrations
of sap extract were tested: 1,000, 500, 250, 125, 63,
32, 16, and 8 ~g/ml and the quantity of HPC present was
determined using purified HPC. Overall, the percentage
of HPC increased with the dilution of the sap extract
(Table 1). Sap was extracted from six plants which had
ELISA readings greater than 0.375 during HPC screening.
Numbers represent the percentage of plant-produced HPC
among total soluble proteins. ELISA readings were
blanked against sap of non-transformed plants and the
percentage of HPC determined against a purified HPC
standard curve. A protein concentration of S ~g/ml was
selected because a lower concentration of soluble
protein would be too close to the lower limit of
detection and a higher concentration would lead to
underestimation of the amount of HPC present.
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Table 1
Determination of optimal soluble protein concentration
in sap extractions for the immunodetection of 8PC
Nghnlleaf82F S84B S82E 84F SS1A SS1B
extract
1000 0.0002 0.0000 0.0001 0.0000 0.0001 0.0001
500 0.0004 0.0002 0.0002 0.0001 0.0001 0.0003
250 0.0006 0.0003 0.0003 0.0001 0.0002 0.0004
125 0.0013 0.0005 0.0006 0.0001 0.0005 0.0007
63 0.0022 0.0011 0.0014 0.0002 0.0012 0.0016
32 0.0034 0.0017 0.0028 0.0003 0.0023 0.0032
18 0.0057 0.0028 0.0050 0.0005 0.0048 0.0054
8 0.0079 0.0031 0.0072 0.0004 0.0068 0.0089
Plant identification starting with "S" were engineen3d with the plasmid pCP2
while those
starting with "SS" were engineered with pLG3.
Quantification of protein C expression
To confirm results from the first ELISA screen-
ing and to obtain a better estimate of the amount of
protein C in transformed plants, an ELISA assay using
the above antibodies and sample dilutions was per-
formed. Sap was extracted from each Rp plant selected
during the screening, and triplicates of the samples
were incubated with the antibodies. Twelve non-trans-
formed plants were used as negative controls to remove
background. due to plant proteins, and ELISA values were
plotted against a purified HPC standard curve. Some R~
plants expressed HPC at almost 0.03$ of their proteins,
others failed to produce significant amounts (Table 2).
The five best plants, SSN, SSR, S2H, SS2D, and SSFF,
were selected for a final quantification of HPC and
verified for the expression of the NPTII marker gene.
The percentage of HPC relative to plant proteins was
determined as well as standard deviation.
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Table Z
Quantification of HPC among ap plaats which were
potentially highly expressing protein C
PLANT % 8PC STD DRV PLANT % 8PC STD DBV
S5N 0.028 0.001 S5F 0.009 0.001
S5R 0.025 0.001 S1B 0.009 0.001
S2B 0.025 0.001 SS7G 0.008 0.002
SS2D 0.023 0.001 S1F 0.008 0.001
SSFF 0.020 0.000 S5W 0.008 0.001
SS7R 0.019 0.001 S5U 0.008 0.001
SSlA 0.019 0.001 SSSG 0.007 0.003
S6B 0.018 0.002 S1I 0.007 0.001
S5B 0.018 0.001 SS1F 0.005 0.002
S8C 0.017 0.003 SSCC 0.004 0.002
SS1B 0.017 0.005 SS4B 0.004 0.000
S7L 0.017 0.002 S4F 0.003 0.001
SS5J 0.017 0.001 S5H 0.002 0.002
SS3F 0.016 0.000 S7F 0.001 0.001
S5L 0.015 0.000 S5X 0.000 0.000
S2F 0.015 0.006 S70 -0.000 0.002
S5G 0.014 0.001 SSSB -0.002 0.001
SSBM 0.014 0.003 S7K -0.003 0.001
SS6B 0.014 0.002 S6H -0.003 0.002
SSSQ 0.013 0.000 S5K -0.005 0.001
SS2E 0.012 0.000 S5I -0.005 0.000
SS6P 0.011 0.002
Plant identfications starting with "S" v~re engineered with the plasmid pCP2
while those
starting with "SS" were engineered with pLG3.
Readings represent the average of three replicates. "96 HPC" is the percent
among total
soluble tobacco proteins.
$xpression of the marker gene NPTII and HPC among the
bast five plants
The same HPC ELISA procedure as above was used
to confirm HPC quantification of the best five HPC-pro
ducing tobacco plants. In addition, NPTII ELISA was
used to verify the expression of the marker gene.
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After removing background (PBS-TP buffer) from ELISA
readings, all five plants had positive NPTII readings
while all four negative control plants had negative
NPTII readings (Table 3). Moreover; HPC percentage
among soluble proteins was slightly negative for con-
trol plants because they consistently had ELISA read-
ings lower than the PBS-TP buffer used to blank read-
ings. Some engineered plants produced 0.02 to 0.03 of
HPC. S2B, SSN, SSR, SSFF were transformed with pCP2,
SS2D was transformed with pLG3 and C1B, C2A, C3A, C3D
were not transformed with a binary vector.
Table 3
Percentage of 8PC and detection of NPTII from
selected Rp plants
NPTII NPTII % HPCj' HPC
RSADINGSa STD DBV. STD DEV.
S2B 0.371 0.015 0.033 0.001
S5N 0.048 0.014 0.024 0.001
85R 0.040 0.010 0.023 0.001
SS2D 0.155 0.030 0.021 0.001
S5Fg 0.134 0.050 0.020 0.002
C1B -0.079 0.017 -0.008 0.001
C2A -0.101 0.018 -0.007 0.001
C3A -0.152 0.007 -0.010 0.001
C3D -0.084 0.002 -0.004 0.003
a: NPTII EIISA readings were blanked against PBS-TP buffer reading.
b: °~6 HPC is the percentage of HPC among tobacco soluble proteins.
Numbers represent the mean of three replicates.
Expression of HPC in R1 families
The ELISA assay for HPC was used to verify the
synthesis of HPC among the progenies of two of the best
five HPC-producing tobacco plants. Seeds of S2B, SSN,
and C3A were collected and germinated in soil. When R1
plants had approximately four leaves, an ELISA was used
to quantify HPC levels (Table 4). Twenty seedlings
were tested per mother plant and ratios of seedlings
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synthesizing HPC versus those not synthesizing HPC were
statistically analyzed. Mother plants were S2B and
SSN, which were transformed with pCP2. The ratio of
S5N progenies expressing HPC to those not expressing
HPC was in agreement with the segregation of one
dominant gene (Table 4). All S2B progeny expressed
HPC, suggesting that two or more T-DNAs were present in
the plant genome of the S2B mother plant.
Table 4
Inheritance of HPC expression in the R1 generation
% HPC S28a SSNb C3A
Detected
0.02 11 0 0
0.01 9 14 0
0.00 0 6 10
Numbers represent the number of R1 plants from which 0.02, 0.01, or 0.0096 of
soluble
HPC protein was detected by ELISA.
a: Chi-square analysis at p = 0.05 for the segregation of iwo or more T-DNA
(15:1,..:1).
b: Chi-square analysis at p = 0.05 for the segregation of one T-DNA (3:1).
Double transformation
The S2B tobacco plants used here for a second
transformation had previously been screened for their
ability to survive on kanamycin medium. It was there-
fore not possible to use kanamycin selection to iden-
tify plants that were transformed a second time, since
the same plasmid was used for both transformations.
Plants capable of producing larger amounts of HPC (as a
result of a second transformation event) were identi-
fied by comparing the putative double transformants to
three single transformed plants (F1 generation of S-2B
plant) for the level of HPC using a DAS-ELISA.
First screen
Figs. 3 to 6 show the relative HPC content of
various second transformants; of 104 plants, eight
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produced significantly higher amounts of HPC: A 9-4
(#37). B 4-5 (#80), B 5-1 (#81), B 5-2 (#82), B 8-4
(#94), B 9-2 (#97), B 9-4 (#99) and B 10-1 (#100). To
avoid eliminating plants that were high producers but
were not among the eight best identified above, the 17
best plants (DAS-ELISA units / Bradford units higher
than 7) were transferred to the greenhouse for further
analysis (indicated by arrows on Figs. 3 to 6).
Second and third screens
Following two additional quantitative evalu-
ations, three plants showed consistently higher HPC
content:' plants B 2-2, B 5-1 and B 8-1. During the
second and the third evaluations, their average HPC
production were respectively 42~ and 23~ higher than
the three controls.
The plasmid pCP2 contained the marker gene NPTII
and the cDNA of HPC under the control of the CaMV 35S
promoter. Plasmid pLG3 contained NPTII and the cDNA of
HPC was controlled by a dimer of CaMV 35S promoter with
an AMV leader sequence. Growing non-transformed
tobacco plants on kanamycin-containing medium indicated
that the antibiotic selection was efficient while
engineering leaf discs with pBI121 showed that the T-
DNA was transferred properly to plant cells. The best
HPC-producing tobacco plants were shown to express
NPTII and HPC at levels representing 0.02 to 0.03 of
their soluble proteins. The expression of HPC in R1
plants was transmitted with a 3:1 ratio for S5N progeny
while all S2B progeny expressed HPC, suggesting that
the mother plant had two or more T-DNA inserts in its
genome.
Some plants, transformed a second time, produced
more HPC than the original transformants, with the best
"twice-transformed" plant producing 43~ more than the
original mother plant.
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5. Partial purification of plant-produced
protein C
A series of experiments were designed to par
tially purify HPC in order to eventually characterize
the protein and assay for its activity. All manipula
tions were performed either on ice or in a cold room at
4°C .
Anion-exchange chromatography
HPC expressed in tobacco plants was partially
purified using an affinity chromatography purification
protocol. Approximately IO g of tobacco leaves with
positive HPC ELISA readings were homogenized in a
WaringT"~ blender with 30 ml of extraction buffer ( 20 mM
Tris-C1 pH 7.4, 150 mM NaCl, 4 mM EDTA pH 7.4, 5 mM
benzamidine-HCl). Most debris was removed by filtra-
tion through MiraclothT"~ and by centrifugation at 15,000
g for 10 minutes. A 16 X 200 mm column (Pharmacia) was
filled with 10 ml of Fast Flow Q'~'~ Sepharose (FFQ) anion
exchanger (Pharmacia). FFQ resin was washed with 30 ml
of equilibration buffer ( 20 mM Tris-C1 pH 7 .4, 150 mM
NaCl, 2 mM EDTA pH 7.4, 2 mM benzamidine-HC1). The
sample was applied at the surface of the resin followed
by another 30 ml of equilibration buffer. HPC was
eluted by injecting 30 ml of elution buffer (20 mM
Tris-C1 pH 7.4, 150 mM NaCl, 10 mM CaCl2, 2 mM
benzamidine-HCl). A high salt elution was then applied
( 20 mM Tris-C1 pH 7. 4, 400 mM NaCl, 2 mM benzamidine-
HC1) and the resin was cleaned with a solution of 2 M
sodium acetate.
Sephadex desalting
Salts were removed from sap extracts immediately
after centrifugation, using PD-10'''M columns (Pharmacia)
according to the manufacturer's instructions.
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Complete debris removal
After centrifugation of the sap extract at
15,000 g, the solution was centrifuged for 1 hour at
100,000 g.
6. Partial purification of plant-produced HPC using
ion-exchange chromatography
Following tests to optimize the pH of buffers
(as described above in section 5.), three preliminary
tests were made on the automated liquid chromatography
system (BioRad) in order to set larger scale working
conditions.
During experiments, the relative amount of total
protein was monitored using the system's UV monitor,
whereas the relative amount of HPC was determined on
eluted samples using DAS-ELISA. All experiments were
conducted under the following conditions .
Samples: Approximately 5.0 g of HPC+ tobacco leaves
were homogenized with 15.0 ml of extraction
buffer and removed from debris using Miracloth
T"~ and 10 minutes of centrifugation at 15,000
g.
Buf f ers
Extraction buffer: 20 mM Tris-C1, 150 mM NaCl, 4 mM
EDTA, 5 mM benzamidine-HC1, pH 8.
Equilibration buffer:20 mM Tris-C1, 150 mM NaCl, 2 mM
EDTA, 2 mM benzamidine-HC1, pH 5
or 8.
Elution buffer: 20 mM Tris-C1, 150 mM NaCl, 2 mM
benzamidine-HC1, pH 8.
Ca2+ elution buffer: 20 mM Tris-C1, 150 mM NaCl, 10 mM
CaCl2, 2 mM benzamidine-HC1, pH
8.
Ion-exchange conditions
Column: Sepharose'"d Q (Sigma), 5 ml
Buffers: Extraction and equilibration at pH 8
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Elution at pH 8
mM Ca2+ elution at pH 8
10 mM Ca2+ salt elution with 200 and
400 mM NaCl at pH 8
5
7. Analysis of plant-produced 8PC
7.1 SDS-PAG$ aad Western immunoblot
Human Protein C is a 62,000 Da protein made of
10 two subunits (41,000 and 21,000 Da) linked by a disul
fide bridge. The addition of mercaptoethanol to the
sample before loading onto the gel has the effect of
destroying that disulfide bridge. Gel electrophoresis
and Western blots were used to characterize the protein
produced in tobacco plants.
Western immunoblot
Immunodetection was conducted by blocking 30
minutes with PBS and 5$ powdered milk. Rabbit anti-HPC
antibody (1:7,000) was added to the blocking solution.
After an overnight incubation, the nitrocellulose was
washed three times for 10 minutes each with PBS + 0.5~
Triton's followed by a 10 minutes wash in TBS. The
nitrocellulose was then transferred to TBS + 5~ pow-
dered milk supplemented with a 1:14,000 dilution of
goat anti-rabbit IgG conjugate (Bio-Rad) for 60 min-
utes. The nitrocellulose was washed four times in TBS
for 15 minutes each. Colorimetric detection of HPC was
carried out using alkaline phosphatase activity.
Using these parameters, the samples used were:
S 25 ng of standard HPC (Sigma);
T+ HPC+ tobacco plant (S-2B);
T- HPC- tobacco plant; and
T++ HPC+ tobacco plant (second transformant).
This immunoblot confirmed the presence of HPC in
transformed tobacco plants ( Fig. 7 ) . Both transformed
plants displayed a major protein reacting with anti-HPC
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serum (lanes T+ and T++: 61,300 MW) whereas no band was
detected in the control (non-transformed) tobacco plant
(lane T-). Lane S contained 25 ng of Sigma HPC. Two
major proteins were detected corresponding to the heavy
and light chains (40,100 and 23,700 Da, respectively).
From these results, it was concluded that: 1-
HPC was produced in transformed plants; 2- HPC was not
completely processed and possibly not cleaved into the
heavy and light chain.
7.2 HPC location in tobacco plant
HPC concentration was measured in different tis-
sues of a twice-transformed tobacco plant: 1- roots;
2- stem; 3- primary vein of the leaf ; 4- leaf without
the primary vein. An equal amount of each part of the
plant (5.0 g) was homogenized with 15.0 ml of extrac
tion buffer (20 mM Tris, 10 mM benzamidine-HC1, pH
7.4), filtered and centrifuged 10 minutes at 15,000 g.
These four extracts were analyzed for HPC content using
DAS-ELISA.
HPC content of different tissues of tobacco
plants was measured using DAS-ELISA. Leaves showed a
higher concentration than other parts tested (Table 5).
HPC concentration was lowest in the roots.
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Table 5
HPC content of various tissues measures using
DA8-BLISA
Plant tissue Amount of HPC
(Ng HPC / g fresh tissue)
Leaves 0.388
Stem 0.274
Veins 0.235
Roots 0.173
7.3 Biological activity using delay in coagulation
time
The Acticlot'~'M assay kit (from American Diagnos-
tica) was used. HPC+ and HPC- sera, dilution buffer
and solutions were prepared according to the manufac-
turer's instructions. Tubes, ActiclotT"~ activator and
CaCl2 stock solution were prewarmed to working tempera-
ture (37°C).
Prior to testing, HPC samples were prepared as
follows: 50 ~,1 undiluted sample, 50 ~tl HPC deficient
plasma, 400 ~,1 American Diagnostica's dilution buffer.
A 50 ~.~,1 volume of this prepared sample was mixed with
an equal amount of HPC deficient plasma and incubated
for 2 minutes. A volume of 50 ~,.~,1 of Acticlot activator
was mixed with the sample solution and incubated five
more minutes. Finally, 50 ~1 of calcium chloride stock
solution was added and clotting time was monitored by
the tilt-tube technique.
Changes in coagulation times were observed in
several experiments when tobacco extracts were tested
for clotting activity. Some of the clotting assays
indicated that biological activity was present.
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
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Coagulation times of a tobacco extract using
American Diagnostica's Acticlot assay kit
HPC+ HPC chromato chromato
plasma #9 #8
HPC neg.
tobacco control
Assay 1 60 sec. 44 sec. 52 sec. 107 sec.
Assay 2 - - 145 sec. 130 sec.
Assay 3 - - 40 sec_ 60 sec.
Assay 4 - - 140 sec. 130 sec.
The present invention will be more readily un
derstood by referring to the following examples which
are given to illustrate the invention rather than to
limit its scope.
Expression vector pCP2 construction for the production
of human protein C
A 1420 by BclI fragment, which contained the
cDNA of HPC, was cut out from pLPC and cloned into the
BamHI site of vector pBI524. pBI524 is a derivative of
pUC9 with, (5' to 3'). a dimer of the CaMV 35S pro-
moter, an alfalfa mosaic virus (AMV) leader sequence, a
polylinker (NcoI, XbaI, BamHI), and a NOS-T. The new
construct was called pCPl. In order to verify the ori-
entation of the cloned HPC cDNA, a BglII restriction
digestion was performed on plasmid DNA isolated from
recovered E. coli colonies. BglII is expected to
cleave at the 3' end of the double CaMV 35S promoter
and 210 by away from the 5' end of HPC cDNA thus gen-
erating two DNA fragments of approximately 300 by and
4800 by if the cDNA was well oriented, that is the ATG
codon from the HPC cDNA was immediately downstream of
the AMV leader sequence.
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
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An XbaI-EcoRI cassette was isolated from pCPl
and ligated in place of the Xbal-EcoRI cassette from
pBI121. Therefore, the GUS gene with the NOS-T was
replaced by the cDNA of HPC with its accompanying~NOS-T
forming the plasmid pCP2. The HPC cDNA is under the
control of the constitutive CaMV 35S promoter from
pBI121.
Plasmid pCP2 was transferred into A. tumefaciens
LBA4404 using the freeze/thaw method. In order to
verify whether the plasmids were successfully trans
ferred, a non-radioactive Southern hybridization was
performed. The cDNA of HPC was observed to hybridize
to plasmid DNA isolated from A. tumefaciens and E.
coli .
Seeds of Nicotiana tabacum cv. Xanthi were ster-
ilized for 15 minutes in a 10$ bleach solution with a
drop of detergent (TweenTM-20). Seeds were washed at
least five times with sterile distilled water and
allowed to germinate and grow on artificial medium
composed of the basal MS salts, B5 vitamins, 3$
sucrose, and 0.6~ agar (Anachemia) at pH 5.7-5.8. Seed
were grown under a 16 hour photoperiod with a light
intensity of 50 ~E and a temperature of 24°C.
A. tumefaciens was grown in Luria Broth (LB) (1$
tryptone, 0.5$ yeast extract, 85 mM NaCl, pH 7.0)
medium supplemented with 50 ~g/ml kanamycin and
25 ~g/ml of streptomycin for 18 hours or until the
optical density at 595 nm reached 0.5 to 1Ø Cells
were spun down at 3,000 g for 5 minutes and the pellet
was resuspended to its initial volume with MS-104
medium (MS basal salts, B5 vitamins, 3$ sucrose,
1.0 ~g/ml benzylaminopurine (BAP), 0.1 ~g/ml naph-
taleneacetic acid (NAA), pH 5.7-5.8, and 0.8~ agar)
without agar.
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
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Leaf squares of about 64 mm2 were dissected
using a sharp scalpel, immersed in the inoculum for 15-
30 minutes and plated onto MS-104 medium for 2 days
under a 16 hour photoperiod, under low light intensity
(20 ~E) at 24°C for cocultivation. Leaf discs were
washed alternately three times with sterile distilled
Water for 1 minute and sterile distilled water supple-
mented with 500 ~g/ml of carbenicillin for 5 minutes.
Leaf discs were transferred to MS-104 medium with 500
~g/ml of carbenicillin for another 2 days under the
same environmental conditions.
Leaf discs were washed as above, plated on MS-
104 medium with 500 ~g/ml of carbenicillin and
100 ~g/ml of kanamycin, and grown using the above envi-
ronmental conditions until calluses appeared. The
light intensity was increased to 50 ~E and the explants
were allowed to grow until development of well-formed
shoots. Shoots were excised and transferred onto MS-
rooting medium (MS-104 but with 0.6$ agar and no plant
growth regulators) with 500 ~g/ml of carbenicillin and
100 ~g/ml of kanamycin. Surviving plantlets with well-
formed roots were removed from the artificial medium,
dipped alternately in a 0.06$ 50WP Benlate solution and
in a rooting powder (Stim-root #1) containing indole-3
butyric acid, and transplanted into pasteurized Promix
soil mixture. Plantlets were covered with a transpar-
ent cover which was gradually lifted during the follow-
ing 7 days.
LE II
Expression vector phG3 construction for the production
of human protein C
Vector pBI524 contained an undesirable ATG which
is part of the NcoI restriction site. The ATG was
deleted by cleaving pBI524 with NcoI, removing single
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
- 37 -
stranded sticky ends with mung bean nuclease, and
ligating the modified vector which was named pLGl. The
removal of the NcoI restriction site was verified with
a double digestion with NcoI and ScaI. Two bands were
observed when pBI524 was digested with the two restric-
tion enzymes while only one band appeared for pLGl
indicating that the NcoI site was missing.
The HPC cDNA BclI fragment was cloned into the
BamHI site of pLGl to create pLG2. Then, a HindIII
EcoRI cassette from pLG2 was cloned in place of the
HindIII-EcoRI from pBIl2l. Therefore, the CaMV 35S
promoter along with the GUS gene and a NOS-T were
replaced by a dimer of the CaMV 35S promoter with an
AMV leader sequence, the cDNA of HPC, and a NOS-T.
Plasmid pLG3 was transferred into A, tumefaciens
LBA4404 using the freeze/thaw method. In order to
verify whether the plasmids were successfully trans-
ferred, a non-radioactive Southern hybridization was
performed. The cDNA of HPC was observed to hybridize
to plasmid DNA isolated from A. tumefaciens and E.
coli .
Seeds of Nicotiana tabacum cv. Xanthi were ster-
ilized for 15 minutes in a 10$ bleach solution with a
drop of detergent (TweenTM-20). Seeds were washed at
least five times with sterile distilled water and
allowed to germinate and grow on artificial medium
composed of the basal MS salts, B5 vitamins, 3~
sucrose, and 0.6$ agar (Anachemia) at pH 5.7-5.8. Seed
were grown under a 16 hour photoperiod with a light
intensity of 50 ~E and a temperature of 24°C.
A. tumefaciens was grown in Luria Broth (LB) (1~
tryptone, 0.5$ yeast extract, 85 mM NaCl, pH 7.0)
medium supplemented with 50 ~g/ml kanamycin and
25 ~g/ml of streptomycin for 18 hours or until the
optical density at 595 nm reached 0.5 to 1Ø Cells
CA 02300383 2000-02-09
WO 99109187 PCT/CA97/00590
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were spun down at 3,000 g for 5 minutes and the pellet
was resuspended to its initial volume with MS-104
medium (MS basal salts, B5 vitamins, 3~ sucrose,
1.0 ~g/ml benzylaminopurine (BAP), 0.1 ~g/ml naph-
taleneacetic acid (NAA), pH 5.7-5.8, and 0.8~ agar)
without agar.
Leaf squares of about 64 mm2 were dissected
using a sharp scalpel, immersed in the inoculum for 15-
30 minutes and plated onto MS-104 medium for 2 days
under a 16 hour photoperiod, under low light intensity
(20 ~E) at 24°C for cocultivation. Leaf discs were
washed alternately three times with sterile distilled
water for 1 minute and sterile distilled water supple-
mented with 500 ~g/ml of carbenicillin for 5 minutes.
Leaf discs were transferred to MS-104 medium with 500
~g/ml of carbenicillin for another 2 days under the
same environmental conditions.
Leaf discs were washed as above, plated on MS
104 medium with 500 ~g/ml of carbenicillin and
100 ~g/ml of kanamycin, and grown using the above envi
ronmental conditions until calluses appeared. The
light intensity was increased to 50 NE and the explants
were allowed to grow until development of well-formed
shoots. Shoots were excised and transferred onto MS-
rooting medium (MS-104 but with 0.6$ agar and no plant
growth regulators) with 500 ~g/ml of carbenicillin and
100 ug/ml of kanamycin. Surviving plantlets with well-
formed roots were removed from the artificial medium,
dipped alternately in a 0.06$ 50WP Benlate solution and
in a rooting powder (Stim-root #1) containing indole-3
butyric acid, and transplanted into pasteurized Promix
soil mixture. Plantlets were covered with a transpar-
ent cover which was gradually lifted during the follow-
ing 7 days.
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
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BXAMPLE III
i~xpression vector construction for the production of
chicken nuclear oncoprotein p53.
Proceeding as for Example I, but using a cDNA
sequence coding for chicken nuclear oncoprotein p53
instead of the cDNA of the human protein C gene.
Vector pBI524 contained an undesirable ATG which
is part of the NcoI restriction site. The ATG was
deleted by cleaving pBI524 with NcoI, removing single
stranded sticky ends with mung bean nuclease, and
ligating the modified vector which was named pLGl. The
removal of the NcoI restriction site was verified with
a double digestion with NcoI and ScaI. Two bands were
observed when pBI524 was digested with the two restric-
tion enzymes while only one band appeared for pLGl
indicating that the NcoI site was missing.
The chicken cDNA for nuclear oncoprotein p53 is
excised using EcoRI restriction digestion. T4 DNA
polymerase is used to fill in the 3' recessed end and
therefore eliminate the EcoRI site. BamHI linkers (5'-
CGGATCCG-3'.) are added by ligation with T4 DNA ligase.
The BamHI ends are then digested with BamHI and ligated
into the BamHI site of pLGl to create pLG53. Then, a
HindIII-EcoRI cassette from pLG2 is cloned in place of
the HindIII-EcoRI from pBI121. Therefore, the CaMV 35S
promoter along with the GUS gene and a NOS-T were
replaced by a dimer of the CaMV 35S promoter with an
AMV leader sequence, the cDNA of HPC, and a NOS-T.
Plasmids pLG53 is transferred into A. tumefaciens
LBA4404 using the freeze/thaw method.
Seeds of Nicotiana tabacum cv. Xanthi are ster-
ilized for 15 minutes in a 10~ bleach solution with a
drop of detergent (TweenTM-20). Seeds are washed at
least five times with sterile distilled water and
allowed to germinate and grow on artificial medium
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
- 40 -
composed of the basal MS salts, 85 vitamins, 3~ sucr-
ose, and 0.6$ agar (Anachemia) at pH 5.7-5.8. Seed are
grown under a 16 hour photoperiod with a light inten-
sity of 50 ~E and a temperature of 24°C.
A. tumefaciens is grown in Luria Broth (LB) (1$
tryptone, 0.5$ yeast extract, 85 mM NaCl, pH 7.0)
medium supplemented with 50 ~g/ml kanamycin and
25 ~.g/ml of streptomycin for 18 hours or until the
optical density at 595 nm reaches 0.5 to 1Ø Cells
are spun down at 3, 000 g for 5 minutes and the pellet
is resuspended to its initial volume with MS-104 medium
(MS basal salts, B5 vitamins, 3~ sucrose, 1.0 ~g/ml
benzylaminopurine (BAP), 0.1 ~g/ml naphtaleneacetic
acid (NAA), pH 5.7-5.8, and 0.8~ agar) without agar.
Leaf squares of about 64 mm2 are dissected using
a sharp scalpel, immersed in the inoculum for 15-30
minutes and plated onto MS-104 medium for 2 days under
a 16 hour photoperiod, under low light intensity (20
~E) at 24°C for cocultivation. Leaf discs are washed
alternately three times with sterile distilled water
for 1 minute and sterile distilled water supplemented
with 500 ~.g/ml of carbenicillin for 5 minutes. Leaf
discs are transferred to MS-104 medium with 500 ~g/ml
of carbenicillin for another 2 days under the same
environmental conditions.
Leaf discs are washed as above, plated on MS-104
medium with 500 ~g/ml of carbenicillin and 100 ~g/ml of
kanamycin, and grown using the above environmental con-
ditions until calluses appeared. The light intensity
is increased to 50 ~E and the explants allowed to grow
until development of well-formed shoots. Shoots are
excised and transferred onto MS-rooting medium (MS-104
but with 0.6~ agar and no plant growth regulators) with
500 ~g/ml of carbenicillin and 100 ~g/ml of kanamycin.
Surviving plantlets with well-formed roots are removed
CA 02300383 2000-02-09
WO 99/09187 PCT/CA97/00590
- 41 -
from the artificial medium, dipped alternately in a
0.06 50WP Benlate solution and in a rooting powder
(Stim-root #1) containing indole-3 butyric acid, and
transplanted into pasteurized Promix soil mixture.
Plantlets are covered with a transparent cover which is
gradually lifted during the following 7 days.
While the invention has been described in con-
nection with specific embodiments thereof, it will be
understood that it is capable of further modifications
and this application is intended to cover any varia-
tions, uses, or adaptations of the invention following,
in general, the principles of the invention and
including such departures from the present disclosure
as come within known or customary practice within the
art to which the invention pertains and as may be ap-
plied to the essential features hereinbefore set forth,
and as follows in the scope of the appended claims.
