Note: Descriptions are shown in the official language in which they were submitted.
2Q78657
D E S C R I P T I 0 N
Human Protein C Expression Vector
Technical Field
The present invention relates to DNA fragments encoding
human protein C and to expression vectors of human protein C
lQ including them. The human protein C which is produced using
the expression vector accordin~ to the present invention or
the activated human protein C which is prepared by activa-
tion thereof can be used as an anticoagulant or fibrinolytic
accelerator.
In the present invention, the sequence of` DNAs is
abbreviated with bases constituting individual deoxyribonu- ~ :
cleotides and. for example, the bases are abbreviated as
follows:
Aadenine (representing deoxyadenylic acid)
Ccytosine ~representing deoxycytidylic acid)
Gguanine (representing deoxyguanylic acid)
Tthymine (representing deoxythymidylic acid)
:
Background Art
2~ Protein C is one of ~ymogens of plasma serine protease
and activated through limited proteolysis with a complex of
thrombin and thrombomodulin on the surface of blood plate-
lets or endothelial cells of blood vessels to convert into
serine protease, namely activated protein C, (abbreviated to
APC hereinafter).
APC exhibits its anticoagulative action by selective
hydrolysis of the activated factor V and activated factor
VIII in the blood coagulation system. This activity i.s known
to be enhanced by protein S. Further, APC has been thought
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to get its fibrinolysis-accelerating activity by cleaving
PAI-1, an inhibitor of tissue plasminogen activators.
The nucleotide sequence of the gene for human protein C
was already described by Foster DC (Proc. Natl. Acad. Sci.
USA, 82, 4673-4677 (1985) and the nucleoti~e sequence of
human protein C cDNA was shown in Nucleic Acids Res., 13,
5233-5247, (1985).
The amino acid sequence of human protein C is composed
of, as shown evidently in Proc. Natl. Acad. Sci. USA, 82,
4673-4677 (1985), the light chain (molecular weight: about
21,000) having Gla domain and epidermal growth factor (EG~)-
like domain on the amino terminus and the heavy chain
(molecular weight: about 41,000) comprising the activation
peptide and catalytic domain where both of the chains are
1~ disulfide-bonded (two chain type).
The light chain of human protein C comprises 153 amino
acids from Ala as the amino terminal to Leu as the carboxyl :
terminal, while the heavy chain includes 262 amino acids
from Asp as the amino terminal to Pro as the carboxyl termi-
20 nal, and they are biosynthesized in the form of (H2N-)-light - .
chain-Lys-Arg-heavy chain-(-COOH) in the cells and Lys-Arg
is cleaved into two strands while it is secreted from the
cells.
The light chain and the heavy chain are linked through
a disulfide bond at 141 Cys from the amino terminal of the
light chain and 120 Cys from the amino terminal of the heavy
chain.
Disclosure of Invention
An object of the present invention is to provide D~'A
fragments which are useful for expression of human protein
C, the expression vectors using said fragments and the
mammalian cells transformed with said expression vectors.
Another object of the present invention is to provide
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human protein C-encoding DNA fragments of high expression
efficiency.
These objects can be achieved by DNA fragments which
are prepared by removing n introns (where n = 1 to 6) from
the seven introns between eight exons in the pro'ein C gene
and ligating the remaining before and behind sequences.
The present invention is DNA fragments encoding human
protein C which are prepared by removing n (n = 1 to 6)
introns from 7 introns distributing between 8 exons in human
protein C gene and ligating the remaining before and behind
sequences.
The above-stated DNA fragment which is prepared by
removing 2 introns between exons 6 and 7 and between exons 7
and 8 in human protein C gene is preferred. Further, the
above-stated DNA fragment which is prepared by removing 5
introns between exons 3 and 4, exons ~ and 5, exons 5 and 6,
exons 6 and 7, and exons 7 and 8 is preferred.
The present invention also includes a DNA fragment
encoding human protein C that is constructed by incorporat-
ing at least one of the following DNA fragments into any one
intron, a plurality of introns or partially deleted introns
among n (n = 1 to 7) introns included in human protein C
2~ gene or the DNA fragments mentioned above:
a. a DNA fragment which accelerates transcription activity
in mammalian cells.
b. a DNA fragment which can be a replication origin of D~A
in mammalian cells and
c. a DNA fragment of adnovirus VA gene.
Moreover, the present inventi.on is an expression vector
which can be transduced into mammalian cells and contains a
promoter, the above-descri.bed DNA fragment and. when needcd,
the polyadenylation signal wehre the transcription of the
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above-stated DNA fragment is commanded by the promoter.
The present invention further includes human protein C-
producing mammalian cells which has been transformed by the
expression vectors. The cells are suitably selected from the
group consisting of BHK cells, C~0 cells, HeLa cells, C].27
cells and 293 cells.
The cells to be transformed may undergo other transfor-
mations with other expression vectors, before or after
transformation with the expression vectors according to the
present invention. For example, after transduction of human
protein C expression vector, pKEX2-gpt or p~A-gpt to be
stated later can be transduced.
The human protein C gene according to the present
invention has almost the same base sequence as the sequence
of 8975 bases from 1st to 8975th described in Fig. 4 of
Japanese Patent Specification Laid-open No. Tokkaisho 62-
111690 (Proc. Natl. Acad. Sci. USA., 82, 4674 (1985)), but
partially differs.
The gene comprises eight exons (called exon 1, 2, ...... 8,
respectively) and seven introns (called intron 1, 2, ... 7,
respectivel~
The exon 1 is the sequence of seventY bases from 1 A to
70 G in the base sequence shown in Fig. 4.
The intron 1 has the same base sequence as the sequence
of 1263 bases from 71 G to 1333 G given in Fig. 4 of the
above-stated patent specification except that
(1) there is G between 127 G and 12B C;
~2) there is A between 532 G and 533 C;
(3) there is C between 733 C and 734 A;
(4) 761 is not C but G;
(5) there are G and A in this order between 981 G and
982 C;
(6) there is C between 1300 T and 1301 C;
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(7) there is C between 1318 C and 1319 T;
The exon 2 is the sequence of 167 bases from 1334 A to
1500 A in the base sequence shown in Fig. 4.
The intron 2 has the same base sequence as the sequence
5 of 1462 bases from 1501 G to 2962 G given in Fig. 4 of the
above-stated patent specification except that
(1) there is C between 1739 C and 1740 A; .
(2) there is G between 2141 G and 2142 C;
(3) there is G between 2398 G and 2399 C;
(4) Ihere is C between 2672 G and 2673 C;
(5) there is no 2927 G.
The exon 3 is the sequence of 25 bases from 2963 C to
2987 G in the base sequence shown in Fig. 4.
The intron 3 is the sequence of 92 bases from 2988 G to
3079 G.
The exon 4 is the sequence of 138 bases from 3080 A to
3217 G in the base sequence shown in Fig. 4.
The intron 4 is the sequence of 102 bases from 3218 G
- to 3319 G.
The exon 5 is the sequence of 135 bases from 3320 A to
3454 G except that 3342 T is replaced with G in the base se-
quence shown in Fig. 4.
The intron 5 is the sequence of 2668 bases from 3455 G ~-
to 6122 G.
The exon 6 is the sequence of 143 bases from 6123 T to
6265 G in the base sequence shown in Fig. 4.
The intron 6 is the sequence of 873 bases from 6266 G
to 7138 G.
The exon 7 is the sequence of 118 bases from 7139 G to ~ `
7256 G i-n the base sequence shown in Fig. 4.
The intron 7 is the sequence of 1129 bases from 7257 G --
to 8385 G.
The exon 8 is the sequence cf 590 bases from 8386 G to
8975 G in the base sequence shown in Fig. 4.
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The DNA sequence according to the present invention can
be built up from the gene for human protein C.
The removal of an arbitrary intron can be attained by
effecting deletion mutagenesis in the intron region employ-
S ing the site-directed mutagenesis or the so-called cassette
mutagenesis techniques.
Further, it can be attained, as will be shown in the
examples, by substituting a part of *he human protei C
genomic DNA with the corresponding region of its cDNA uti-
lizing restriction sites in common.
As an example of the DNA sequence according to the
present invention, can be cited, as will be given in exam- ~
ples, the se~uence in which two introns between exons 6 and ~ -
7, and between e~ons 7 and 8 are deleted, respectively or
the sequence in which five introns are deleted between exons
3 and 4, 4 and S, S and 6, 6 and 7, and 7 and 8, respective-
ly.
In general, the partial mutation of intron base se-
quence causes no mutation at the protein level and the
possibility is low that the bodies having such mutation are
excluded in comparison with the mutation in the protein-
encoding regions. Therefore, it is thought that there are a
few differences in the intron sequences between human races,
individual bodies and so on. The DNA fragments according to
the present invention are permissible in such partial dif-
ferences of intron sequences and must not be limited to the
above-described intron sequences.
The D~A fragments encoding human protein C according to
the present invention includes at least one intron. This
intron can include DNA fragments which exert some actions or
effects in mammalian cell nuclei. The present invention
covers such recombinant DNA fragments, too.
The D~A fragments having some actions and effects in
mammalian cell nuclei mean, for example, enhancer: the DNA
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fragments accelerating transcription activity. replication
origin from an animal virus or adenovirus VA gene.
These incorporations. however, must not exert any
influence on the DNA sequence acting as the splicing signal
in the intron. Accordingly, such incorporation must be done
in the other regions than the splicing signal sequence.
Additionally, the DNA fragments to be incorporated must not
have the sequence functioning as a splice acceptor.
In the meantime, the partial removal of the sequence
can be permitted for convenient recombination before the
incorporation, unless the sequence is for splicing signal in
the intron. The simultaneous incorporation of DNA fragments
which have no special funtion is also permitted.
This DNA fragment incorporation is done by ~ltilizing
1~ appropriate restriction sites in the intron and further, by
creating suitable restriction sites through the site-
directed mutagenesis, when needed. Further, *he incorpora-
tion can be achieved by general recombination technology,
for example, utilizing a snthetic DNA as an adaptor.
As a DNA fragment accelerating transcription activity
in mammalian cells, is cited, for example, enhancers in
animal viruses such as SV40, human cytomegalovirus, BK
virus, EB virus, herpes simplex virus, or enhancers included
in animal genes such as antibody genes. The incorporation is
2~ not always limited only to the terminology "enhancer" but
any D~'A fragments having such activities can be utilized in
the present invention. For example, the DNA fragment in
HTLV-1 LTR responding to P40x of HTLV-1 to accelerate the
transcription activity, the D~'A fragment in the promoter
region of metallothionein-1 gene, responding to heavy metal
ions or glucocorticoid hormones to promote the transcription
activity or the like are cited.
As the DNA fragments which can be a DNA replication
origin in mammalian cells, are cited, for example, the
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replication origins in the D~As of animal viruses such as
SV40 or BK virus.
There are two genes, VAI and VAII in the adenovirus VA
gene and both of them or either of them can be used. It has
been thought that the VA gene is transcribed into VA RNA,
which is presumed to increase the e~pression efficiency.
In order to obtain human protein C using the DNA frag-
ments according to the present invention, the DNA fragments
are incorporated into an expression vector which is suitably
selected depending on the host vector system. Such expres-
sion vectors are, for example, vectors which are prepared by
incorporating suitable controlling regions into plasmids
originating from bacteria, DNA originating from bacterio-
phage, or DNAs of animal virus. Such controlling regions are
selected from, for example, adenovirus major late promoter,
SV40 early promoter, SV40 late promoter, mouse metallothio-
nein-I promoter, M~TV (mouse mammary tumor virus) promoter,
RSV (Rous sarcoma virus) promoter, human ~-actin promoter,
chicken ~-actin promoter. The vectors originating from
viruses such as bovine papilloma virus `are also preferable.
As a polyadenylation signal according to the present
invention, may be used any signal as long as it works in
mammalian cells. Accordingly it may originate from animal
viruses or a polYadenylation signal from animal genes (class
2~ 2). For example, the polyadenylation signal existing in the
; early gene region of SV40 can be cited. Or the signal in
human protein C gene itself may be used.
The integration of the above-stated DNA fragments into
such vectors can be carried out by a known method per se,
for example, the method described by Miura 0. et al~, J.
; Clin. Invest., 83, 1958-1604 (1989) and others.
The mammalian cells for the host may be either human
cells or cells of any animals other than human and, for
example, CHO, C127, LHK, Cosl, Cos7, LM, NIH3T3, 293, HeLa
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or the like may be cited. Particularly, C~O, BHK and 293 are
preferred.
The transfection of the above-stated expression vector
into these cells also can be effected by the well~known
methods per se in the prior art, for example, described in
the follo~ing literature: Spandidos D. A. and ~'ilkie ~T, ~
Expression of Exogenous DNA in Mammalian Cells, Ed. Hames B.
D. and Higgins S. J., Transcription and Translation, IRL
Press Oxford pp l - 48 and others.
The transformed cells are cultured by a usual method
under conditions suitable for the cells so that the target
protein can be recovered from the culture supernatant.
The concentration of human protein C obtained according
to the present invention can be determined by the method ..
disclosed in Japanese Patent Specification Laid-open No.
Tokkaisho 61-283868. This method can also determine only
human protein C having Gla.
The barium adsorption method and the ion-exchange
chromatography can be utilized for purification of the
protein C produced according to the present invention, but
the affinity column chromatography using an anti-human
protein C monoclonal antibody ~hich recognizes the conforma-
tion change in the Gla domain caused by calcium ion is
particularly preferablY used (see Japanese Patent Specifica-
tion Laid-open No. Tokkaisho 64-85091).
The method is an excellent process because it can pu-
rifiy only human protein C having Gla and can use EDTA, a
mild eluent.
Human protein C is converted into APC by removal of the
activation peptide r-anging from the 12th amino acid to the
amino terminus (abbreviated to N-terminus hereinafter) from
the heavy chain by the protein C activation enzyme.
The protein C activation en~yme is, for example. throm-
bin, thrombin-thrombomodulin complex, snake venom or the
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like.
The use of the expression vector of human protein C
solves the problems on contamination with viruses which may
cause troubles. Moreover, stabilized supply can be achieved
because it becomes unnecessary to demand human blood as a
starting substance.
In general, the e~pression of cDNA in mammalian cells
is effected by artificial addition of the splice donor and
the acceptor to the noncoding region on the 5' or 3'-side,
but it becomes unnecessary, when the DNA fragments according
to the present invention are used.
So far, a variety of modified human protein C have been
prepared by partial modification of the sequence o* cDNA in
the system for expression of human protein C cDNA in mamma-
la lian cells.
For example, a cDNA modified at the connecting peptide[Lys (156) Arg (157)] region (see Japanese Patent Laid-open
No. Tokkaisho 64-85084) is cited for the purpose of in-
creased two chain processing efficiency, another cD~A where
the activation peptide region is deleted for direct expres-
sion of activated protein C, and one where the neighborhood
is further modified (see Japanese Patent Specificatlon Laid-
open No. Tokkaihei 2-2338).
As these modified cDNAs correspond to partial modifica-
2a tion of exon 6 sequence, the part of exon 6 in the D~TAfragment according to the present invention is replaced with
a DNA fragment modified so as to correspond to the above-
stated modification instead of the naturally occurring DNA
fragment so that the same modified human protein C as these 30
proteins can be obtained. In other words, direct expression
of human protein C of increased two chain processing effi
ciency and of activated human protein C can be realized.
Additionally, modified human protein C which are pro-
duced by other modifications in human protein C cDNA than
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above-stated examples also can be prepared by Usillg the
correspondingly modified DNA fragments in the D~A fragments
according to the present invention.
Each gene in mammalian cells is not always expressed
constantly at a certain level. Some genes are expressed only
in the cells differentiated in a specific tissue, while some
other genes are expressed only at a specific differentiation -.
stage or only before the start of differentiation.
Further, some genes change the expression depending on
the outside environments. For example, the expression is
increased or decreased depending on nutrient conditions such
as glucose concentration, temperature, metal ion concentra-
tion or signal transduction substances in the living bo~y. A
living body copes with the surrounding environments by con-
trolling the gene expression to maintain its life activity.
Multistage fine mechanisms participates in such controlof gene expression in mammalian cells. One of them is the
control at the transcription stage. The transcription is
controlled through mutual action between the promoter/
enhancer region of the gene and the DNA-binding proteins in
the nuclei.
In this case, the kinds and amounts of DNA-binding
proteins vary reflecting the outside environments and the
direction and degree of differentiation. Further, the ex-
2~ pression is also controlled by varying the half-life of
mR~A. For example, it has been known that the presence of
estrogen largely prolongs the half-life of some kind of
mRNA.
In addition, the expression is presumably controlled at
the stages of mRNA transport from the nucleus to the cyto-
plasm, translation and intracellular transport of the formed
protein, too.
By the way, almost all genes of mammalian cells have
introns. The sequences are spliced out in the nuclei after
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transcription, and this stage is also thought to give mamma-
lian cells -the chance to control the gene expression.
Thus, it is more advantageous than the expression with
cDNA to use genomic DNA in the Production of a substance in
mammalian cells because the expression can be controlled
through the modification of the splicing efficiency or the
transporting efficiency from the nuclei into the cytoplasm
of the participating mRNA.
The control of expression efficiency can be utilized so
that the protein production is inhibited and the protein-
synthesizing mechanism is directed to other functions such
as cell division at the stage in no need of the protein
synthesis, for example, a cell number-increasing step, while
the mechanism is directed to the protein synthesis, when
lS becomes necessary. In general, however, the expression
efficiency of genomic DNA is lower than that of cDNA in many
cases. The DNA fragments according to the present invention
has enabled extracellular control of the expression effi-
ciency to secure a practical level of the efficiency.
The DNA fragments according to the present invention is
also to provide "a place where the sequence relating to
transcription control is to be ~ut". The DNA sequence con-
trolling the transcription is placed near the promoter to
develop its effect and it has been known that the site is on
2~ the upper stream of the promoter, but in some cases, it is
effective even on the down stream of the transcription
initiation point. An antibody enhancer is cited as an exam-
ple.
In the case of the expression with cDNA, the primary
structure of protein is not changed and such DNA sequence
needs to be placed between the transcription initiation site
and the translation initiation point. In this case, however,
there is the possibility of decreased translation efficienc~
because the distance between the transcription initiation
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site and the translation initiation site is long.
On the contrary, the DNA fragments according to the
present invention do not change the primary structure of
protein even ~hen modified by partial substitution or incop-
oration as long as modification is done in introns and thereis no problem even when a transcription-controlling sequence
such as an enhancer is incorporated.
In this case, the sequence from the transcription
startin~ point to the translation-starting point can be made
optimal length and sequence order for translation efficien-
cy .
Meanwhile, similar things are also possible in the caseof genomic D~A, but low expression efficiency is an inherent
problem, as stated above. ~'hen the DNA fragments according
to the present invention is used, the sequence of an enhanc-
er or the like can be integrated, as a practical level of
expression efficiency is secured.
The similar conception of a place where the DNA se-
- quence is to be placed also can be applied to incorporation
of the replication origin of polyoma viruses such as SV40,
or VA gene of adenovirus. The VA gene is a sequence which
can increase the protein production. when the major late
promoter of adenovirus is used as a promoter.
An expression vector which is prepared by incorporating
2~ the sequence into an intron of the DNA fragments according
to the present invention is transfected into mammalian cells
and screened using the expression of the target protein as
an indication. At this time, the cells expressing the target
protein can be regarded almost always as to include the VA
gene in the chromosome DNA .
On the contrary, in the case of an expression vector
where the VA gene is incorPorated into the outside of the
protein transcription unit, the cells producing the tar~et
protein do not always inc].ude the VA gene, even when the
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gene is on the same vector. It is why a complicated recombi-
nation may happen during the integration of the expression
vector into the chromosome DNA in some cases.
As a matter of cQurse, it cannot be accurately concluded
that VA gene is incorporated into the chromosome of the
protin-expressing cells on the reason that the target pro-
tein has been expressed, in the case of cotransfection of an
expression veclor having the transcription unit of the
target protein and a plasmid having VA gene, too.
By the way, the judgment on whether VA gene is incorpo-
rated into a cell or not can be made by the Southern hybri-
di~ation method or the like. However, in the expression
vectors prepared by incorporation of the VA gene into the
introns of the DNA fragments according to the present inven-
tion, the ,judgment can be made by examining the expression
of the target protein as an indication. and so a method
which can simply investigate a plurality of specimens simul-
taneously such as ELISA can be advantageously used.
Particularly, when the target protein is secretory one,
the Southern hybridization method always requires the de-
struction of the cells for extraction of the DNA, while only
collection of the supernatant of cell culture mixture is
required enough for the method.
It is a matter of course that VA RNA is formed by tran-
~5 scription even in the case where the VA gene integrated in
intron is incorporated into the chromosome DNA of the ce]]s,
and it is independent from splicing of the mRNA encoding the
target protein.
It can be also applied to the case of incorporation of
replication origin in*o the introns of the DNA fragments
according to the present invention. For example, when an ex-
pression vector including a DNA fragment as a replication
origin of SV40 is integrated into the chromosome DNA of
mammalian cells and T-antigen of SV40 is expressed, the
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replication of DN~ can be started from the replication
ori~in, thus the copies of the transcription units of the
target protein nearby are increased, resulting in increase
of the expression.
In the case, when an expression vector prepared by
incorporation of such a D~IA fragment as a replication origin
into the introns of the DNA fragments according to the
present invention is used, it can be concluded that the
replaction origin has been also incoporated into the chro-
mosome DNA of mammalian cells on the reason that the target
protein has been expressed, as in the case of ~A gene.
VA gene can work so long as it is incorporated into an
arbitrary place in the chromosome of mammalian cells, but -
the replication origin requires the location near the tran-
1~ scription unit (a set of DNA sequence series of promoter
region, protein-encoding region, polyA addition signal and
so on).
But, the Southern hybridization method or the like
gives only information that the replication origin is some-
where in the chromosome of mammalian cells. On the contrary,
the use of the DNA fragments according to the present inven-
tion substantially always ensures the location of the repli-
cation origin in the transcription unit of the target cells,
every when the target protein has been expressed.
In addition, use of the DNA fragments according to the
present invention enables the replication origin to locate
near the center of the transcription unit of the target
protein and more efficient gene amplification can be expect-
ed. By the way, even when a replication origin in introns is
incorporated into the chromosome DNA of mammalian cells, it
functions as the replication origin in the chromosome, as a
- matter of course, and it is indifferent from splicing of the
mR~rA encoding the target protein.
The above-described effects can be also expected in the
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case where VA gene of adenovirus or the replication origin
is incorporated into the introns of genomic DNA, but there
is a problem of low expression ef-~iciency of the target
protein. But, the application of DNA fragments according to
present invention makes the above-stated things possible as
ensuring a practical level of expression.
Example
The present invention willed illustrated in more detail
with the following examples, but the present invention is
not limited to them at all.
Following methods are used in the examples according to
present invention.
Cleavage of DNA
The cleavage of 1 ~ g of plasmid DNA or M13 phage repli-
cative form (RF) DNA or DNA fragments was carried out in 10
J/l of buffer solution using 4 to 10 units of a restriction
enzyme and keeping them at the temperature indicated by the
maker for 2 hours. The buffer solution was accompanied by
the restriction enzyme.
Separation and Collection of DNA fragments
The DNA fragments cleaved with the restriction enzyme
were separated by agarose gel electrophoresis with a subma-
rine type electrophoresis system. The agarose gel containing
the target DNA fragments is cut out and the fragments were
recovered with GENECLEAN II (Registered Trade Mark) (Bio 101
Co.). The procedure followed the manual appended to the
reagent. Unless otherwise noted, 0.8 % agarose gel was used.
Ligation of DNA fragments
; It was carried out with the DNA ligation kit (TAKARA
SHUZ0 C0. LTD.). The procedure followed the manuals appended
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to the kit.
B nting of D~TA fragments
It was effected using a blunting kit (TAKARA SHUZ0 C0.
LTD.). The procedure followed the manual appended.
Transformation of Escherichea coli
Less than 20~ l of DNA solution was added to the compe-
tent cells of E. coli HB101 strain (TAKARA SHUZ0 Co. Ltd.),
placed on ice for 1 hour, and dipped in the water bath at
42 C for 1 minute, and placed on ice, again for 5 minutes.
The mi~ture was added to 1 ml of L-broth and subjected to
shake culture for 1 hour, then a part (50 to 300 ~ l) of the
culture mixture was spread on an ampicillin plate (L-broth,
agar 15 g/l, ampicillin 50 llg/ml) and cultured at 37 C one
overnight to form colonies.
Small-scale preparation of plasmid DNA
The alkaline lysis method was employed for preparation
(see [Molecular Cloning] T. Manuals, Cold Spring Harbor
Laboratory (1982), P 368). ~'hen needed, further purification
was effected with GENECLEAN II (Registered Trade Mark).
Large-scale preparation of plasmid DNA
2~ The alkaline lysis method (see [Transcription and
Translation] B. D. Hames, IRL press, (1384) P 8) and the
CsCl equilibrium density-gradient centrifugation were used.
The rotor for ultracentrifugation was Hitachi's RP-67VF
vertical rotor. CsCl was removed not by dialysis but 4-time
dilution.with TE (10 mM Tris-HCl pH 8.0 1 mM EDTA) followed
by twice precipitation with ethanol.
Determination of nucleotide sequence ,
A single-stranded DNA was obtained by incorporatin~ the
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DNA fragment to be determined into bacteriophage M13.
The procedure is described in the manual appended to
"M13 cloning kit" made by Amasham Co. Ltd. A large amount of
single-stranded DNA was prepared by the me~hod described in
the manual appended to "oligonucleotide-directed in vitro
mutagenesis system" (Amasham Co.). The resultant single-
stranded DNA was used as a temperate and a synthetic 20mer
DNA near the region to be examined whose synthetic method
will be explained later T~as used as a primer to carry out
the sequencing reaction using Dye DeoxyTM Terminator Taq Se-
quencing kit (Applied Biosystems Co. Ltd.).
The sample obtained by the reaction was analyzed with
Applied Biosystem's 373A DNA sequencer to determine the
nucleotide sequence.
Chemical synthesis and purification of DNA fragments
The synthesis was effected using the Applied Biosys-
tem's 380A DNA synthesizer under the "Tr ON, Auto condi-
tions. The products were purified using "oligonucleotide
purification cartridge" of Applied Biosystem in accordance
with the appended manual.
On the preparation of the DNA fragments and the expres-
sion vectors according to the present invention, following
2~ plasmids and DNA fragments were used.
.
pDX/PC
This is a human protein C expression vector having a
~ full-length protein C cD~A under the control of the adenovi-
j 30 rus type-2 major late promoter, which is described by Fos-
ter, D.C. et al., Biochemistry 26, 7003-7011 (1987) or in
Japanese Patent Specification Laid-open No. Tokkaisho 62-
11690.
The functional part constituting the e~pression vector
2~7S~7
- 19 - ,
is described by Busby S. et al., Nature, 316, 271-273 (198~)
and Berkner, K.L. et al., Nuc. Acids Res., 13, 841-8~7
(1985).
.
Human protein C genomic DNA
Human protein C genomic DNTA was disclosed in Japanese
Patent Specification Laid-open ~'o. Tokkaisho 62-111690.
In the following examples, the DNA fragment spanning
from the EcoRI site in the intron between exon 2 and exon 3
to the EcoRI site which is located at 4.4 kbp upstream of it
(called R4.4-1 hereinafter), and the 6.2 kbp DNA fragment
spanning from the EcoRI site in the intron between exon 2
and exon 3 to the EcoPI site in the intron between exon 7
and exon 8 (called R2-14 hereinafter) were used after sub-
cloning them into the EcoRI site of PUC9.
Zem228 and Zem229
These expression vectors are disclosed in JapanesePatent Specification Laid-open No. Tokkaihei 2-2338. In
these vectors, the promoter of mouse metallothionein I gene
is used and neomycin resistant gene or dihydrofolate reduc-
tase gene is used as a selection marker.
pSV2-dhfr
It is registered in American Type Culture Col]ections
(ATCC) as No. 37146.
228/PC594
It is a human protein C expression vector which is
prepared by incorporating human protein C cDNA into Baml~I
site of the above-stated Zem228. The human protein C cDNA is
identical with that in the above-stated PDX/PC.
229/PC962
, . - , : , ,
. . ~ :
'. . . :'
. .
~: i
2~7~7
- 20 -
It is a modified human protein C expression vector
which is prepared by incorporating a modified human protein
C cDNA PC962 into the Bam~lI site of the above-stated Zem229.
The PC962 is disclosed in Japanese Patent Specification
Laid-open No. To~kaisho 64-85084.
Rc/CMV
It is commercially available from Invitrogen Co..
KEX2
The KEX2 gene is a region shown as KEX2 in a plasmid
which is disclosed in Japanese Patent Specification Laid-
open ~o. Tokkaihei 2-2338.
pSV2-gpt
It is registered in ATCC as ~o. 37145.
Example 1
Preparation of human protei.n C expression vector
(1) Preparation of 228/AC-PC9001
In order to destroy the BalI site in the neomycin
resistant gene in 228/PC594, 228/PC594 was digested with
AatI and ClaI and the resultant D~'A fragments were blunted
at their ends.
The larger fragment was separated, subjected to intra-
molecular ligation, then transduced into E. coli HB 101 to
increase the amount of the plasmid. Then, the plasmid was
digested with BalI and SacII (both of them have the cleavage
sites onlY in protein C cD~A) and the larger BalI-SacII
fragment was recovered.
Two plasmids which were prepared by subcloning the
- - . . . . . , , . . -
- : . .
.
, ~ .
- 21 - 2 9 rl ~ ~ ~ ri
above-stated R4.4-1 and R2-14 in PUC9 were digested with
BalI and PstI and EcoRI, and SacII and EcoRI, respecti~ely
to collect the BalI-EcoRI fragment of about 1.65 kbp and the
EcoRI-SacII fragment of about 4.5 kbp.
These two DNA fragments and the above-stated BalI-SacII
fra~ment ~ere ligated termolecularly to form 228/AC-PC9001.
Thus, the expression veclor has human protein C gene from
which two introns between exons 6 and 7 and exons 7 and 8
were removed. The expression vector lose most of neomycin
resistant gene.
(2) Preparation of 229-PC9002
The above-stated plasmid resulting from subcloning
R4.4-1 in PUC9 was digested with BalI, EcoRI and PstI to
recover a BalI-EcoRI DNA fragment of about 1.65 kbp (A
fragment)
Meanwhile, the above-stated plasmid resulting from
subcloning R2-14 in PUC9 was digested with XcyI, dephospho-
. rylated with alkaline phosphatase, further digested with
EcoRI to recover an EcoRI-XcyI DNA fragment of about 1.22
kbp (B fragment~.
Subsequently, the A fragment and the B fragment were
ligated and digested with BalI to recover a BalI-XcyI DNA
fragment of about 2.87 kbp, then the fragment was phosphory-
2~ lated with T4 polynucleotide kinase (TAKARA SHUZ0 Co.Ltd.)(C fragment).
The DN~ fragment from XcyI (No.2902 residue) to BstEII
(No.3082 residue) in the human protein C gene where the
intron between exon 3 and exon 4 had been removed was chemi-
cally synthesized. The sequence was disclosed in the abo~e-
stated specification of Japanese Patent Laid-open No. Tok-
kaisho 62-111690 (or Proc. Natl. Acad. Sci. USA, 82, 4674
(198~)).
After on~ the antisense strand was phosphor~lated wi.th
\
- 22 - 2~ 7~ ~g 7
T4 polynucleotide ~inase, both strands were annealed (D
fragment). 228/PC594 was digested with Sac I~ and BstEII and
a BstEII-SacII DNA fragment of about 0.36 kbp was separated
by means of 2% agarose-gel electrophoresis (E fragment).
The ligation between D fragment and E fragment was
followed by digestion with SacII to recover a XcyI-SacII D~A
fragment of about 0.45 kbp through the 2 % agarose gel
electrophoresis. Then, the fragment was phosphor~lated with
T4 polynucleotide kinase (F fragment).
Further. 229/PC962 was digested with BalI and SacII and
the larger fragment was collected (G fragment). Finally, the
C fragment, the F fragment and the G fragment were termolec-
ularly ligated to form 229-PC9002. This vector contains a
human protein C gene where other introns than two introns
between exon 1 and exon 2, and between exon 2 and exon 3 are
removed under the control of the mouse metallothionein 1
promoter and has the transcription unit of dhfr gene on the
same plasmid.
(3) Preparation of TZml-PC9002
Zem228 has two EcoRI sites and the partial digestion
with EcoRI and agarose gel electrophoresis gave the only one -.
site-digested product.
The DNA lragment was blunted and subjected to self-liga-
2~ tion. The product was transduced into E. coli, proliferated,recovered, then completely digested with EcoRI, blunted
again, further self-ligated, and transduced into E. coli to
proliferate ~he plasmid. Thus, two EcoRI sites were deleted.
Then, a short double-stranded synthetic DNA having
BamHI cleavage terminals on both ends and EcoRI site inside
was incorporated into unique BamHI site in the plasmid to
prepare an expression vector ZemZ28R having the EcoRI cleav-
age site immediatelY downstream o~ the mouse metallothionein
1 promoter.
- !
- - . :
`.
: ,, : :~ , . . . :
: - , -: . ,. , , . `.: , ~ :
. :.
:, . .
.
- 23 - ~ ~r~ 7
The Zem228R was digested with EcoRI and HindIII and
subjected to agarose-gel electrophoresis to collect the
large fragment. Further, the HindIII-EcoRI fragment of 1.03
kbp was excised from the above-stated pDX/PC and both of
fragments were ligated to give ZmB3. This expression vector
has the transcription unit of neomycin resistant gene as
well as adenovirus major late promoter.
The ZmB3 was digested with EcoRI and ClaI to separate
the large fragment having neomycin resistant gene (H frag-
ment). Fllrther, 229-PC9002 was digested with BalI and ClaI
to recover the larger fragment (I fragment).
229/PC962 was digested with EcoRI and BalI and subject-
ed to agarose-gel electrophoresis to recover the smaller
fragment (J fragment). The ~ fragment, the I fragment and
the J fragment were termolecularly ligated to give TZml-
; PC9002.
This expression vector contains a human protein C genewhere other than two introns between exons 1 and 2, and
exons 2 and 3, respectively, are removed under the control
of the adenovirus major late promoter and has the transcrip-
tion unit of neomycin resistant gene on the same plasmid.
(4) Preparatio of 228-PC9001
228-PC9001 was obtained by termolecular ligation be-
tween the smaller fragment resulting from digestion of228/AC-PCsool with KpnI and SacII, the smaller fragment by
digestion of 229-PC9002 with SfiI and KpnI and the larger
fragment from digestion of 228/PC594 with SfiI and SacII.
The expression vector expresses a human protein C gene
where two introns between exons ~ and 7, and exons 7 and 8
were removed by the mouse metallothionein I promoter and has
the transcription unit of neomycin resistant gene on the
same plasmid.
- ~
:;
-- 24 - 2~7~
(5) Preparation of 228-PC9002
` 22~-PC9002 was formed by ligation between the smaller
fragment from digestion of 229-PC9002 with SfiI and SacIl
and the larger fra~ment from digestion of 228/PC594 with
SfiI and Sac II.
This e~pression vector expresses a human protein C gene
where other than two introns between exons 1 and 2, and
exons 2 and 3 were removed by the mouse metallothionein 1
promoter, and has the transcription unit of neomycin resist-
ant gene on the same plasmid.
(6) Preparation of TZm4-PC9002
TZm4-PC9002 corresponds to the sequence of the TZml-
PC9002 from which the sequence from the top of the second
branch of the tripartite leader sequence of adnovirus to the
37th residue upstream of the translation initiation site of
human protein C is deleted. The upstream sequence of human
protein C initiation codon is shown by Beckmann et al., Nuc.
: .. Acids Res., 13, 5233 (1985).
In other words, TZm4-PC9002 has only the first branch
of the tripartite leader sequence and lacks the sequences of
the second and third branches, the sequence containing the
splice donor and the splice acceptor on the downstream of
them, and a part of S'-noncoding region of human protein C
~5 cDNA.
This deletion mutagenesis aims at increased translation
efficiency by shortening the distance from the transcription
start site to the translation start site and increased
expression efficiency of human protein C by removal of
introns.for reducing the splicing load.
Since the DNA sequence encoding human protein C accord-
ing to the present invention includes introns, it should be
noted that there is in no need of new intron incorporation
into the untranslated regions. The deletion mutagenesis was
:
- , . : . .~, ,. , .: . ............................... -
.
~ ," ' ,
- ~ - 2 ~
effected by the so-called cassette mutagenesis ~hich ~Yill be
stated in the following:
TZml-PC9002 was digested with SacII, then PmaCI to
separate the larger ~iNA fragment (K frag~ient). The double-
stranded DNA fragment where the part to ~ie deleted wasremoved from the sequence from the PmaCI site in the ade-
novirus major late promoter to the BalI site in the DNA
sequence encoding human protein C in TZml-PC9002 was chemi-
cally synthesized.
The synthesis was carried out in three blocks. These
fragments were phosphorylated at the 5' terminals, annealed
and ligated to give the desired DNA fragment.
The upstream terminal of this fragment had PmaCI
cleaved end, and the downstream terminal had 6 base BalI
recognition site followed by two base spacer (G, C) and
SacII cleaved end.
The synthetic PmaCI-SacII fragment and the K fragment
were ligated and the product was transduced into E. coli
~B101. The plasmid obtained was digested with BalI and the
produced smaller fragment was recovered (L fragment).
The two BalI sites on this plasmid are in the neomycin
resistant gene and in the synthetic DNA. Then. TZml-PC9002
was digested with BalI, dephosphorylated with al~aline
phosphatase and the larger DNiA Iragment was recovered and
ligated with L-fragment to give TZm4-PC9002.
(7) Preparation of TZm5-PC9002
TZml-PC9002 was digested with BalI, XbaI and ClaI to
prepare a DNA fragment of 4.66 kbp (M fragment). A DNA
fragment.ranging from the BalI site at about 30 bp down-
stream of the initiation codon to the 103 bp upstream of
BalI site in human protein C was synthesi~ed in two blocks
with ~indIII cleaved end on the upstream terminus. Four
strands of synthetic DNA were phosphorylated on the 5'
.
-
.
.~
- 26 - ~7~67 -
terminals, annealed and ligated. The product was digested
with BalI and HindIII and subjected to 2% agarose-gel elec-
trophoresis to collect a DNA fragment of 0.11 kbp ~N frag-
ment). The M fragment and the N fragment were termolecularly
ligated with HindIII-XbaI large fragment of Rc/CMV to give
TZmS-PC9002. This is an expression vector whose transcrip- ~-;
tion is driven by cytomegalovirus IE enhancer/promoter.
:, .
(8) Preparation of TZm9-PC9002
TZm5-PC9002 was digested with HindIII and MluI, de-
phosphorylated, and blunted (O fragment). TZml-PC9002 was :
digested with KpnI and XhoI and subjected to 2% agarose-gel
electrophoresis to collect a DNA fragment of 0.42 kbp, which
was blunted. The fragment was ligated with-the 0 fragment
and the product was analyzed by restriction enzyme cleavage
to select the plasmid into which the adenovirus major late
promoter was incorporated in such a direction that it can
command the expression of human protein C as TZm9-PC9002.
~! . .
(9) PreParation of TZml6-PC9002 .:
TZmS-PC9002 was digested with Asp700 and HindIII to
collect the larger fragment (P fragment). Further, TZm9-
PC9002 was digested with NruI and HindIII to collect the
smaller fragment (Q fragment). Rc/CMV was digested with BanI
1`~ 25 to collect a DNA fragment of 1.69 kbp, which was digested
¦ with Asp700 to collect the larger fragment. This DNA frag-
~: ~ ment, P fragment and 4 fragment were termolecularly ligated
. to prepare TZml6-PC9002, an expression vector having human
' cytomegalovirus IE enhancer and Adenovirus major late pro-
. 30 moter.
~ I .. ~;. .
'~ (10) Preparation of TZm20-PC9002
~ TZmS-PC9002 was digested with SfuI, blunted, additi.on-
- aLly digested with SfiI to recover the larger fragment. This
..
29~g~7
- 27 -
DNA fragment was ligated with the smaller fragment which is ~:
obtained by digestin~ pSV2-gpt with BamHI, bluntin~ the
digestion product followed by additional digestion with SfiI
to give TZm20-PC9002. This has the same transcription unit
as in TZm5-PC9002, but has Eco-gpt gene as a selection
i marker.
(11) Preparation of TZm31-PC9002
A DNA fragment ranging -from adenovirus tripartite leader
leader sequence to the BalI cleavage site at about 30 bp
downstream of the initiation codon of human Protein C gene
in TZml-PC9002 was chemically synthesized in four blocks
with HindIII cleaved end on the upstream terminus. Six syn-
thetic D~s other than two synthetic DNAs whose ~' terminal
1~ was to constitute both terminals of the DNA fragment were
phosphorylated on their 5' terminals. After annealing of the
complementary chain, ligation was effected and a DNA frag-
ment of 0.53 ~bp was recovered by 2 % agarose-gel electro-
phoresis. The HindIII-BalI fragment was to be called R frag-
', 20 ment. TZm5-PC9002 was digested with BalI and SfiI to collect
-~ the smaller fragment (S fragment). Further, TZm5-PC9002 was
digested with HindIII and SfiI to collect a smaller frag-
ment. The DNA fragment, the R fragment and the S fragment
were termolecularlY ligated to give TZm31-PC9002.
This has the same basic structure as that of TZm5-
PC9002, but includes the adenovirus tripartite leader se-
quence in the 5' untranslated region.
(12) Preparation of TZmS-PC9004
Human adenovirus II DNA (about 36 kbp) was digested with
HindIII to recover a DNA fragment of 5.32 kbp, which was
~ digested with HpaI to collect a DNA fragment of 1.33 kbp.
; The DNA fragment was subcloned into the HindIII-HincII site
in PUC8 to gl~-e PUC-VA. The PUC-VA was digested with EcoRI
,
- . .;
,
. .
~ . . .
2~7~S7
- 28 -
and NruI to collect the smaller fragment (T-fragment). TZm5-
PC9002 was digested with NruI and SfiI to collect the small-
er fragment (U fragment). Further, TZm5-PC9002 was digested
with SfiI and EcoRI to collect the larger fragment. This DNA
fragment, the T fragment and the U fragment were subjected
to termolecular ligation to give TZm5-PC9004.
This has the same basic structure as that of TZm5-
PC9002, but includes adenovirus VAI and VAII gençs in the
second intron of human protein C.
,
(13) Preparation of TZm31-PC9004
The smaller fragment which was prepared by digestion of
TZm5-PC9002 with HindIII and SfiI, the larger fragment from
digestion of T2m5-PC9004 with SfiI and Bgl II, and the
medium fragment from digestion of TZm31-PC9002 with HindIII
and BglII were termolecularly ligated to give TZm31-PC900~.
This has the same basic structure as that of TZm31-
PC9002, but includes adenovirus VAI and VAII genes in the
. second intron of human protein C.
(14) Preparation of TZm5-PC9005
TZm~-PC9002 was digested with BstXI to recover the
larger fragment, which was ligated with a single stranded
' synthetic adaptor DNA which converts the upstream terminal
of the uppermost BstXI site in the first intron of human
protein C gene into the pairing terminal of MluI, then the
terminals were phosphorylated using T4 polynucleotide ki-
nase. The product was digested with SfiI to recover the
larger fragment (V fragment). TZm5-PC9002 was digested with
SfiI and ScaI to recover the larger fragment (W fragment).
Rc/CMV was digested with BanI to collect a DNA fragment of
1.69 kbp, further digested with MluI to give a DNA fragment
of 0.49 kbp.
This DNA fragment. the V fragment and the W fragment
- .. . , ,. : ~,
;:
2~786~7
- 29 -
were subjected to termolecular ligation to give TZm5-PC9005,
an expression vector including IE enhancer of human cytome-
galovirus in the first intron of human protein C gene.
(lS) Preparation of TZm5-PC9006
.
Rc/CMV is digested with BanI to recover a DNA fragment
of 1.69 ~bp followed by digestion with NruI to obtain a DNA
fragment of 0.51 kbp (X fragment). Further, TZmS-PC9002 was
digested with BstXI to recover the iarger fragment, ~hich
was ligated with a single stranded synthetic adaptor DNA
which converts the upstream terminal of the uppermost BstXI
site in the first intron of human protein C gene into the
pairing terminal with the BanI terminal of the X fragment,
then the terminals were phosphorylated using T4 polynucleo-
tide kinase. The product was digested with SfiI to recoverthe larger fragment (Y fragment). TZm5-PC9002 was digested
with ScaI and SfiI to recover the larger fragment. This DI~A
fragment, the ~ fragment and the Y fragment were termolecu-
.. larly ligated to give TZm5-PC9006, an expression vector
including human cytomegalovirus I~ enhancer in the first
intron of human protein C gene. It is different from TZmS-
PC9005 in the direction of the human cytomegalovirus IE
enhancer.
(16) Preparation of TZml-PC9005 and TZml-PC9006
The larger fragment from digestion of TZmS-PC900S with
I TthlllI was digested with ApaI to recover the smaller frag- ~-
ment. This D~A fragment, the smaller fragment from digestion
of TZml-PC9002 with ClaI and ApaI and the larger fragment
from digestion of TZml-PC9002 with TthlllI and ClaI were
subjected to termolecular ligation to give TZml-PC900S.
This has the same basic structure as that of TZml-PC9002 but
includes human cytomegalovirus IE enhancer in the first
intron of human protein C gene. TZmS-PC900S was replaced
,
. ,...... , ~ .
," . ~
. .
,
- ~ .. ' '
, :, . .:
, ,
2B7~fi~7
- 30 -
with TZmS-PC9006 to give TZrn5-PC9006.
(17) Preparation of TZm9-PC9005 and TZm9-PC9006
The larger fragments from digestion of TZm5-PC9005 and
5 TZm5-PC9006 with HindIII and SfiI were ligated with the .-
smaller fragment from digestion of TZm9-PC9002 with HindIII
and SfiI, respectively, to give TZm9-PC9005 and TZm9-PC9006.
~ They have the same basic structure as that of TZm9-PC9002,
i but include human cytomegalovirus IE enhancer in the first
intron of the human protein C gene.
(18) Preparation of 228-PC9005 and 228-PC9006
The medium fragment from digestion of TZm5-PC9005 or .
TZm5-PC9006 with BalI and SacII, the larger fragment from
digestion of 228-PC9002 with SacII and SfiI and the smaller
fragment from digestion of 228-PC9002 with BalI and SfiI
were termolecularly ligated to give 228-PC9005 and 228-
PC9006, respectively. They are egual to 228-PC9002 in their
- ......... basic structure, but have human cytomegalovirus IE enhancer
in the first intron of the human protein C gene.
.:
(19) Preparation of pkEX2-gpt
KEX2/Zem228 was digested with BamHI to cut out the KEX2
gene and cloned into the Bam~I site of the ZmB3. The frag-
2~ ment into which the gene is incorporated in the directionthat the transcription of KEX2 gene is commanded by the
adenovirus promoter was selected as ZmB3-KEX2. The Zm~3-KEX2
was digested with KpnI and AatII and the resultant smaller
; fragment was blunted (Z fragment). pSV2-gpt was digested
- 30 with EcoRI, dephosphorylated and blunted. The DNA fragment ~-
was ligated with the Z fragment.to give pKEX2-gpt.
(20) Preparation of pVA-gpt
The P~C-VA was digested wi.th SmaI and ~'ruI and the
. .
.
r
2~7~7
- 31 -
resultant smaller fragment was ligated with the fragment
which was prepared by digestion of pSV2-gpt with EcoRI,
followed by dephosphorylation and blunting to give pVA-gpt.
Example 2
Expression of human protein C in mammalian cells
BHK-21 cells (ATCC CCL-10) or 293 cells (ATCC CRL1573)
were cultured in a Falcon's 3003 petri dish containing 10 ml
of the culture medium which is prepared by adding streptomY-
cin and penicillin G to inactivated 10 % FCS-eRDF (Kyokuto
Pharmaceuticals Co. Ltd.)-5 I/g/ml vitamin K1 so that they
reach 100 ~g/ml and 100 units/ml. respectivelY-
The expression vector was used 10 ~g each petri dish.
Only in the case of 228/AC-PC9001, a mixture of 8 ~g of the
expression vector and 2 ~g of pSV2-dhfr was employed. The
expression vector was mixed with 10 ~g of salmon sperm D~TA,
25 J~l of 2M CaCl2, and was adjusted to 200 ~ l with TE (lmM
Tris-~ICl 0.0~ mM EDTA, pH 7.5).
To the solution, was added dropwise under stirring 200
~1 of 2x~BS (280 mM NaCl, ~0 mM ~epes, 1.5 mM NaH2P04, pH
7.12) and the mixture was allowed to stand at room tempera-
ture for 30 minutes. The medium was removed from the petri
2~ dish in which the cell confluency reached 60 to 80 % and 3
ml of the culture medium containing 100 ~M of chloroquine
was added.
The D~A-containing mixture was added dropwise to the
petri dish and it was kept at 37 C-in 5 % C02 incubator for
4 hours. Then, the culture medium was removed, 1 ml of
glycerol solution (prepared by adding 15 % of glycerol to
eRDF medium) was added-to the dish, it was allowed to stand
at room temperature for 1 minute, then the glycerol solution
was aspirated off, the dish was rinsed with 3 m] of PBS (-)
.
.:
,,:
. . ~ .
. .
:: , . . .
.~ . , , ~.
.: .
- 32 - 2 ~7 8 ~ 7
(Nissui Pharmaceuticals Co. Ltd.) twice, added to 10 ml of
the medium and the culture was continued in the ~ % C02
incubator at 37 C.
The method stated here is known as the calcium phos- :
phate coprecipitation method and its basic technology is
shown by Wigler, et. al., Cell., 14, 725 (1978) and Van der
Eb, et.al., Virology 52, 4S6 (1973). The next day, the cells
were trypsinized, diluted 30 to 90 times and the culture was
continued in the above-stated medium in Falcon 3025 dishes.
At that time, 1 mg/ml of G418 (Gibco) was added to the
medium when neomycin resistant gene was used as a marker; 1
~g/ml of methotrexate ~Sigma), to dhfr gene; 6 I/g/ml of
mycophenolic acid, 15 ~g/ml of hypoxanthine and 10 J/g/ml of
thymidine, to Eco-gpt gene. Thereafter the selective medium
was used for the culture.
Colonies were formed in 12 days (BHK cells) or 18 days
(293 cells) and they were transferred to Coaster 3424 dishes
using a cloning cylinder. They were transferred to Falcon
.. 3003 dishes and the culture solution was exchanged, when the
cells became confluent, the cultivation was continued for
additional 24 hours to collect the culture supernatant.
When needed, the cloning of the cell strain was carried
out by the limiting dilution mehtod. In other words, a -
coaster 35g9 dish (96 wells) was used, 200 ~l of the selec-
tive medium was added to each well, one cel]. was added to :-
each well, and cultivation was continued as the medium
solution was exchanged once every 4 days. The cells which
formed one colony in one well were transferred to a Coaster -
3424 dish to continue cultivation, further transferred to :~
Falcon 3003 dish, and the medium solution was exchanged,
when the cells reached confluence, cultured for additionally
24 hours to recover the culture supernatant.
Further, pVA-gpt was transfected into the cloned cell
strain. The procedure was the same as in the transfection of
. -
- : . ~, . .~ . :
- - , , ,~ . . .
. : , ,.. :, .. : ,, : . , :
. - .: ~:,: . . . ; . ..
`,' ' ' ~ ., "'' :~ ' ' .:' :
:~ ~:;, -
2978~7
- 33 -
strain. The procedure was the same as in the trans~ection of
human protein C expression vector except that e~DF medium
containing 500 ~g/ml of G418, 6 ~g/ml of mycophenolic acid,
15 ~g/ml of hypoxanthine, 10 I/g/ml of thymidine, 250 ~g/ml
of xanthine, 5 ~g/ml of vitamin K1, 100 J~g/ml of streptomy-
cin, lO0 units/ml of penicillin G and 10 % FCS. The trans-
fection of pKEX2-gpt was also carried out by the same proce-
dure.
Then, the total concentration of human protein C and the
concentration of the Gla-containing human protein C were
determined by the ELISA. In this ELISA, the heavy chain-
recognizing anti-human protein C monoclonal antibody, JTC-4
was used on the plate side, while JTC-5 (for the activated
peptide recognition and determination of the total human
protein C) or JTC-1 (for recognition of Gla domain depending
on Ca2' and determination of the human protein C bearing Gla
normally) were used as a horse radish peroxidase (H~P0)-
labeled antibody.
These monoclonal antibodies are described by ~aka-
bayashi, et. al., J. Biol. Chem., 261, llOY7 (1987). Among
the results, the examples which gave the highest values will
be given in Table 1.
., ~ , ' .
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,: . ' , ,~, i ... .
- '~.` 2078667
- 34 -
Table 1
Expression vectors Cells Concentration Concentration of
of Total ITPC HPC bearing Gla
228/AC-PC9001 BllK 0.29 ju g/ml 0.29 ~ g/ml
~pSV2-dhfr .
TZml-PC9002 293 2.17 1.87 .:
: -.
TZml-PC9002 293 3.37 3.09 : :
(after cloning) ~;~
TZml-PC9002 293 _ 6.41
. (after cloning)
lncluding pVA-gpt .
228-PC9002 293 _ 1.47
TZm4-PC9002 293 _ 3.38 :
,
~ 15 TZm5-PC9002 2934.70 4.71 .-
i TZm5-PC9002 2938.99 6.33
i (after cloning) .~ .
T%ml6-PC9002 2938.14 6.52 :- :
. TZm20-PC9002 293 7.40 :
.
TZm31-PC9002 2937.64 7.59
TZm5-PC9OOS - 293 _ 5.19 -... .
. . .~ : . .
..
5 ~ IIPC means human protein C. ::
, ~ ~
~: 25 ~urther. another series of cxpression experiments gave :~
3~ the results shown in Table 2.
., ~ - :,. ..
,
~ ' " ' '
1 . . '
., ~
'I . _ --
5.
'1
~5,~ . .
2~78~7
- 35 -
Table 2
Expression vectors Cells Concentration of HPC
bearing Gla
,
TZml-PC9002 293 0.73 /Ig/ml
TZml-PC9006 293 . 1.81
__
228-~C9002 293 0.~1
228-PC9005 293 1.64
,
HPC means human protein C.
Example 3
Increase in two chain processing efficiency in human protc~n
The strain which was obtained by transfection of TZm5-
PC9002 into 293 cells was cloned by the above-stated proce-
dure and pKEX2-gpt was transfected by the above-stated meth-
.. od. The cells before and after transfection of pKEX2-gpt
were cultured in Falcon 3003 dishes until the cells bccamc
confluent, rinsed with PBS(-) twice, then 10 ml of a ser~lm-
free medium (5 ~ g/ml vitamln Kl-eRDF) was added. Two days
latcr, the culture supernatant was recovered, passed through
a mcmbrane filter (Millipore, pore si%e: 0.22 ~ m) and
concentrated with Centricon 10 (Amecon, Registered Trade
Mark) into 1/20 volume. The samp].e was subjected to SDS
polyacry]amide-gel electrophoresis (10 - 20 %) under reduc-
tive conditions to examine the two chain processing effi-
ciency by the immunoblotting method using anti-human protcin
- 30 C antiserum (American Diagnostic Co.). The efficiency was
about 80 ~ bcfore transfection of pKEX2-gpt and found to be
almost 100 % in almost all clones after transfection. ~o
reduction in exprèssion efficiency of human protein C was
was caused b~ transfectlon of pKEX2-gpt.
' . :. . .: ' '~ . ....... :. . ..
. ~', : , . . :,
t ` :
2~78~7
- 36 -
Example 4
Puri~ication of human protein C and measurement of the
activity
A human protein C-producing strain prepared from TZml-
PC9001 was cultured in the selective medium stated in Exam-
ple 2 in Falcon 3025 dishes to collect about 600 ml of the
culture supernatant.
The supernatant was passed through a filter of 0.45 ~ m
pore size, then CaCl~ was added so that the final concentra-
tion became 5 mM, and an anti-human protein C monoclonal
antibody-immobilized column was used to purify human protein
C.
The antibody used here is described as 6H2 in Japanese ~ :
Patent Specification Laid-open No. Tokkaisho 61-134399.
Further, the purification is disclosed in Japanese Patent
Specification Laid-open ~lo. Tokkaihei 2-163085. Human pro-
tein C was eluted as a single peak and the amount of the
protein was found to be about 300 ~ g calculated from the
absorbance.
The purified human protein C showed a single band on
the non-reduced SDS polyacrylamide gel electrophoresis.
Further, it showed a single chain band in addition to the
heavy chain band and the light chain band on the reduced ~S
polyacrylamide gel electrophoresis. The densitometry showed
that the fraction of single chain human protein C was about
20%.
Three micrograms of the purified human protein C were
activated with 0.3 ~ig of bovine thrombin (Mochida Pharma-
ceuticals Co.) at 37 C, then 9-time volume of antithrombin
III (150 1lig/ml)-heparin (2 units/ml) was added 5, 15, 30
and 60 minutes later to terminate the reaction.
Fifty microliters of the reaction mixture and 50 lli ]. of
2mM S-2366 (Cabi) were added to each well of a 96 wcll micro-
. : ' - - . : - . .
2078~7
- 37 -
plate and the absorbance change at 405 nm wavelength was
measured with ETY-96 analyzer (TOYO SOKKI Co.).
As shown in Table 3, time de~endent increase in hydrol-
ysis of synthetic substrate S-2366 was observed.
Table 3
Activation time Change in absorbance (~ A405xlOOO/minntc)
_
O minute 0.4
6.6
_ 17.8
30.0
48.5
~urther. the activated human protein C mentioned above
was diluted to 10 - 200 ng sample/50 ~ l 0.1% BSA-TBS (pll
7.4). The sample and 50~ l of Sysmex ~PTT reagent were addcd
to 100 ~ 1 of Sysmex control serum I which was kept at 37 C
for 2 minutes, stirred, maintained at 37 C for 2 minutes,
then stirred together with 100 /~l of 25 mM CaCl2 and APTT
was determined by ~eans of Sysmex CA-100 type blood coagu~a-
tion analyzer. The activity was compared with that of the
- activated human protein C which was obtained by purification
wi.th the above-stated affinity column from human plasma and
activation through a similar mcthod and the product was
found to have equal or higher specific activity.
: ~ . . .: -, . :. . . - . . : . .
; . .: .
