U.S. patent application number 10/944810 was filed with the patent office on 2005-03-17 for method for purifying calcium ion-binding protein.
This patent application is currently assigned to JURIDICAL FOUNDATION THE CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE. Invention is credited to Furukawa, Shinichi, Komatsu, Kazuhiro, Koyanagi, Satoshi, Mizokami, Hiroshi, Onchi, Tatsufumi, Sugawara, Keishin, Yoshizaki, Hideo.
Application Number | 20050059805 10/944810 |
Document ID | / |
Family ID | 18715177 |
Filed Date | 2005-03-17 |
United States Patent
Application |
20050059805 |
Kind Code |
A1 |
Mizokami, Hiroshi ; et
al. |
March 17, 2005 |
Method for purifying calcium ion-binding protein
Abstract
The present invention relates to a method for purifying a
calcium ion-binding protein by cation exchange chromatography. The
present invention provide a method for isolating and purifying a
calcium ion-binding protein in a simple and efficient manner from a
liquid sample containing a calcium ion-binding protein and
contaminants without any pretreatment such as addition of a
chelating agent. More specifically, the present invention relates
to a method for purifying a calcium ion-binding protein which
comprises contacting said protein with a cation exchange carrier in
the presence of calcium ions to let the said protein be adsorbed to
the carrier, and after washing, eluting said protein, and to a
calcium ion-binding protein having substantially no contaminants
obtained by the method of the present invention.
Inventors: |
Mizokami, Hiroshi;
(Kumamoto-shi, JP) ; Furukawa, Shinichi;
(Kumamoto-shi, JP) ; Sugawara, Keishin;
(Kumamoto-shi, JP) ; Onchi, Tatsufumi;
(Kumamoto-shi, JP) ; Komatsu, Kazuhiro;
(Kumamoto-shi, JP) ; Koyanagi, Satoshi;
(Kumamoto-shi, JP) ; Yoshizaki, Hideo;
(Sayama-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
JURIDICAL FOUNDATION THE
CHEMO-SERO-THERAPEUTIC RESEARCH INSTITUTE
KOWA COMPANY, LTD.
|
Family ID: |
18715177 |
Appl. No.: |
10/944810 |
Filed: |
September 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10944810 |
Sep 21, 2004 |
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10088588 |
Mar 21, 2002 |
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10088588 |
Mar 21, 2002 |
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PCT/JP01/06209 |
Jul 18, 2001 |
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Current U.S.
Class: |
530/350 |
Current CPC
Class: |
C07K 14/4721 20130101;
C12Y 304/21006 20130101; C12N 9/6432 20130101; C07K 14/4728
20130101 |
Class at
Publication: |
530/350 |
International
Class: |
C07K 014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 2000 |
JP |
2000-220600 |
Claims
1. A calcium ion-binding protein selected from the group consisting
of Annexins I, II, III, IV, V, VI and VII having a purity of 80 to
100% as determined by gel filtration chromatographic analysis,
obtained by a method, comprising: contacting a sample containing
said protein with a cation exchange carrier in the presence of
calcium ions to let the protein be adsorbed to the exchange
carrier; and eluting the adsorbed calcium ion-binding protein from
the exchange carrier by (a) decreasing or removing the
concentration of said calcium ions (b) adding counter ions other
than said calcium ions or (c) both (a) and (b).
2. A calcium ion-binding protein selected from the group consisting
of Annexins I, II, III, IV, V, VI and VII having a purity of 80 to
100% as determined by gel filtration chromatographic analysis or
SDS-PAGE analysis, obtained by a method, comprising: contacting a
sample containing said protein with a cation exchange carrier in
the presence of calcium ions to let the protein be adsorbed to the
exchange carrier; and eluting the adsorbed calcium ion-binding
protein from the exchange carrier by (a) decreasing or removing the
concentration of said calcium ions (b) adding counter ions other
than said calcium ions or (c) both (a) and (b).
3. A calcium ion-binding protein selected from the group consisting
of Annexins I, II, III, IV, V, VI and VII having a purity of 80 to
100% as determined on a molecular size basis, obtained by a method,
comprising: contacting a sample containing said protein with a
cation exchange carrier in the presence of calcium ions to let the
protein be adsorbed to the exchange carrier; and eluting the
adsorbed calcium ion-binding protein from the exchange carrier by
(a) decreasing or removing the concentration of said calcium ions
(b) adding counter ions other than said calcium ions or (c) both
(a) and (b).
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to a method for purifying a
calcium ion-binding protein by a cation exchange process. More
specifically, the present invention relates to a method for
purifying a calcium ion-binding protein which comprises contacting
said protein with a cation exchange carrier in the presence of
calcium ions to render said protein be adsorbed to the cation
exchange carrier, and after washing, eluting said protein from the
cation exchange carrier, and to a calcium ion-binding protein
obtained by said method which contains substantially no
contaminants.
BACKGROUND OF THE INVENTION
[0002] For isolation and purification of a protein of interest from
contaminants, physico-chemical properties such as a molecular size,
an electric charge on the surface or solubility of said protein is
utilized. A process for purification commonly used in the field of
protein chemistry includes, for instance, salting out,
ultrafiltration, isoelectric precipitation, electrophoresis, ion
exchange chromatography, gel filtration chromatography, affinity
chromatography, and the like. In case that a protein of interest
must be purified from the living tissues, cells, or blood, where an
enormous variety of different proteins exist, these processes need
often be combined in a manifold manner. However, it is possible to
provide a method for purification with much more specificity by
utilizing a property commonly shared by a certain kind of
protein.
[0003] By way of example, a unique method for purification using
anion exchange chromatography is known wherein a divalent
cation-binding protein is adsorbed to an anion exchange resin and
then eluted therefrom with a divalent cation to specifically purify
said divalent cation-binding protein as disclosed in Japanese
patent publication No. 200180/1990. According to this method, a
chelating reagent such as ethylenediaminetetraacetic acid (EDTA) is
added to a solution containing a divalent cation-binding protein to
first remove divalent ions. Then, the resulting solution is
contacted with an anion exchange resin such as MonoQ to render the
divalent cation-binding protein be adsorbed to the anion exchange
resin. Finally, addition of sodium chloride and calcium chloride
elutes the divalent cation-binding protein from the anion exchange
resin. However, most of naturally occurring proteins are negatively
charged under physiological conditions and hence numerous
contaminants other than a protein of interest are preferentially
adsorbed to an anion exchange resin, thus hampering efficient
purification of the desired protein. Therefore, this method for
purification through adsorption of a desired protein to an anion
exchange resin is preferably used for a small amount of a protein
solution or at an advanced stage of purification processes.
[0004] Japanese patent publication No. 258286/1995 discloses a
method for purifying a calcium ion-binding, vitamin K-dependent
protein by an anion exchange process wherein calcium chloride is
added to a solution containing a vitamin K-dependent protein and
the resulting solution is passed through an anion exchange resin to
isolate the desired protein from contaminating proteins. This
method, however, is disadvantageous in that a large volume of
fractions containing the desired protein must be passed through and
hence subsequent procedures will become troublesome especially when
conducted in a large scale.
[0005] Annexin V, one of calcium ion-binding proteins, is a simple
protein of about 34 kDa molecular weight bearing no sugar chain
that has a physiological activity such as anti-coagulating
activity, corneal epithelium-extending activity, and phospholipase
A.sub.2 inhibitory activity. It is known that Annexin V distributes
in a variety of tissues and secretions within the living body
including human placenta (Chem. Pharma. Bull., 38, 1957-1960,
1990). Annexin V is called a calcium ion-binding protein since it
has an ability to bind with a lipid membrane via calcium ions.
[0006] Annexin V has been extracted from organs of human or animals
(Japanese patent publication No. 174023/1987). Nowadays, however,
it can be produced in E. coli and yeast by the use of the genetic
recombination technique (Japanese patent publications No.
20095/1989 and No. 219875/1991).
[0007] Annexin V has conventionally been purified, after
pretreatment of an Annexin V-containing solution with
precipitation, membrane filtration and centrifugation, by a
combination of ammonium sulfate fractionation, anion exchange
chromatography, hydrophobic chromatography and affinity
chromatography (Jurgen Romisch et al., Biochem. J. 272, 223-229,
1990; T. R. Hawthorne et al., Journal of Biotechnology 36, 129-143,
1994).
DISCLOSURE OF THE INVENTION
[0008] However, these processes are disadvantageous in that
purification steps are complicated and troublesome requiring a
great deal of labor and time and hence possibly meet an obstacle in
view of reproducibility and yield, rendering them not be suitable
for purification of Annexin V in an industrial scale. Moreover, as
purification process in a large scale, these processes are
disadvantageous in economical point of view as well since they used
heparin Sepharose, which is rather expensive, for enhancing
purification degree of Annexin V. Previously, the present inventors
have provided a method for preparing Annexin V by pretreating a
protein solution to remove contaminants to some extent and then
performing anion exchange chromatography on the resulting solution
(Japanese patent publication No. 219875/1991). This method might
possibly enables purification of Annexin V in an industrial scale
but would not exceed the method of the present invention.
[0009] As described above, for use in an industrial scale, the
conventional processes are problematic in view of cost, efficiency
and handling.
[0010] An object of the present invention is to provide a method
for isolating and purifying a calcium ion-binding protein in a
simple and efficient manner from a liquid sample containing a
calcium ion-binding protein and contaminants without any
pretreatment such as addition of a chelating agent.
[0011] Another object of the present invention is to provide
Annexin V of high purity obtained by the method of the present
invention.
[0012] Under the circumstances, the present inventors investigated
for attaining the above objects and have found that Annexin V, one
of calcium ion-binding proteins, is adsorbed to SP-Sepharose cation
exchange carrier in the presence of calcium chloride and at pH of
around neutrality. The present inventors have also noted that the
adsorption of Annexin V to SP-Sepharose cation exchange carrier
occurred specifically in the presence of calcium ions but could
scarcely observe the adsorption in the presence of other divalent
ions than calcium ions, e.g. magnesium ions. With this finding, the
present inventors added calcium chloride to a large amount of
homogenate of Annexin V-producing cells produced by the genetic
recombination technique and contacted the resulting homogenate with
SP-Sepharose cation exchange carrier which has been equilibrated
with ammonium chloride buffer containing calcium chloride. After
washing, elution was performed by decreasing or removing calcium
chloride level or with ammonium chloride buffer containing sodium
chloride in the presence of calcium ions to successfully purify
Annexin V with high purity. Moreover, a trace amount of remaining
proteases could successfully be removed by performing said cation
exchange chromatography at pH 9.0.
[0013] Thus, the present invention encompasses a method for
purification of a calcium ion-binding protein, either naturally
occurring or produced by the genetic recombination technique, by
cation exchange chromatography using SP-Sepharose cation exchange
carrier in the presence of calcium chloride.
[0014] The present invention also encompasses a calcium ion-binding
protein, either naturally occurring or produced by the genetic
recombination technique, thus obtained by the method of the present
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIGS. 1A and 1B are a schematic illustration of cloning of
Annexin V structural gene and preparation of yeast cells
transformed with said gene.
[0016] FIG. 2 shows results of gel filtration chromatography for
(a) samples prior to cation exchange chromatography, (b) fractions
passed through cation exchange chromatography, and (c) fractions
eluted from cation exchange chromatography after washing.
[0017] FIG. 3 shows results of gel filtration chromatography for
fractions eluted from cation exchange chromatography after
washing.
[0018] FIG. 4 shows an elution pattern of Annexin VI from
SP-Sepharose.
[0019] FIG. 5 is a photograph showing results of SDS-PAGE of
Annexin VI. Lane 1: BioRad prestained marker proteins;
phosphorylase B (116,000), BSA (80,000), ovalbumin (52,500),
carbonic anhydrase (34,900), soybean trypsin inhibitor (29,900),
lysozyme (21,800); Lane 2: Annexin VI standard; Lane 3: elution
with DEAE-Toyopearl (sample); Lane 4: SP-Sepharose, fraction No. 5;
Lane 5: fraction No. 7; Lane 6: fraction No. 9; Lane 7: fraction
No. 16; Lane 8: fraction No. 51; Lane 9: fraction No. 54; and Lane
10: fraction No. 57.
[0020] FIG. 6 shows an elution pattern of coagulation factor X from
SP-Sepharose.
[0021] FIG. 7 is a photograph showing results of SDS-PAGE of
coagulation factor X. Lane 1: BioRad prestained marker proteins;
phosphorylase B (116,000), BSA (80,000), ovalbumin (52,500),
carbonic anhydrase (34,900), soybean trypsin inhibitor (29,900),
lysozyme (21,800); Lane 2: commercially available coagulation
factor X; Lane 3: fraction No. 4; Lane 4: fraction No. 7; Lane 5:
fraction No. 11; and Lane 6: fraction No. 12.
BEST MODE FOR CARRYING OUT THE INVENTION
[0022] The method of the present invention comprises a step in
which a liquid sample containing a calcium ion-binding protein is
contacted with a cation exchange carrier in the presence of calcium
ion, followed by a step in which a concentration of calcium ion is
decreased or removed and/or a concentration of counter ions (salts)
is increased to elute and recover said protein. The method of the
present invention enables production of a calcium ion-binding
protein with high purity. The contacting process with the cation
exchange carrier may be performed either in a batch or by
chromatography. When chromatography is used, a column size may
appropriately be selected depending on a production scale.
[0023] A cation exchange carrier used herein includes, but not
limited to, SP-Sepharose, CM-Sepharose, CM-cellulose, SE-cellulose,
S-Spherodex, SP-Spherosil, and the like, all of which are
commercially available. Among these, SP-Sepharose is preferably
used.
[0024] An amount of a protein solution to be contacted with the
carrier may vary depending on a concentration of the solution or an
ability of the carrier for adsorption. In case of SP-Sepharose, for
instance, 0.1 to 30 g/L carrier of the protein may be used.
Preferably, 15 to 20 g/L carrier of the protein is used.
[0025] A flow rate while adsorption to the cation exchange carrier
may be 1 to 150 cm/h, preferably 15 to 100 cm/h, more preferably 50
to 80 cm/h. On the other hand, a flow rate while elution of the
adsorbed protein from the cation exchange carrier may be 1 to 150
cm/h, preferably 30 to 100 cm/h, more preferably 30 to 80 cm/h.
[0026] A buffer that may be used for adsorption to or elution from
the cation exchange carrier includes any buffer conventionally used
in ion exchange chromatography, including ammonium chloride buffer,
citrate buffer, acetate buffer and Tris-HCl buffer. Among these,
ammonium chloride buffer is preferably used. A concentration of a
buffer may be in a range of 5 to 100 mM, preferably 10 to 40 mM. A
buffer may be used at pH 5 to 10, preferably at pH 8 to 9.5,
conditions where proteases are removed. More preferably, 20 mM
(around pH 9.0) ammonium chloride buffer is used.
[0027] As a source of calcium ions, any substance that can afford
calcium ions may be used, including calcium chloride, calcium
carbonate, preferably calcium chloride.
[0028] When a large quantity of calcium chloride is added to
homogenate of tissues or cells or plasma, hydrophobic proteins or
high molecular weight compounds are sometimes deposited as a result
of salting-out. Thus, calcium ions may preferably be used in such
an amount that not only renders a calcium ion-binding protein be
bound to and isolated from the cation exchange carrier but also
forms no precipitation from a liquid sample containing a calcium
ion-binding protein.
[0029] For adsorption of a calcium ion-binding protein to the
cation exchange carrier, calcium ions at a concentration of 5 to
100 mM may preferably be used. More preferably, calcium ions at a
concentration of 10 to 30 mM may be used. In combination with a
buffer to be used for adsorption to and elution from the cation
exchange carrier, 20 mM ammonium chloride buffer (pH 9.0)
containing 20 mM calcium chloride may preferably be used.
[0030] The adsorbed calcium ion-binding protein may be eluted from
the carrier by removing or decreasing calcium ion level in the
buffer or adding other counter ions than calcium ions, or both. A
counter ion includes Na.sup.+, Li.sup.+, K.sup.+ ions, and the
like. Preferably, elution may be performed by adding 1 to 500 mM,
more preferably 50 to 500 mM, still more preferably 50 to 300 mM
sodium chloride to the ammonium chloride buffer, and most
preferably by adding 200 mM sodium chloride to 20 mM ammonium
chloride buffer (pH 9.0) containing 20 mM calcium chloride.
Alternatively, the adsorbed calcium ion-binding protein may be
eluted from the carrier merely by decreasing the calcium chloride
level to less than 5 mM.
[0031] The method of the present invention, even when used solely,
can afford to provide purification of a calcium ion-binding protein
of 80% purity or more. It may more efficiently be used, however, in
combination with other purification processes. For example, in case
that a liquid sample containing a calcium ion-binding protein is
contaminated with insoluble substances, pretreatment for removing
such substances, e.g. centrifugation, salting-out, membrane
filtration, etc., is preferably carried out prior to the method of
the present invention.
[0032] In addition to the above-described processes, other
purification processes of various chromatographic procedures,
including anion exchange chromatography, hydrophobic
chromatography, gel filtration chromatography, affinity
chromatography, adsorption chromatography, etc. may be performed
together with the method of the present invention to provide a
calcium ion-binding protein of higher purity. The method of the
present invention may be used at any stage of the above-described
processes. Preferably, after a sample containing a calcium
ion-binding protein is pretreated to remove insoluble substances,
the method of the present invention is used and then anion exchange
chromatography is followed. More specifically, an Annexin
V-containing fraction obtained by the cation exchange
chromatography is applied to Q-Sepharose column equilibrated with
10 mM sodium phosphate buffer (pH 7.4) containing 50 mM sodium
chloride, and after washing, elution is performed with linear
gradient of concentration from 50 mM to 500 mM sodium chloride to
give Annexin V with much higher purification.
[0033] A calcium ion-binding protein to be purified by the method
of the present invention typically includes Annexins I, II, III,
IV, V, VI and VII but may be any protein that has an ability to
bind to calcium ions, such as coagulation factor X.
[0034] The method of the present invention may efficiently be
applied to blood, body fluid and tissue homogenate from an animal
either naturally occurring or genetically engineered that produces
a calcium ion-binding protein, as well as cell homogenate and
culture supernatant of recombinant cells, including plant cells,
bacterial cells, yeast cells, insect cells and animal cells.
preferably, the method of the present invention may be used in
recombinant yeast cells producing a calcium ion-binding protein.
More preferably, the method of the present invention may be used in
cell homogenate or culture supernatant of yeast cells producing
Annexin V.
[0035] Annexin V thus prepared, having special physiological
activities, may be formulated into a pharmaceutical preparation in
any conventional dosage form such as injections, eye drops, oral
preparations, suppositories, etc. alone or in combination with a
pharmaceutically acceptable carrier, diluent, stabling agent or
preservative.
[0036] According to the present invention, an efficient method for
purifying a calcium ion-binding protein with high purity is
provided. Also provided is the calcium ion-binding protein thus
obtained by the method of the present invention having
substantially no contaminants.
[0037] According to the method of the present invention, most
proteins negatively charged under physiological conditions are
passed through the cation exchange carrier whereas a calcium
ion-binding protein, which can form a complex with calcium ions, is
preferentially adsorbed to the carrier. Thus, the method of the
present invention enables handling of a large quantity of a sample
at one time without deterioration of the adsorption capacity of the
cation exchange carrier by contaminating proteins other than the
desired protein.
EXAMPLE
Preparation Example
Preparation of Recombinant Yeast Cell Producing Annexin V
[0038] Recombinant yeast cells producing Annexin V were prepared as
described in a publication of patent application (Japanese Patent
Publication No. 219875/1991). FIG. 1 schematically shows
preparation of the recombinant yeast cells wherein the term "CPB-I"
is used for referring to "Annexin V".
[0039] (1) Cloning of Annexin V Structural Gene
[0040] From human placenta cDNA library (Clontech Laboratories,
Inc.), phage that bears Annexin V structural gene was insolated by
immunoscreening using anti-Annexin V monoclonal antibody. DNA was
then prepared from phage and digested with restriction enzyme EcoRI
to produce a fragment, which was then inserted into the EcoRI site
of pUC118 vector to construct pMKT7.
[0041] (2) Construction of Expression Plasmid
[0042] The plasmid pMKT7 was digested with restriction enzymes NcoI
and SacI and a DNA fragment containing Annexin V structural gene
was separated by agarose electrophoresis. Addition of a synthetic
linker converted both ends of the DNA fragment into XhoI and BamHI
sites. The resulting DNA fragment was inserted into XhoI and BamHI
sites of the expression vector pPS1 to construct expression vector
pAPCPBI.
[0043] (3) Preparation of Recombinant Yeast Cells
[0044] Host yeast cells (Saccharomyces cerevisiae AH22) were
transformed with the expression plasmid pAPCPBI by the lithium
acetate technique. After transformation, colonies appeared on an
agar medium deprived of leucine were isolated and an expression
level was measured. Those clones with higher expression level were
selected and subjected to repetition of plating to the agar medium,
isolation of colonies and measurement of expression level to give
recombinant yeast cells with stability.
Example 1
Purification of Recombinant Annexin V
[0045] (1) Culture of Annexin V-Producing Recombinant Yeast
Cells
[0046] Annexin V-producing recombinant yeast cells were cultured on
2L synthetic selection medium at 28.degree. C. for 3 days. The
recombinant yeast cells were then inoculated to 88L selection
medium and cultured at 28.degree. C. for 2 days. The recombinant
yeast cells were then transferred to 810L semisynthetic medium (40
g sucrose, 5 g yeast extract, 5 g ammonium sulfate and 0.5 g
magnesium sulfate septahydrate in 1L medium) and culture was
continued at 28.degree. C. for 24 hours.
[0047] (2) Pretreatment of Annexin V in Large Quantity Prior to
Purification
[0048] The large culture solution was filtered with a 0.1 .mu.m
membrane filter to collect the recombinant yeast cells, which were
physically ruptured with a French press-type cell homogenater. The
ruptured cell suspension was filtered with the membrane filter and
the filtrate was concentrated with a ultrafiltrater. To the
concentrate was added acetic acid for isoelectric precipitation (pH
5.0). Precipitates formed were filtered with the membrane filter to
remove the precipitates. Then, pH of the filtrate was adjusted to
9.0 with ammonia and the filtrate was again concentrated with a
ultrafiltrater (pretreated solution).
[0049] (3) Cation Exchange Chromatography (Elution by Decreasing or
Removing Calcium Chloride Level)
[0050] To the pretreated solution was added a calcium chloride
solution to a final concentration of 20 mM and was subjected to
cation exchange chromatography with SP-Sepharose (Pharmacia).
Specifically, the pretreated solution supplemented with calcium
chloride was applied to a column equilibrated with 20 mM ammonium
chloride buffer (pH 9.0) containing 20 mM calcium chloride and 50
mM sodium chloride. After washing with the buffer, the column was
further washed with 20 mM ammonium chloride buffer (pH 9.0)
containing 20 mM calcium chloride. Then, Annexin V was eluted with
20 mM ammonium chloride buffer (pH 9.0).
[0051] (4) Cation Exchange Chromatography (Elution by Increasing
Sodium Chloride Level)
[0052] As in the step (3), the pretreated solution was added with a
calcium chloride solution to a final concentration of 20 mM and was
subjected to cation exchange chromatography with SP-Sepharose.
Specifically, the pretreated solution supplemented with calcium
chloride was applied to a column equilibrated with 20 mM ammonium
chloride buffer (pH 9.0) containing 20 mM calcium chloride and 50
mM sodium chloride. After washing with the buffer, Annexin V was
eluted by a linear gradient of concentration of sodium chloride
from 50 mM up to 300 mM with 20 mM ammonium chloride buffer (pH
9.0) containing 20 mM calcium chloride (flow rate at adsorption and
elution: 56.7 cm/h).
[0053] FIG. 2 shows elution patterns obtained by gel filtration
chromatography of (a) sample prior to cation exchange
chromatography, (b) fractions passed through cation exchange
chromatography, and (c) fractions eluted from cation exchange
chromatography, respectively. Gel filtration chromatography was
performed wherein 20 .mu.L sample was applied to TSKgel G3000
SW.times.1 (7.8 mm (ID).times.30 cm) equilibrated with 10 mM
phosphate buffer (pH 7.2) containing 0.14 M NaCl at a flow rate of
125.6 cm/h. The results of gel filtration chromatography for
fractions eluted from cation exchange chromatography are shown in
Table 1 wherein purity was obtained from the elution pattern.
[0054] (5) Anion Exchange Chromatography (Comparison With
Conventional Technique)
[0055] Anion exchange chromatography was performed for the
pretreated solution. The pretreated solution was applied to
Q-Sepharose (Pharmacia) column equilibrated with 10 mM sodium
phosphate buffer (pH 7.4) containing 50 mM sodium chloride. After
washing, Annexin V was eluted by a linear gradient of concentration
of sodium chloride from 50 mM up to 300 mM. The results of gel
filtration chromatography for the eluted fractions are shown in
Table 1.
[0056] Annexin V obtained by the conventional technique and that
obtained by the method of the present invention were measured for
their anti-coagulating activity after further purification with
additional purification processes. Measurement of anti-coagulating
activity was made in the same manner as the quantification of
sodium heparin described in the Japanese Pharmacopoeia (the 13th
revision, p.900-901). Anti-coagulating activity was calculated
wherein prolongation in time for coagulation induced by 1 mg of
standard sample (Annexin V purified by the conventional technique)
was defined as one unit (U). As a result, no inactivation of
Annexin V obtained by the method of the present invention was
observed (Table 1).
1 TABLE 1 Anion exchange Cation exchange chromatography
chromatography Purity (%) 81.86 100 Activity (U/mg) 0.9 to 1.0 0.9
to 1.0
Example 2
Purification of Recombinant Annexin V
[0057] A recombinant Annexin V was purified as in Example 1 except
that 20 mM citrate buffer (pH 6.0) containing 20 mM calcium
chloride and 50 mM sodium chloride was used in place of 20 mM
ammonium chloride buffer (pH 9.0) containing 20 mM calcium chloride
and 50 mM sodium chloride, and 20 mM citrate buffer (pH 6.0) was
used in place of a linear gradient concentration of sodium chloride
from 50 mM up to 300 mM with 20 mM ammonium chloride buffer (pH
9.0) containing 20 mM calcium chloride, in the cation exchange
chromatography of the step (4). Flow rate was also altered to 15.6
to 54.6 cm/h at adsorption and 39 cm/h at elution.
[0058] FIG. 3 shows an elution pattern obtained by gel filtration
chromatography of fractions eluted from cation exchange
chromatography.
Example 3
Purification of Annexin VT From Placenta
[0059] One placenta (about 500 g) excepting the anion and an
umbilical cord was sliced into pieces, washed with 2 L
physiological saline, and minced with a meat grinder. After adding
400 mL of 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM calcium
chloride, 0.1% Triton X-100 and 5 mM benzamidine, the mince was
homogenated with a whirling blender. The homogenate was centrifuged
at 10,000 rpm for 20 minutes to collect precipitates, which were
again suspended in 300 ml of 50 mM Tris-HCl buffer (pH 7.4)
containing 50 mM EDTA and homogenated. The homogenate was again
centrifuged at 10,000 rpm for 20 minutes and an extract of
supernatant was recovered (about 300 mL), to which 63 g ammonium
sulfate was added to prepare a 30%-saturated solution of ammonium
sulfate. After centrifugation to remove precipitates, to the
supernatant was added 54 g ammonium sulfate (60%-saturated ammonium
sulfate) and precipitated Annexin VI fraction was recovered.
Annexin V could preferentially be recovered in precipitates formed
when the salt concentration was raised to 80% saturation of
ammonium sulfate.
[0060] The precipitates formed with a 60%-saturated solution of
ammonium sulfate were dissolved in 50 mM Tris-HCl buffer (pH 7.4)
and dialyzed against the same buffer. A dialyzed solution (80 mL)
was adsorbed to DEAE-Toyopearl (3.times.20 cm) equilibrated with
the same buffer. After washing with the same buffer, elution was
performed by a linear gradient from the same buffer (180 mL) to 50
mM Tris-HCl buffer (pH 7.4) containing 0.3 M NaCl (180 mL)(each
fraction: 4 mL/tube). Annexin VI of interest was eluted in
fractions No. 46 to No. 50. These Annexin VI fractions were
dialyzed against 20 mM ammonium chloride (pH 9.0), to which was
added calcium chloride to make finally 20 mM ammonium chloride
buffer (pH 9.0) containing 20 mM calcium chloride. The dialyzed
solution was adsorbed to SP-Sepharose FF (1.5.times.8 cm)
equilibrated with 20 mM ammonium chloride buffer (pH 9.0)
containing 20 mM calcium chloride (FIG. 4; fractions No. 1 to No.
15). After washing with the same buffer, elution was performed with
the same buffer supplemented with 0.5 M NaCl (FIG. 4; fractions No.
45 to No. 70). Samples at each stage were analyzed by non-reductive
SDS-PAGE and the results are shown in FIG. 5.
Example 4
Purification of Coagulation Factor X
[0061] Commercially available coagulation factor X (about 1 mg) was
dialyzed against 20 mM Tris-HCl buffer (pH 8.0), to which was added
one tenth amount of 200 mM calcium chloride to make finally 20 mM
Tris-HCl buffer (pH 8.0) containing 20 mM calcium chloride. The
dialyzed solution was adsorbed to SP-Sepharose FF (0.8.times.7 cm)
equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 20 mM
calcium chloride. After washing with 20 mM Tris-HCl buffer (pH 9.0)
containing 20 MM calcium chloride, elution was performed with 20 mM
Tris-HCl buffer (pH 9.0) containing 0.3 M calcium chloride. In all
stages, 5 mM benzamidine was added (Since benzamidine per se
exhibits UV absorption at A280, an elution pattern is not explicit;
FIG. 6). Fractions at each stage were analyzed by non-reductive
SDS-PAGE and the results are shown in FIG. 7.
* * * * *