JPH05211874A - Recombinate human osteocalcin - Google Patents

Abstract

PURPOSE:To produce human osteocalcin, especially tau-Carboxylated Gla human osteocalcin by recombination technique. CONSTITUTION:A DNA sequence for manifesting human osteocalin, a vector for manifesting human osteocalin and a transformant transduced with the vector. The transfromant is used to produce human osteocalcin. tau-Carboxylated human osteocalcin can be provided.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to recombinant human osteocalcin. In particular, the invention relates to a series of techniques for expressing human osteocalcin using bacterial cells. More specifically, the present invention relates to a DNA sequence for expressing human osteocalcin, a vector for expressing human osteocalcin, a transformant into which the vector is introduced, and human osteocalcin or a protein physiologically equivalent thereto using the transformant. To a method of manufacturing.

[0002]

2. Description of the Related Art Osteocalcin is one of the Gla proteins found in the bone matrix and is a vitamin K-dependent bone calcium-binding protein. Human osteocalcin has a molecular weight of 580
It is composed of 0 to 49 amino acids (JW Poster
U. Biol. Chem. 255 , 8685-8691, 1980). The amino acid sequence of this human osteocalcin is D of SEQ ID NO: 1.
It is shown at the bottom of the NA sequence. The amino acid sequence of SEQ ID NO: 1 is shown as a non-Gla-ized amino acid sequence.

Osteocalcin has a Gla residue (γ-carboxyglutamic acid residue) and has a strong affinity for hydroxyapatite, and therefore it is presumed that it has an important role in bone matrix formation.

It is necessary to provide a large amount of human osteocalcin in order to further study the clinical significance of such human osteocalcin or to research and develop its potential as a medicine.

However, in order to obtain human osteocalcin at present, human bone must be used as a raw material and highly purified, and it is extremely difficult and problematic to obtain not only a small amount but a large amount.

As one method for solving this problem, application of genetic engineering technology can be considered. For example, it was reported that fusion expression of the human osteocalcin gene was attempted in Escherichia coli (N.OYAMA et al, 11th session).
Proceedings of the Molecular Biology Society of Japan, page 161, 3D-04 (19
88)).

This report was made under the heading "Expression of human osteocalcin precursor gene in Escherichia coli" and the content is extremely simple. According to the report, a human osteocalcin gene was connected in a 4-copy tandem downstream of the pstS gene derived from Escherichia coli, and a fusion gene in which a DNA sequence encoding a Lys (lysine) residue was inserted between the genes was prepared, Performing fusion expression. However, in this report, the expression was confirmed only by Western blotting using a monoclonal antibody that recognizes the C-terminal of bovine osteocalcin, and purification of the fusion gene product and monomerization by lysyl endopeptidase were not performed.

Even if a protein which is a gene product is obtained by the above method, it has no physiological activity, that is, Gl.
It is considered that it is almost difficult to obtain the humanized osteocalcin which is not a-modified and has the physiologically active Gla-modified human osteocalcin by this method.

Therefore, it is desired to provide a significant amount or a large amount of human osteocalcin having physiological activity.

On the other hand, a protein containing a Gla residue is known for prothrombin, factor IX, factor VII, protein C, protein S, protein Z, and other coagulation / fibrinolytic proteins. These proteins are converted to Gla (Glu → Gl) in vivo by the action of γ-carboxylase (vitamin K-dependent carboxylase).
a) [JW Suttie et al., J.
Biol. Chem. 251, 5827-5830 (1976)].

Further, the following reports have been made that a propeptide is required for factor IX, protein C, prothrombin, etc. to work efficiently with γ-carboxylase (LC Pan et al, PNAS, 8).
2 , 6109-6113 (1985); JW Suttie et al, PNAS,
84, 634-637 (1987); BE Furie et al, WO 88-3926;
DC Foster et al, Biochemistry 26 , 7003-7011 (19
87)).

[0012] However, no knowledge has been obtained so far about the propeptide and its action for Gla conversion of osteocalcin.

[0013] Therefore, the present inventors diligently studied a series of techniques for obtaining a large amount of physiologically active human osteocalcin using bacterial cells. That is, in order to enable efficient expression of a protein containing human osteocalcin in a bacterial cell, and to monomerize human osteocalcin from the obtained protein and to convert it into human osteocalcin in the form of Gla. DNA of
The present invention has been intensively studied for the purpose of providing a plasmid containing a sequence and a DNA sequence, and the expression and purification of transformed cells and human osteocalcin by the plasmid.

[0014]

The inventors of the present invention have reached the present invention as a result of extensive research to achieve the above-mentioned object of the present invention.

According to the present invention, first, according to the following formula [I] ABCD ... [I], D is a DNA sequence (D sequence) encoding human osteocalcin or a protein physiologically equivalent thereto. Where C is the DNA sequence encoding the cleavage sequence peptide (C
Sequence), where B is B-1 and / or B-2, wherein B-1 is a DNA sequence encoding an additional polypeptide for the large expression of human osteocalcin in a host cell ( B-1 sequence), B-2 is a DNA sequence encoding the human osteocalcin Gla recognizing enzyme recognition polypeptide (B-2) sequence, A is a DNA encoding a signal peptide which may be optionally bound. A DNA sequence for expressing human osteocalcin represented by the formula (A sequence) is provided.

Human represented by the above formula [I] of the present invention
A DNA sequence for expression of osteocalcin is prepared by preparing a plasmid containing the same according to the method described below, transforming a host cell with the obtained plasmid, and culturing the transformed host cell. It is possible to obtain osteocalcin.

Human represented by the above formula [I] of the present invention
DNA sequence for expression of osteocalcin, plasmid containing the same, transformation of host cell with this plasmid,
The expression, isolation, purification and confirmation of human osteocalcin will be described in detail below.

In the above-mentioned DNA sequence for human osteocalcin expression of the formula [I] of the present invention, the D sequence represents a DNA sequence encoding human osteocalcin or a protein physiologically equivalent thereto. As such a DNA sequence, for example, the DNA sequence shown in SEQ ID NO: 1 can be mentioned. The amino acid sequence corresponding to the DNA sequence in SEQ ID NO: 1 is shown in the lower row.

Such a protein physiologically equivalent to human osteocalcin means (i) an amino acid which has the same physiological activity as human osteocalcin itself or does not have the same physiological activity. Glu in the sequence
If all or part of the
It refers to a variant of human osteocalcin having the same physiological activity as that of osteocalcin, or (ii) a substance that can be detected by an immunological assay method for fully human osteocalcin.

Here, a modified human osteocalcin means, for example, in the 49 amino acid sequence of human osteocalcin shown in SEQ ID NO: 1, some amino acids, preferably 1 to 5 amino acids are other amino acids. Or a deletion of 1 to 5 amino acids, insertion of another 1 to 5 amino acids, or a combination thereof.

Further, the above-mentioned immunological assay method for fully human osteocalcin may be a method capable of immunologically assaying and detecting the intact human osteocalcin in a sample containing human osteocalcin separately from its fragment. For example, the PCT /
The method described in JP90 / 00155 application (Japanese application date February 10, 1989: application number 30003/89) can be mentioned. The fully human osteocalcin enzyme immunoassay method proposed by the present inventors is described in Reference Example 1 to
4 will be described.

The C sequence in the above formula [I] represents a DNA sequence encoding a cleavage peptide. Examples of the sequence that can be used as such a DNA sequence include, for example, an amino acid sequence cleavable by human osteocalcin propeptidase, an amino acid sequence cleavable by thrombin, and an amino acid sequence cleavable by Factor Xa (FXa),
An amino acid sequence cleavable by the CNBr method (Met),
Examples thereof include a DNA sequence encoding an amino acid sequence (Lys) that can be cleaved by lysyl endopeptidase. Among these, a sequence cleavable by thrombin, for example, a DNA sequence represented by SEQ ID NO: 3 is preferable. The lower row in SEQ ID NO: 3 is such DNA
The amino acid sequence corresponding to the sequence is shown.

The B sequence is a DNA sequence encoding a peptide necessary for expressing human osteocalcin or a protein physiologically equivalent thereto.

B sequence is B-1 sequence and / or B-2
It consists of an array. The B-1 sequence is a DNA sequence encoding an additional polypeptide for expressing human osteocalcin in a large amount (significant amount or efficiently) in a host cell, and it exists stably in the host cell, or It is preferably a DNA sequence which encodes a polypeptide forming an inclusion body. The B-1 sequence is preferably selected depending on the type of host cell. Specific examples of the B-1 sequence include β-galactosidase, β-lactamase, chloramphenicol acetyltransferase, alkaline phosphatase, and Escherichia coli as host cells.
Staphylococcal protein A, β-interferon, α
Examples thereof include DNA sequences encoding ₁ -antitrypsin, human growth hormone and the like. Particularly, a DNA sequence encoding a human growth hormone-derived polypeptide is preferable.

On the other hand, the B-2 sequence is a DNA encoding a human osteocalcin Gla-recognizing enzyme recognition polypeptide.
It is an array. As a sequence that can be used as such a DNA sequence, a Gla protein-derived propeptide or a derivative thereof (for example, 1 to 5 amino acid residues of the propeptide are deleted or inserted or substituted so that γ-carboxylase can work efficiently). DNA sequence (B-2 sequence) that encodes Of these, a DNA sequence encoding a human osteocalcin propeptide or a prothrombin propeptide is preferable. Such a D
As the NA sequence, for example, the human sequence shown in SEQ ID NO: 2
The DNA sequence encoding the propeptide of osteocalcin may be mentioned. DNA in SEQ ID NO: 2
The amino acid sequence corresponding to the sequence is shown in the lower row.

In the above formula [I] of the present invention, B may be either the B-1 sequence or the B-2 sequence, but preferably the B-1 sequence and the B-2 sequence are combined. It is a DNA sequence.

In this case, it is preferable that the B-1 sequence and the B-2 sequence are arranged in this order.
It also includes one having a structure in which 1 sequence and B-2 sequence are linked by a DNA sequence encoding a cleavage peptide as shown in the above C sequence.

In the present invention, such D, B, C
By arranging the sequences in the order of B-C-D represented by the above formula [I], DN for expressing human osteocalcin is obtained.
It can be an A array.

Further, to the DNA sequence of the above formula [I] of the present invention, a DNA sequence (A sequence) encoding a signal peptide can be ligated to the N terminus of the BCD sequence described above. The A sequence may be linked at any time as necessary, and a mode in which it is not linked is also included in the present invention.

The A sequence may be a DNA sequence encoding a signal peptide that can be secreted by the host cell, and an appropriate sequence is selected depending on the type of host cell. When Escherichia coli is used as a host, a DNA sequence encoding a signal peptide such as OmpA, PhoA, OmpF, protein A, β-lactamase (penicillinase), endotoxin, and human growth hormone can be used as the A sequence. The signal peptide (pre-peptide) of human osteocalcin has an N-terminal charge of +1 (arginine residue). On the other hand, in signal peptides such as prolipoprotein, it was reported that it secretes most when the charge of the N-terminal region is +2 (Inouye, S. et al PNAS 79, 3438-
3441, 1982).

The signal peptide of the present invention includes
Among the above, the signal peptide of human osteocalcin or its N-terminal charge of +2 (modified) is preferable. As such an A sequence, for example, SEQ ID NO:
The DNA sequence shown in 4 can be mentioned. Sequence number:
The lower row of 4 shows the amino acid sequence corresponding to such a DNA sequence.

Further, according to the present invention, a human osteocalcin expression vector for expressing a DNA sequence for expressing human osteocalcin represented by the above formula [I] in a host cell, and a set transformed with the vector. Altered host cells are provided.

DN for expressing human osteocalcin
A vector capable of expressing the A sequence in a host cell (human osteocalcin expression vector), and recombinant host cells transformed with this vector can express human osteocalcin. In that case, human osteocalcin is expressed. It is desirable to provide a DNA sequence having an expression control function (expression control sequence). As such a sequence, a sequence having a function of promoting expression or, in some cases, suppressing expression,
Examples include promoters and enhancers,
Among them, expression control sequences capable of functioning in E. coli, such as tryptophan operon promoter and tac promoter, are preferable.

B-C-D in the above formula [I] of the present invention
Alternatively, the ABCD sequence is a suitable promoter,
By linking to the downstream of the SD (Shine-Dalgarno) sequence, an intracellular expression type gene can be obtained.

[0035] as an available promoter, tryptophan operon promoter (trp promoter), lactose operon promoter (lac promoter), tac promoter, P _L promoter, such as lpp promoter, and the like, and especially trp promoter, tac A promoter is preferred.

In order to efficiently express human osteocalcin, which is the object of the present invention, a promoter, an SD sequence, a translation initiation codon, the DNA sequence of the above formula [I], a translation termination codon, and a terminator (transcription termination sequence) are used. Those linked in order are preferred.

By inserting the sequence thus ligated into an appropriate expression vector such as a plasmid, human
An osteocalcin expression plasmid can be created.

As the expression plasmid vector in E. coli, pUC18, pHSG398, pHSG399, p
RIT2T, pUEX1-3, pKK223-3, pI
NIII 1, pTTQ18, pGEMEX-1, pGH-
L9, pKK233-2, etc. can be used.

In the present invention, the tac promoter,
An SD sequence, a translation initiation codon, a cloning site, and a terminator are included in this order, and pKK233-2 having the sequence inserted at an appropriate cloning site is human.
Preferred as an osteocalcin expression plasmid. Furthermore, pGH-L9 having a trp promoter, an SD sequence, a translation initiation codon, a DNA sequence encoding human growth hormone, and a terminator in this order is particularly preferable as an expression plasmid.

In particular, in the case of the latter expression plasmid, the trp promoter can be obtained by including the DNA sequence portion coding for human growth hormone of the expression plasmid into a BCD sequence which is a DNA sequence for human osteocalcin expression. SD sequence, translation initiation codon, BCD sequence,
A human osteocalcin expression plasmid containing and terminator in this order is obtained.

By introducing the expression plasmid into a host cell, a transformed host cell capable of producing human osteocalcin can be obtained.

As the host cell to be transformed,
Examples thereof include microorganisms, cultured animal cell lines, and animal cells, but are not particularly limited as long as they are host cells suitable for expressing human osteocalcin. However, the use of microorganisms, especially E. coli, is preferred. When E. coli is used as a host cell, HB101, D derived from E. coli K12 strain
H1, C600, SCS1, JM109, XL1-Blue
Among them, HB101 is preferable, and the XL1-Blue F ⁻ strain from which the F factor of XL1-Blue (including the lacI repressor gene) is removed is also preferable.

Further according to the present invention, the host cell transformed as described above is cultured, and in the culture, the following formula [II] B'-C'-D '... [II] wherein D'is human. -Osteocalcin or a physiologically equivalent protein thereof, C'indicates a cleavage sequence peptide, B'indicates B'-1 and / or B'-2, where B'-1 is of human osteocalcin Showing a Gla-recognizing enzyme-recognizing polypeptide and B'-2 showing an additional polypeptide for expressing human osteocalcin in a host cell in a large amount, and producing a polypeptide-containing protein represented by (a) This protein is cleaved and human osteocalcin or a protein (D ') physiologically equivalent thereto is isolated, or (b) Glu in the D'protein in the protein is converted to Gla and further cleaved. A method for producing human osteocalcin or a protein physiologically equivalent thereto is provided, which comprises isolating human osteocalcin or a protein physiologically equivalent thereto.

Further, according to the present invention, there is provided human osteocalcin obtained by the above production method or a protein physiologically equivalent thereto.

The D'protein, B'peptide and C'peptide are, for example, amino acid sequences corresponding to the DNA sequences represented by the B, C and D sequences, more specifically, SEQ ID NO: 1 to SEQ ID NO: : 3 can be mentioned.

In the production method of the present invention, the culture of the recombinant host cells is preferably 2XYT medium or M9CA.
The culture is performed in a medium or the like, and such a culture produces a protein containing the polypeptide represented by the above formula [II] in the culture. The term "cultured product" as used herein refers to a product containing the inside of the cells and / or the medium, depending on whether the host cells used for the culture produce the target protein outside the cells.

In the present invention, the D'protein portion of the protein is then converted to Gla. For example, in the case of a host cell that secretes into the cells, preferably, the cells are collected in the late logarithmic growth phase to obtain a periplasmic fraction by a known method such as an osmotic shock method. The protein is purified by the method described above, for example, antibody affinity column method. Then, this purified or unpurified protein was subjected to in vitro γ-
G by carboxylase (eg from liver)
la conversion. Then, the B'peptide and the Gla'ed D'protein are cleaved at the C'peptide portion to isolate the D'protein. Isolation can be performed by a known method, for example, using a hydroxyapatite column or an antibody affinity column to purify Gla-modified human osteocalcin.

Alternatively, in the present invention, for example, AB
2-Using the expression DNA sequence represented by the C-D sequence (the C sequence is preferably a sequence that can be cleaved by propeptidase), it is expressed in an animal cell (CHO cell), and the Gla-modified D protein is intracellularly expressed. Alternatively, the D'protein can be isolated and purified to obtain Gla-modified human osteocalcin.

On the other hand, in the present invention, the protein obtained by the above method can be purified to make the D'protein portion Gla. For example, in the case of a host cell forming an inclusion body, it is preferable to collect the cells at the final stage of growth and perform a known method,
For example, the cells are disrupted by ultrasonic disruption, and then the inclusion bodies are obtained as a precipitate by performing centrifugation, and the inclusion bodies are solubilized using a protein denaturing agent such as urea, and the crude protein is subjected to dialysis. A purified aqueous solution is obtained. Further, the protein is purified by a known method such as reverse phase high performance liquid chromatography. This purified human
The osteocalcin fusion protein was labeled with γ-carboxylase (for example, from liver) in vitro by γ-carboxylase.
(Γ-carboxylation).

The Gla conversion method can be carried out by a method known per se.

Specifically, JE Knobloch and JW Sutti
e, J. Boil. Chem. 262: 15334-15337 (1987) or C.
Carboxylation is carried out by the method described in Vermeer JP-A-63-502956. For example, a microsomal fraction was prepared from rat or bovine liver (JA Sad
owski et al, J. Biol. Chem. 251: 2770-2776 (197
6)), 0.25 M sucrose-0.025 M imidazole-0.5 M KCl (pH 7.2), 1.5% T
Suspend in a solution with the composition of the Ritton X-100. To this suspension, add 2mM NADPH, 1mM DTT, 1m
M EDTA, 1 mM MnCl ₂ , 0.01-mM
The B'-C'-D 'peptide (carbonate buffer solution) is added, and 100 μg · ml Vitamin K ₁ H ₂ is further added to start the reaction, which is incubated at 17 ° C to carry out the carboxylation reaction. Thereafter, the Gla-modified B'-C'-D 'peptide was used as an antibody (WO-90
09587) Purify by affinity column and cleave B'-C 'peptide by treating with thrombin.

Further, a Gla-modified D'peptide (human osteocalcin) is obtained by an antibody against an N-terminal 20 residue peptide (WO-9009587) affinity column and hydroxyapatite column.

The non-Gla-ized human osteocalcin and the Gla-ized human osteocalcin thus obtained are not only the standard substance of the enzyme immunoassay, the antigen for antibody production, or the purification of the antibody, but also those associated with bone metabolism. It can be used as a medicine.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

The various reagents and genetic engineering techniques adopted in the examples are as follows.

(1) Cleavage of DNA by restriction enzyme (method
1) Restriction enzymes EcoRI, BamHI, XbaI, Dr
aI, AvaIII (EcoT22I), XhoI, Kp
For nI and PstI, a universal buffer manufactured by Takara Shuzo was used.

For NheI and BglI, 10X buffer manufactured by Nippon Gene was used.

NcoI, PvuI, and BclI are One-Phor-All manufactured by Pharmacia.
Buffer PLUS was used.

(2) Agarose electrophoresis (method 2) After digestion with a restriction enzyme, 3 μl of 0.25% bromophenol blue / 50% glycerol aqueous solution was added, and agarose gel electrophoresis (gel concentration 0.8-2%). I went. As the electrophoresis buffer, 40 mM Tris
-Acetate-1 mM EDTA aqueous solution was used.

(3) DNA fragments (number
100 bp-) (Method 3) Agarose electrophoresis was performed using low melting point agarose (manufactured by Sigma), and a band corresponding to the desired DNA fragment was cut out and recovered by GENECLEAN Kit (manufactured by Funakoshi). ..

(4) DNA fragment from agarose gel
Recovery of (up to several hundred bp) (Method 4) Agarose gel electrophoresis is performed, and separation is performed to obtain the desired D
The DEAE membrane (NA & 45, S & S Co.) is inserted immediately before the band corresponding to the NA fragment, and electrophoresis is performed again to adsorb the target DNA fragment on the membrane. NE this
T Buffer (0.15M NaCl-0.1 mM
EDTA-20 mM Tris.HCl (pH 8))
After washing with, cut into small pieces and cut with High Sal + NE
T Buffer (1.0 M NaCl-0.1 mM
EDTA-20 mM Tris.HCl (pH 8)) was added, the mixture was incubated at 65 ° C. for 1 hour, the DNA fragment was eluted, and the target DNA fragment was recovered by ethanol precipitation.

(5) Inversion by T4-DNA ligase
Response (Method 5) 1 using a DNA ligation kit (Takara Shuzo)
Ligation was performed by reacting at 6 ° C. for 3 hours or more.

(6) Repair reaction (blunting reaction of protruding ends)
(Method 6) A DNA blunting kit (manufactured by Takara Shuzo) was used according to the manual of the kit.

(7) Competent cell preparation and transformation
(Method 7) Transformation of Escherichia coli is carried out by the usual CaCl ₂ method (MV Norga
rd et al.). Ie 5 ml LB
Medium (1% tryptone, 0.5% yeast extract, 0.5%
NaCl, pH 7.2) was inoculated with a culture medium of E. coli for 18 hours, and the turbidity (OD) at 600 nm of the culture solution containing the bacterial cells was
₆₀₀ ) grown to 0.5-0.6.

30 ml of culture solution was centrifuged to collect the cells, and 15 ml
Wash with ice-cold 0.1M MgCl ₂ , and centrifuge again to collect the cells, suspend in 15 ml of 0.1M CaCl ₂ , and leave in ice water for 20 minutes or more. Then centrifuge again to collect the bacteria,
Resuspend with 1.5 ml 0.1 M CaCl ₂ and 0.2 ml
DNA solution after the ligation reaction (~ 10 μl (~ 1
μg)) was added and the mixture was incubated at 0 ° C. for 30 minutes or more. Then after incubating at 42 ℃ for 2 minutes,
Cool again at 0 ° C, add 0.8 ml of LB medium, and add 37 ° C.
And incubated for 1 hour. This culture solution was inoculated into a selective medium (LB agar medium containing 50 μg / ml of ampicillin or 30 μg / ml of chloramphenicol) and cultured at 37 ° C. overnight to grow a transformant.

From the obtained drug resistant colonies, Birnboim
Et al. (Birnboim, HC and J. Doky, Nucleic Acids
Res., 7, 1513 (1979)), the plasmid DNA was prepared, hydrolyzed using an appropriate restriction enzyme, and the pattern was analyzed by agarose gel electrophoresis.
The desired clone was obtained.

(8) Determination of DNA base sequence (method 8) Chen et al.'S method (Chen, EY and Seeburg, PH, DNA,
4 , 165 (1985)), using plasmid DNA as a template, M13 primer M3 and RV (manufactured by Takara Shuzo) as primers, and Deaza T7 Sequencing Mixes (manufactured by Pharmacia) and T7 Sequencing Kit (manufactured by Pharmacia). went.

(9) Other methods All DNA manipulations are performed by the method of Maniatis et al. (Molecular Cl
onings Cold Spring Harbor Laboratory, New York, 19
82).

(10) Peripla by osmotic method
Collection of the sham fraction (method 10) The culture solution was centrifuged to collect the cells. Sedimented cells (cell volume = (O
D ₆₀₀ value) x (culture medium (ml)) = 2-3) 0.15
ml of solution A (20% sucrose, 10 mM Tris HC
1 (pH 7.6)), and incubated in ice water for 10 minutes. Next, it was centrifuged at 7,000 rpm at 4 ° C. for 10 minutes, and the precipitated bacterial cells were resuspended in 0.15 ml of ice-cooled distilled water and incubated in ice water for 10 minutes. At this time, the mixture was vigorously stirred several times with vortex etc. (the outer membrane of the bacterium is ruptured by the Osmotech shock, and the protein in the periplasm comes out). Then, it was centrifuged again at 7,000 rpm at 4 ° C. for 10 minutes, and the supernatant was collected as a periplasmic fraction.

The precipitate was added to 0.15 ml of 50 mM Tris.
It was suspended in HCl (pH 8) and crushed using an ultrasonic crusher (Tosoh Corp., UCD-200T type). This was centrifuged at 15,000 rpm at 4 ° C. for 10 minutes, the supernatant was subjected to cytoplasmic fractionation, and the precipitate was used as (outer membrane + inner membrane + inclusion body) fractionation. The precipitate was 0.15 ml of 50 mM Tris HCl (pH
8), suspended in 1 / SDS 10% Glycerol, 9
Incubate at 5 ° C for 5 minutes to dissolve.

(11) Complete human osteocalcin enzyme
Immunoassay Method (Method 11) Hereinafter, a method for preparing an antibody and a configuration of a measurement system will be described.

Reference Example 1 Preparation of N-Terminal Peptide Antibody (A) Synthesis of N-Terminal 20-Residue Peptide; Cysteine at 20th and 21st Residues of Amino Acid Sequence Specific to N-Terminal of Human Ostecalcin The inserted peptide represented by SEQ ID NO: 7 was synthesized. A peptide synthesizer manufactured by ABI was used for the synthesis. The name of this peptide is Ost-N
(20). (B) Preparation of antigen (conjugate of Ost-N (20) and carrier-protein); keyhole limpet hemocyanin (KLH) was used as carrier protein, and N- (m-
KLH was MBSed with maleimidobenzoic acid) -N-succinimide ester (MBS). On the other hand, Ost
The SH group of -N (20) was made free by 2-mercaptoethanol, and Ost-N (20) was added dropwise to MBS-form KLH to carry out the reaction while maintaining the pH of the reaction solution at 6.0 to 6.5. After reacting for 3 hours, it was dialyzed and the obtained product was used as an antigen. (C) Preparation of antibody: KLH conjugate of Ost-N (20) was immunized into a rabbit at 500 μg / 1 shot. Two
After immunization 6 times at weekly intervals, the antibody titer increased, so blood was collected and the antibody was purified with protein A-Sepharose to obtain the desired antibody.

Reference Example 2 Preparation of C-Terminal Peptide Antibody- (1) (A) Synthesis of C-Terminal 7-Residue Peptide; Amino Acid Sequence 7 Specific to C-Terminus of Human Osteocalcin Shown in SEQ ID NO: 8 A peptide having a residue was synthesized using a peptide synthesizer manufactured by ABI. The name of this peptide is Ost-
It was set to C (7). (B) Preparation of antigen (conjugate of Ost-C (7) and carrier protein); Ost-C (7) and KLH are mixed in equal weights, and dicyclohexylcarbodiimide (DCC)
Was reacted to prepare a conjugate of Ost-C (7) and KLH. The resulting product was produced using HPLC gel filtration. (C) Preparation of antibody; The KLH conjugate of Ost-C (7) was immunized into a rabbit at 500 μg / 1 shot. Thereafter, the target antibody was obtained in the same manner as in Reference Example 1.

Reference Example 3 Preparation of C-Terminal Peptide Antibody- (2) (A) Peptide Synthesis of C-Terminal 15 Residue; Amino Acid Sequence 15 Residue Specific to C-Terminal of Human Osteocalcin Shown in SEQ ID NO: 9 Was synthesized.

For the synthesis, a peptide synthesizer manufactured by ABI was used. The name of this peptide was designated as Ost-C (15). (B) Binding of Ost-C (15) with carrier protein;
Using the same method as in Reference Example 2 (B), the Ost-C
A conjugate of (15) and KLH was prepared. (C) Preparation of antibody: Using the same method as in (C) of Reference Example 2, the KLH conjugate of Ost-C (15) was used as an antigen to obtain the desired antibody.

Reference Example 4 Configuration of Measurement System (A) Horseradish Peroxidase (HR)
P) Preparation of labeled antibody; (1) HRP labeling of anti-Ost-N (20) antibody (IgG); 1 mg / ml of anti-Ost-N (20) antibody, 0.01M
2 ml of phosphoric acid, 0.15 M NaCl (pH 7.4) solution
50 μl of a dimethylformamide solution having a concentration of 10 mg / ml of MBS was added to the reaction mixture, and the mixture was reacted at a temperature of 25 ° C. for 30 minutes. Then, using a column packed with Sephadex G-25, 0.1M phosphate buffer (0.1M PB) (pH
Gel filtration was performed at 6.0) to separate the maleimidated antibody and unreacted MBS.

On the other hand, 10 mg / ml HRP 0.1 M P
To 2 ml of B (pH 6.5) solution, 60 mg / ml dimethylformamide solution of S-acetylmercaptosuccinic anhydride 12
0 μl was added, and the mixture was reacted at 25 ° C. for 2 hours. Then 0.1
800 μl of M Tris-HCl buffer (pH 7.0),
160 μl of 0.1 M EDTA and 1.6 ml of 1 M hydroxylamine were added, and the mixture was reacted at 0 ° C. for 4 minutes. afterwards,
The reaction solution was placed in a collodion bag, and 0.1M PB (p
H6.0), using a solution containing 5 mM EDTA, 4 ° C
After dialysis for 3 days, thiolated HRP was obtained.

Next, 2 mg of the maleimidated antibody and 4 mg of thiolated HRP were mixed and concentrated to a protein concentration of 4 to 10 mg / ml under ice cooling using a collodion bag.
Left overnight at ~ 20 ° C. The solution is Ultrogel Ac
Gel filtration was performed using a column packed with A44 (Pharmacia LKB) to obtain an HRP-labeled anti-Ost-N (20) antibody.

(2) Anti-Ost-N (20) antibody (F (a
b ′) ₂ ) HRP labeling; 1 ml of 2.0 mg / ml anti-Ost-N (20) antibody in PBS was added with 100 μl of 1M acetate buffer (pH 4.2) and 40 μg of pepsin.
It was dissolved in 0 μl of the same buffer and added, and the mixture was reacted at 37 ° C. for 4 hours. After completion of the reaction, use a Sephadex G25 column (φ2 cm × 45 cm) equilibrated with PBS to separate F
(Ab ') ₂ was collected. HRP-labeled anti-Ost-N (2
0) (F (ab ′) ₂ ) antibody was prepared by referring to Reference Example 1 (A).
It carried out according to (1).

(3) Anti-Ost-N (20) antibody (Fa
b ′) HRP labeling; F of anti-ost-N (20) antibody
(Ab ') ₂ fraction was prepared according to Reference Example 1 (A) (2), and this was reduced with mercaptoethylamine to purify Fab' by gel filtration HPLC using Tosoh G3000SW column.

On the other hand, HRP was prepared according to the maleimidization of the anti-Ost-N (20) antibody of Reference Example 1 (A) (1).
P was maleimidated. Next, 2 mg of anti-Ost-N (20) antibody (Fab ′) and 4 mg of maleimidated HRP were mixed, concentrated using a Filtron (ultrafiltration device), and reacted at 4 ° C. for 16 hours. This is Toso G3
It was isolated and purified by gel filtration HPLC using a 000SW column to obtain an HRP-labeled anti-Ost-N (20) (Fab ′) antibody. (B) Preparation of antibody-immobilized beads; (1) Antibody immobilization; polystyrene beads (diameter 6 m
m) was thoroughly washed, and then anti-Ost-C (7) and anti-O
P with a concentration of 20 μg / ml of st-C (15) antibody
After leaving it in the BS solution at a temperature of 4 ° C for 1 day and night, wash it with PBS, and leave it in a PBS solution of 1% bovine serum albumin (BSA) at a temperature of 4 ° C for 1 day and post-coat it. , Anti-Ost-C (7), anti-Ost-C (1
5) An antibody-immobilized bead was obtained.

(2) Activity evaluation of antibody-immobilized beads; anti-Ost-C (7) antibody and anti-Ost- prepared in (1) above
The activity of the C (15) antibody was examined by the following method. 0.05M Tris-HCl, 0.15 containing 1% BSA containing 1 bead on which each antibody was immobilized and purified human osteocalcin (standard substance) in the range of 0 to 100 ng / ml.
M NaCl buffer (0.05 MTBS) (pH 8.
0) 0.05 M containing 1% BSA containing 200 μl and Fab ′ fraction of HRP-labeled anti-Ost-N (20) antibody
200 μl of TBS (pH 8.0) solution was added to the test tube and incubated at a temperature of 4 ° C. for 17 hours. Next, after removing the solution in the test tube by suction, 0.05M TBS
After washing with (pH 8.0) 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride, 0.02% H ₂ O ₂ 2.
Add 0.4 ml of 0.1 M phosphate / citrate buffer (pH 4.3) containing 5 mM to each test tube at 25 ° C.
After reacting at the temperature of 30 minutes, 1N as a reaction terminator
The enzymatic reaction was stopped by adding 1 ml of a sulfuric acid aqueous solution.

Then, this solution was mixed with a spectrophotometer to 4 times.
The absorption intensity at a wavelength of 50 nm was measured, and this was blotted at a standard substance concentration of 0 to 100 ng / ml. Results are shown in FIG. (C) Configuration of measurement system; 0 to 20 ng / ml of one anti-Ost-C (7) -immobilized bead prepared in Reference Example 1 (B) (1) and purified human osteocalcin (standard substance)
0.05% TBS containing 1% BSA
(PH 8.0) 200 μl and Reference Example 4 (A) (1)-
200 μl of a 0.05 MTBS (pH 8.0) solution containing 1% BSA of each HRP-labeled antibody prepared in (3),
Immobilized antibody beads and HRP-labeled antibody IgG, F (a
b ') Added to each tube in combination with ₂ fractions and incubated for 2 hours at a temperature of 25 ° C. Next, after removing the solution in the test tube by suction, 0.05M TBS
After washing with (pH 8.0) 3,3 ′, 5,5′-tetramethylbenzidine hydrochloride, 0.02% H ₂ O ₂ 2.
0.1 M phosphate / citrate buffer containing 5 mM (p
0.4 ml of H4.3) was added to each test tube and reacted at a temperature of 25 ° C. for 30 minutes, and then 1 ml of 1N sulfuric acid aqueous solution was added as a reaction terminator to stop the enzymatic reaction. Then, the absorption intensity of this solution was measured at a wavelength of 450 nm using a spectrophotometer, and the concentration of the standard substance was determined to be 0 to 20 ng.
N / S ratio by the fractionation of the HRP-labeled antibody (absorbance at 0 ng / ml of antigen was determined by
The value was divided by the absorbance of 0 ng / ml). The results are shown in Table 1.
Shown in.

[0084]

[Table 1]

As shown in Table 1, depending on the type of HRP-labeled antibody,
Measurement sensitivity changes and F (ab ') ₂, IgG fraction
Has a low background and constitutes a highly sensitive measurement system.
It is clear that HRP-labeled antibody (F (a
b ')₂13 shows a calibration curve using ().

The fully human osteocalcin assay system constructed as described above (HRP-labeled antibody is F (ab ') 2).
₂ ) medium fraction, periplasmic fraction, cytoplasmic fraction,
Dilute the outer membrane, inner membrane, and inclusion body fractions 10 to 20-fold (diluent: 0.1% BSA-0.2% Scum milk-
0.02% Tween20-0.05M TBS (pH
7.4) was used. ) And measured.

[0087]

Example 1 Design of DNA sequence for expression of human osteocalcin and
Synthesis and subcloning ; human shown in SEQ ID NO: 5
Amino acid sequence of osteocalcin (AJ Celeste et al. EMBO
Based on J. 5, 1885-1890, 1986), a restriction enzyme recognition site necessary for gene construction was provided, and a DNA sequence for expressing human osteocalcin was designed. The design is shown in FIG. In the figure, the solid line indicates the boundary of the synthetic DNA fragment, and the dotted line indicates the restriction enzyme cleavage surface.

The DNA sequence for expressing human osteocalcin (SEQ ID NO: 6) is roughly divided into a signal peptide (pre-peptide) coding portion, a propeptide portion [a portion coding for a Gla recognizing enzyme recognition portion + a cleavage sequence portion]. Part] and a part encoding the human osteocalcin part. By cleaving with a suitable restriction enzyme, the signal peptide portion,
The Gla recognizing enzyme recognition portion, the cleavage sequence portion, and the human osteocalcin portion can be replaced with those having different sequences in the cassette type.

The gene thus designed is divided at the boundary position of the synthetic DNA fragment shown in FIG.
A total of 12 oligonucleotides up to ~ 1 were chemically synthesized.

Oligonucleotide synthesis is fully automated DNA
Synthesizer (Applied Biosystems, Model 3
81A) was carried out by the phosphoramidite method. Purification of the synthetic oligonucleotide was performed according to the manual of Applied Biosystems. That is, by keeping an aqueous ammonia solution containing a synthetic oligonucleotide at 55 ° C. overnight, the protecting group of the DNA base was removed, and gel filtration using Sephadex G-50 Fine Gel (Pharmacia) was performed to obtain a high molecular weight product. The synthetic oligonucleotide fraction was collected.

Then, after polyacrylamide gel electrophoresis containing 8 M urea (gel concentration 10%), the migration pattern was observed by the ultraviolet shadowing method, and the band corresponding to the oligonucleotide portion of interest was cut out. After finely crushing the gel fragments, 5
ml elution buffer (500 mM NH ₄ OAc-1
mM EDTA-0.1% SDS (pH 7.5)) was added, and the mixture was shaken at 37 ° C overnight. By centrifugation, the desired D
The aqueous phase containing NA was recovered. Finally, the solution containing the synthetic oligonucleotide was applied to a gel filtration column (Ceradex G
-50) to obtain a purified product of synthetic oligonucleotide. If necessary, polyacrylamide gel electrophoresis was repeated to improve the purity of the synthetic oligonucleotide. 1 to 10 μg of the purified synthetic oligonucleotide thus obtained was added to 1 mM A
Polynucleotide kinase reaction is performed in the presence of TP,
The 5'end side was phosphorylated.

Polynucleotide kinase reaction was carried out at 50 mM using 10 units of T4 polynucleotide kinase.
Tris-HCl (pH 9.5) -10 mM MgCl
_It was carried out in a 2-5 mM dithiothreitol aqueous solution. 5 '
Two synthetic oligonucleotides corresponding to the upper and lower strands with phosphorylated ends were mixed and the aqueous solution temperature was adjusted to 95
Annealing was performed by gradually cooling from ° C to room temperature.

Furthermore, the annealed synthetic fragments (g, h), (i, j), (k, l) (these six synthetic fragments are A of the ABCD sequences described above). 3 µg of each of -BC sequences (Fig. 1) was taken and ligated with T4 DNA ligase. The reaction solution was treated with phenol / chloroform, precipitated with ethanol, and then separated by 2% agarose gel electrophoresis, and a band of about 150 bp was recovered from the gel according to Method 4.

For subcloning, modified pBP of cloning vector pHSG399 (Takara Shuzo)
R9 was used. The preparation method was as follows.

First, pHSG399 was cleaved with a restriction enzyme PvuI, which was blunt-ended by a repair reaction according to Method 6, and then subjected to an intramolecular ligation reaction with T4 DNA ligase according to Method 5, to obtain a pH without PvuI site.
SG-PR9 was created. In addition, add pHSG-PR9 to B
It was cleaved with glI, blunt-ended by a repair reaction, and then subjected to an intramolecular ligation reaction with T4 DNA ligase to prepare pBPR9 having no BglI site. (FIG. 2) (pHSG-PR9 and pBPR9 both produce a peptide that maintains the activity of the α chain of β-galactosidase, and the XL1-Blue transformant shows IPTG-Xg.
Form blue colonies on al agar. ) PBPR9 obtained as described above is transformed with EcoRI and Kpn.
It was cleaved with I, separated by 1% agarose gel electrophoresis (method 2), and recovered from the gel according to method 3. Collected D
The NA fragment and the 150 bp synthetic fragment-ligated DNA fragment corresponding to the ABC sequence were subjected to T4 according to Method 5.
Ligation was performed with DNA ligase.

E. coli XL1-B prepared according to method 7.
The ligation reaction solution was added to 200 μl of lu competent cells, transformation was performed, and the cells were plated on a selective agar medium containing chloramphenicol. From the colonies grown on the selective agar medium, pBPR-PRE was prepared as plasmid DNA. (FIG. 3) The plasmid pBPR-PRE having the desired subclone was produced by using the obtained plasmid Av.
It was confirmed by carrying out agarose gel electrophoresis that a fragment of about 150 bp was generated when digested with aIII and digested with EcoRI and PstI.

Using the same procedure as above, the synthetic fragments a, b, c, d, e, and f corresponding to the D sequence part were annealed, ligated, and the DNA fragment was recovered. On the other hand, the cloning vector pBPR9
It was cleaved with I and XbaI, separated by agarose gel electrophoresis, and recovered from the gel according to Method 3.

The recovered DNA fragment and the synthetic fragment-ligated DNA fragment (about 150 bp) were ligated according to method 5, and transformed according to method 7 to obtain chloramphenicol resistant colonies. Several liquid cultures of these resistant colonies were prepared, plasmids were prepared from each, and Kp
After cleaving with nI and XbaI, a plasmid pB having the desired DNA fragment (D sequence) was confirmed by confirming a band of about 150 bp by agarose gel electrophoresis.
PR-MOC was obtained. (Figure 4)

[0099]

Example 2 Clonin of DNA sequence for human osteocalcin expression
And confirmation of the salt DNA sequence ; the plasmid pBPR-PRE in which the ABC sequence portion of the human osteocalcin expression DNA sequence obtained in Example 1 was cloned was digested with restriction enzymes EcoRI and KpnI, and this was digested with agarose. After separation by gel electrophoresis, a DNA fragment of about 150 bp of ABC sequence portion was recovered from the gel according to Method 4. On the other hand, the plasmid pBPR-MOC obtained by cloning the D sequence portion of the DNA sequence for human osteocalcin expression obtained in Example 1 was digested with restriction enzymes EcoRI and Kp.
It was cleaved with nI, separated by agarose gel electrophoresis, and a DNA fragment of about 2350 bp containing the D sequence and the vector was recovered from the gel according to Method 3.

The former approximately 150 bp DNA fragment and the latter approximately 2
The 350 bp DNA fragment was ligated according to method 5, then transformed according to method 7, and a plasmid was prepared from a chloramphenicol resistant colony. Plasmid DNAs obtained from several colonies were cleaved with restriction enzymes EcoRI and PstI, separated by agarose gel electrophoresis, and a fragment of about 300 bp was confirmed to confirm the desired DNA sequence for human osteocalcin expression (A A plasmid pBPR-OC having the (-B-C-D sequence) was obtained. (Fig. 5)

[0101]

Example 3 Preparation of expression plasmid : The plasmid pBPR-OC obtained in Example 2 was cleaved with the restriction enzyme PvuI, ethanol-precipitated, and the end blunting reaction was carried out by the method 6. Then, it is cut with the restriction enzyme PstI,
About 300 bp DNA by agarose gel electrophoresis
The fragment (DNA sequence portion for expressing human osteocalcin) was separated and recovered according to Method 4. On the other hand, the expression vector pKK233-2 (Pharmacia) was used for Nc
After cleavage with oI and ethanol precipitation, a blunt end reaction was performed according to Method 6. Then PstI
Cleavage with agarose gel electrophoresis
The 00 bp DNA fragment was separated and recovered according to Method 3.

The former about 300 bp obtained in this way
The DNA fragment and the latter approximately 4600 bp DNA fragment were used in Method 5
The ligation reaction was performed according to. The ligation reaction solution was transformed according to Method 7, and a plasmid DN was obtained from colonies grown on a selective agar medium containing ampicillin (50 μg / ml).
A was adjusted. The obtained plasmid DNA is treated with restriction enzyme E
The expression plasmid pKK-OC was obtained by digesting with coRI and PstI and confirming an approximately 600 bp DNA fragment by agarose gel electrophoresis. (Figure 6)

[0103]

Example 4 Human Osteo by Colony Hybridization
Confirmation of production of a polypeptide containing calcin ; host cell XL using the expression plasmid pKK-OC obtained in Example 3
1-Blue was transformed. The obtained transformant (recombinant host cell) XL1-Blue [pKK-OC] was added to L
The cells were inoculated in B medium and cultured overnight at 37 ° C. This culture solution (about 10 ⁹ cells / ml) was diluted 10 ^-6 and 0.05 ml was taken and inoculated on a nitrocellulose membrane placed on LB agar medium (ampicillin 50 μg / ml). The next day, when the diameter of the colony became about 1 mm, remove the membrane (the colony is formed on the membrane), spread 2 ml LB medium (containing 2 mM IPTG) on the agar medium, and mount the membrane again to induce. I hung it After 8 hours, the membrane was peeled off and lysed with chloroform vapor to attach the bacterial protein to the membrane. Then 3% BSA (bovine serum albumin) -20 mM
Blocking with PBS was performed overnight at 4 ° C. The next day 0.
2% at room temperature with 05% Tween 20-20 mM PBS
Washed twice an hour. Next, human osteocalcin N-terminal 20
A 1% BSA-20 mM PBS containing a HRP-labeled antibody (obtained in Reference Example 4) of a rat polyclonal antibody (obtained in Reference Example 1) against a synthetic peptide homologous to the residue at a concentration of 1 μg / ml. Soak and incubate overnight at 4 ° C. Then again 0.05% Tween 20
Wash twice with -20 mM PBS for 20 minutes at room temperature, then 2
It was washed with 0 mM PBS for 20 minutes at room temperature. Finally, Konica Immunostain (manufactured by Konica) was used for color reaction for 4 minutes. As a result, in the transformant XL1-Blue [pKK-OC] which retained the expression plasmid pKK-OC, the colony part was stained in dark blue, and the expression vector pKK
Transformant XL1-Blue [p carrying 233-2
KK233-2] (control) did not stain.
As a result, the transformant XL1-Blue [pKK-
OC] was confirmed to produce a polypeptide containing human osteocalcin.

[0104]

Example 5 Human Osteocalci by Enzyme Immunoassay (EIA)
Of the production site and the amount of production of the polypeptide containing a protein ; the transformant retaining the expression plasmid pKK-OC obtained in Example 3 was LB medium (ampicillin 50 μg / ml).
The cells were inoculated and cultured overnight at 37 ° C. 0.4 to this culture
ml, 40 ml of 2 × YT medium (1.6% tryptone-
1.0% yeast extract-0.5% NaCl
(PH 7.6) and cultured at 37 ° C. Wavelength 60
When the absorbance at 0 nm (OD ₆₀₀ ) reached about 0.8 (logarithmic growth phase), 100 mM IPTG aqueous solution was added to 400
Induction was carried out by adding μl (final concentration 1 mM). Immediately before the induction and 1 hour, 3 hours and 5 hours after the induction, a part of the culture solution was taken and stored at 0 ° C.

The transformant used was XL1-Blue.
^{[PKK-OC], XL1-} BlueF - [pKK-O
C] (this is a factor F-deficient strain of XL1-Blue, in which the repressor lacI does not exist), XL1-B
About lu [pKK-OC] IPTG (Isopropyl-
Those with and without induction with β-D-thio-galactopyranoside were used.

[0106] The collected samples were collected ([centrifugation 5000 rpm, 4 ° C, 5 minutes) by collecting [2.4 ÷ (OD ₆₀₀ value)] ml of culture solution so that the numbers of the cells were equal. The supernatant (medium fraction) was stored at -20 ° C. According to Method 10, the pellet (bacteria) was separated into a periplasmic fraction, a cytoplasmic fraction, an outer membrane / inner membrane / inclusion body fraction.

The amount of the human osteocalcin-containing polypeptide (hereinafter, the human osteocalcin-containing polypeptide is referred to as "hOC '") in the sample thus obtained was measured by EIA (method 11). The results are shown in Table 2.

[0108]

[Table 2]

From these results, the transformant XL1-B was obtained.
In the logarithmic growth phase of lue [pKK-OC], medium fraction 0%, periplasm fraction 50%, cytoplasm fraction 35%,
HOC 'exists in a distribution of outer membrane / inner membrane / inclusion body fraction of 15%, and is most abundant in the periplasmic fraction (P). Therefore, hOC' is a signal peptide (encoded by the A sequence). B'-C'-
It was suggested to be secreted in the form of D '(the above general formula [II]).

Also, lacI (repressor protein)
XL1-transformant XL1 of strain XL1-BlueF ⁻
-BlueF - in ^[pKK-OC], it was found that has been produced most often produced in the logarithmic growth phase.

[0111]

[Example 6] Construction of signal peptide variant expression plasmid ; human
+ N-terminal charge of osteocalcin signal peptide
A modified signal peptide of No. 2 was prepared.

FIG. 7 (solid lines show both ends of the synthetic fragment, dotted lines show restriction enzyme cleavage surfaces, and m and n show synthetic DNA.
Synthetic DNA fragments m and n were prepared in the same manner as in Example 1 as shown in (showing a synthetic oligonucleotide when separated at the boundary position of the fragment), phosphorylated and annealed. On the other hand, the plasmid pBPR-OC in which the human osteocalcin expression sequence was cloned was digested with restriction enzymes EcoRI and BglI, separated by agarose gel electrophoresis, and a DNA fragment of about 2500 bp was recovered by Method 3. The synthetic DNA fragment m + n thus obtained and about 2500 bp DNA fragment were ligated according to Method 5. A plasmid having a modified DNA sequence of a signal peptide was obtained by transforming this according to method 7 and digesting the plasmid DNA obtained from the chloramphenicol resistant colony with the restriction enzyme DraI and observing the agarose gel electrophoresis pattern. pOC28 was obtained. (FIG. 8) The obtained plasmid pOC28 was cleaved with restriction enzymes DraI and PstI, and the resulting about 300 bp DNA fragment (containing a DNA sequence for human osteocalcin expression) was recovered by Method 4. On the other hand, as in Example 3, the expression plasmid vector pKK233-2 was cleaved with the restriction enzyme NcoI, then a repair reaction was carried out, and the digestion was carried out with the restriction enzyme PstI to obtain about 4600b.
The pDNA fragment was recovered. These about 300bp and about 46
The 00 bp DNA fragment was ligated according to Method 5, and then transformed according to Method 7 to isolate plasmid DNA from ampicillin-resistant colonies. Plasmid DNAs obtained from several resistant colonies were cleaved with restriction enzymes BamHI, and a DNA fragment of about 430 bp was confirmed by agarose gel electrophoresis to obtain human osteocalcin expression plasmid pKOC28. (Figure 9)
The hOC 'produced by this plasmid has a lysine residue at the N-terminus of the signal peptide and has a charge of +2 together with the arginine residue.

[0113]

Example 7: Plasmid pKK-OC for expressing human osteocalcin
And its modified pKOC28 in transformants
Comparison of amounts ; according to Example 5, for the following transformants, the logarithmic growth phase (logari) of hOC 'of the periplasmic fraction was calculated.
The production amount in the smic phase and stationary phase was measured by fully human osteocalcin EIA.

[0114] Transformants HB101 using [pKK-OC] HB101 [pKOC28 ] XL1-BlueF - [pKOC28] PAM660 [pKOC28] The results are shown in Tables 3 and 4.

From these results, the transformant XLI-Bl was obtained.
ueF ^- [pKOC28] is the logarithmic growth phase (logarismic p
hase) was found to produce the most hOC '. In addition, E. coli K-12 strain ^XL1-BlueF does not include the lacI - With respect to the [pKOC28], is deposited already in the Agency Fermentation Research Institute on June 13, 1990 as FERM BP-2950 ..

[0116]

[Table 3]

[0117]

[Table 4]

[0118]

[Example 8] Examination of immunoreactivity ; transformant XL1-BlueF
^- [PKOC28] was cultured, and the periplasmic fraction was prepared by Method 10. To 1 ml of this periplasmic fraction, add 2 units of beef thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) 37
Incubated at 0 ° C for 1 hour. A dilution test was performed on the thrombin-digested sample thus obtained and the thrombin-untreated periplasmic fraction. The method was carried out using fully human osteocalcin EIA (method 11) as in Examples 5 and 7.

The results are shown in FIG. The dilution curve of the periplasmic fraction containing hOC '(B'-C'-D') produced from the transformant (straight line b in the figure) is the standard substance, that is, Gla-modified human osteocalcin (in the figure, The parallel relationship with the straight line a) suggests that this hOC 'can be used as a standard substance. The thrombin-digested sample in which the B'-C 'sequence was presumed to have been cleaved (containing the D'sequence, ie, human osteocalcin not glaised; line c in the figure) was hOC'.
Since the value in EIA is higher than that in (B'-C'-D '), it is suggested that the B'-C' sequence sterically hinders the reaction of the antibody to the N-terminal of human osteocalcin. Was done.

Human osteocalcin-containing polypeptides
Confirmation of hOC 'by enzyme immunoassay ; in order to confirm that hOC' actually has the structure of B'-C'-D 'having the B'sequence, human osteocalcin propeptide (sequence Rabbit was immunized in accordance with a commonly known method using the propeptide part (No. 5) to obtain a polyclonal antibody against the human osteocalcin propeptide. Using this polyclonal antibody, an enzyme immunoassay of a periplasmic fraction containing hOC 'was carried out by a sandwich system. According to the method of Reference Example 4, a sandwich system was set up using anti-human osteocalcin propeptide polyclonal antibody-immobilized beads and anti-Ost-C (7) (F (ab ′) ² ) HRP label (Reference Example 2). The periplasmic fraction was prepared according to Method 10 from XL1-BlueF ⁻ [pKOC28]. Measurement result Curve b in FIG.
A dilution curve similar to that obtained is obtained and hOC 'is actually B'-C'.
It was confirmed to have a -D 'sequence.

[0121]

Example 9 Human Osteocalc by Immunoaffinity Column
Purification of syn-containing polypeptides ; transformants XL1-B1
ueF ^- 37 [pKK-OC] the in 500mlLB medium
Cultured overnight at ° C. This was mixed with 10 liter M9CA modified medium (2% casamino acid, 0.2% yeast extract, 0.1% NH ₄ Cl, 0.02% MgCl ₂ , 0.
01% CaCl ₂ , 0.0017% vitamin B 1,0.
(25% glucose) and cultured at 37 ° C. by a jar fermenter. After 8 hours, the turbidity of the culture solution was OD _660.
When the culture reached about 6, the culture was stopped and the cells were separated from the culture medium by continuous centrifugation. (15000 rpm, 15 ° C., 1 hour).

When the separated supernatant was measured by EIA according to Method 11, a hOC 'concentration of 5 to 7 ng / ml was obtained.
1 liter of this supernatant was added to human osteocalcin N powder 2
Polyclonal antibody against 0-residue synthetic peptide (PC
A) was applied to a fixed Sepharose 4B column,
Flow at a flow rate of 0.2 ml / min. Then, the column is
It was washed with ml of PBS so that the absorbance at 280 nm of the flowing-out liquid became zero.

Next, 6 ml of 0.1 M glycine-hydrochloric acid (pH
Elution with 2.5) (flow rate 1 ml / 8-9 min) and fractionation by 1 ml. For the test tubes to be collected, use 0.2M Tr in advance.
1 ml of is-HCl (pH 8.5) was added. The concentration of hOC 'in these aliquots was measured by fully human osteocalcin EIA according to Method 11. The results are shown in Fig. 11.

From FIG. 11, it can be seen that human osteocalcin and human osteocalcin-containing peptide (hOC ') were purified by the antibody affinity column. After purification, in vitro, for example, by glazing with γ-carboxylase derived from liver, it is possible to obtain human osteocalcin which is glaified and has physiological activity.

[0125]

Example 10 Human Growth Hormone (hGH) -Human Osteocalcin
Design and ligation of DNA sequence for expressing (hOC) fusion protein
Synthesis of DNA As shown in FIG. 14, the construction of a plasmid for expressing a human growth hormone-human osteocalcin fusion protein was designed. Further, the ligated DNA portion was designed as shown in FIG. The 5'end can be ligated to the BamHI cleavage site and the BglII cleavage site. The 3'end can be ligated with the BbeI cleavage site. In addition, the amino acid sequence Ile Glu G
lyArg is a cleavage sequence by FXa, and Lys
ProSer Gly corresponds to the N-terminal 4 residues of the B sequence. The ligated DNA was divided into + strand and − strand and synthesized by the same method as in Example 1 described above. Purification of the synthesized DNA was performed using an oligonucleotide purification cartridge (manufactured by Applied Biosystems Japan), and the operating procedure was performed according to the manual. Furthermore, + chain 5 μg, -chain 5 μg
And the annealing solution (0.1 M NaCl-
10 mM Tris.HCl (pH 8.0) -1 mM
Annealing was carried out by heating in EDTA) for 5 minutes at 70 ° C., then incubating at 50 ° C. for 2 hours, and then allowing to stand overnight and returning to room temperature. The DNA was evaporated to dryness and stored.

[0126]

Example 11 (Met ¹ -Phe ¹³⁹ ) Human Growth Hormone Fragment Polype
Peptide and human osteocalcin (BCD sequence part)
Min) fusion expression plasmid ; human growth hormone expression plasmid pGH-L9 (M. Ikehara et al, PNA
S., 81 , 5956 (1984)) 1 μg of restriction enzymes BglII and S
It was cleaved with alI and separated by agarose gel electrophoresis (method 2), and a DNA fragment of about 4.1 kb was recovered from the gel (method 3). In addition, the plasmid pBPR-OC obtained in the above Examples 1 and 2 was digested with the restriction enzymes BbeI and Sal.
After cutting with I and separation by agarose gel electrophoresis (method 2), a DNA fragment of about 220 bp was recovered from the gel (method 4).

A ligation reaction was carried out according to Method 5 between the two kinds of DNA fragments thus recovered and the DNA fragment synthesized in Example 10. Further, the reaction solution was transformed according to Method 7 to obtain ampicillin-resistant colonies. The resistant colonies were cultivated in liquid, and plasmids were prepared from each, and the restriction enzyme K
The fusion expression plasmid pGH-POCI was confirmed to be digested with KpnI by digesting with pnI and performing agarose gel electrophoresis (FIG. 1).
6).

Further, a DNA fragment obtained by cleaving pGH-L9 with restriction enzymes BamHI and SalI was used in place of the DNA fragment obtained by cleaving with BglII and SalLI as described above. The fusion expression plasmid pGH-POCII was obtained by the operation (FIG. 17).

[0129]

Example 12 Expression of fusion protein gene: Host cells HB101 and JM109 were transformed with the fusion expression plasmids pGH-POCI and pGH-POCII obtained in Example 11. The resulting four transformants HB101 [pGH-POC
I], HB101 [pGH-POCII], JM109
[PGH-POCI], JM109 [pGH-POCI]
I] was cultured as follows, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (UK
Leammli, Nature, 227, 680 (1970)).

The transformant was inoculated into LB medium containing 50 to 100 μg / ml of ampicillin, and cultured at 37 ° C. overnight.

M9 medium containing 0.2% glucose and 5 mg / ml casamino acid (0.6% Na ₂ HPO ₄ -0.3
% KH ₂ PO ₄ -0.05% NaCl-0.1% NH ₄
Cl aqueous solution (pH 7.4) was sterilized by autoclave, and then separately sterilized by autoclaving MgSO ₄ aqueous solution and CaCl ₂ aqueous solution to a final concentration of 2 mM and 0.
The above overnight culture solution was added at 1/100 to that added to 1 mM), and the mixture was cultured at 37 ° C. When the turbidity of the bacteria reached an OD _{600 of} about 0.5, the final concentration was 40 μg / ml.
3-β-indole acrylic acid (IAA) was added to the culture medium, and the culture was continued until the growth of the bacteria was stopped.
A few ml of the culture solution was collected 0 hours, 2 hours after the addition of IAA, and then every hour thereafter, and stored frozen. These SDS-PAG
The results of performing E are shown in FIGS. FIG. 18 shows the results when the plasmid pGH-POCI was used.
Shows the result when the plasmid pGH-POCII was used. It can be seen that the fusion protein is produced in the portions a and b indicated by the leader lines in FIG. 18 and the portions c and d indicated by the leader lines in FIG. (In Fig. 18, MW is about 2
8,000, and in FIG. 19, the MW was about 26,000. ) The yield was higher in the transformant having the fusion expression plasmid pGH-POCI at a few% of the total protein amount of the bacterial cells, and in the transformant of the HB101 strain, the growth rate was higher than that of the JM109 strain. Was fast. Regarding the transformant HB101 [pGH-POCI],
Contract number, 1990
It has been deposited at the Institute of Microbial Technology, Institute of Industrial Science, on January 27.

[0132]

Example 13 Purification of Fusion Protein ; Transformant HB101 [pGH-PO
CI] was cultivated in the same manner as in Example 12, the cells were collected by centrifugation, and then 0.5 M Tris.HCl was added.
The cells were suspended in (pH 8.0) and subjected to an ultrasonic disruption device (Kubota Shoji Co., Ltd., 200M type) to disrupt the cells, followed by centrifugation to obtain the target fusion protein as an inclusion body in the precipitate portion. .. The precipitate was added again to 0.5 M Tris.HCl (pH 8.
0), sonicated and centrifuged to remove as much of the membrane protein component as possible from the precipitate. The amount of inclusion bodies obtained was about 5% in terms of wet cell weight ratio. The crude product was dissolved in 8M urea solution and purified by reverse phase HPLC. The fusion protein was eluted at an acetonitrile concentration of about 47%.

[0133]

Example 14: Thrombin cleavage reaction of fusion protein and human osteo
Amino acid sequence of calcin D'sequence An 8M urea solution of the fusion protein obtained in Example 13 was treated with H ₂ O.
Diluted 10 times (the protein concentration will be about 1.0 mg / ml),
Bovine thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) with a final concentration of 0,1,
Add 3 to 10 or 30 unit / ml, and add 2 at 37 ℃.
Time processed. The SDS-PAGE of the reaction solution is shown in FIG. The fusion protein (molecular weight about 26000) was cleaved by thrombin, and the target human osteocalcin D '
It was found that a band of the sequence (molecular weight of about 5,500) (portion indicated by leader line a in the figure) was generated.

[0134] Further, the case where thrombin treatment is not performed,
Reverse-phase chromatography was performed on the product subjected to thrombin treatment (incubation at 30 unit / ml for 2 hours at 37 ° C.), and the newly generated main peak (acetonitrile concentration about 42%) was collected after freeze-drying, It was dissolved in trifluoroacetic acid and adsorbed on a polybrene filter according to the manual of Applied Biosystems, and a protein sequencer (Applied Biosystems, 4
70A), the PTH-amino acid is cleaved from the N-terminus, and a PTH analyzer (Applied Biosystems, 1
20A) to determine the amino acid sequence from the N terminus to 5 residues, the amino acid sequence from the N terminus to 5 residues of the D'sequence (Tyr-Leu-Tyr-Gl) was determined.
n-Trp). Further, the molecular weight by SDS-PAGE was also in agreement with the value estimated from the D'sequence. From this result, it was confirmed that the human
It was confirmed that the osteocalcin D'sequence was correctly excised.

[Sequence list]

SEQ ID NO: 1 Sequence length: 147 Sequence type: Nucleic acid Number of strands: Double-stranded tropology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics 1..147 E mat peptide sequence : TAC CTG TAT CAG TGG CTG GGC GCT CCG GTT CCA TAC CCG GAC CCT CTC 48 Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 5 10 15 GAG CCG CGC CGT GAG GTT TGT GAA CTG AAC CCG GAC TGC GAT GAG CTG 96 Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys Asp Glu Leu 20 25 30 GCT GAT CAC ATC GGT TTT CAG GAA GCA TAC CGT CGC TTC TAC GGT CCG 144 Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro 35 40 45 GTA 147 Val

SEQ ID NO: 2 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double-strand tropology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics 1..66 E pro peptide Partial sequence: AAA CCG TCT GGC GCC GAA TCT TCC AAA GCA TTC GTT TCT AAG CAG GAG 48 Lys Pro Ser Gly Ala Glu Ser Ser Lys Ala Phe Val Ser Lys Gln Glu 5 10 15 GGA TCC GAA GTT GTA AAA 66 Gly Ser Glu Val Val Lys 20

SEQ ID NO: 3 Sequence Length: 12 Sequence Type: Nucleic Acid Number of Strands: Double Strand Troposity: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence Features 1..12 E thrombin cleavage site Sequence: CTG GTT CCG CGG 12 Leu Val Pro Arg

SEQ ID NO: 4 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Double-stranded tropology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence characteristics 1..66 E sig peptide sequence : CGT GCT CTG ACC CTG CTA GCC CTG CTG GCA CTG GCT GCA CTA TGC ATC 48 Arg Ala Leu Thr Leu Leu Ala Leu Leu Ala Leu Ala Ala Leu Cys Ile 5 10 15 GCA GGT CAG GCA GGT GCT 66 Ala Gly Gln Ala Gly Ala 20

SEQ ID NO: 5 Sequence length: 98 Sequence type: Amino acid Tropoposity: Linear Sequence type: Protein Origin organism name: Human Direct origin Library name: Human Genomic Library Position in the genome Chromosome: Chromosome 1 sequence features 1..23 S pre peptide 24..49 S pro peptide 50..98 S mat peptide sequence: Met Arg Ala Leu Thr Leu Leu Ala Leu Leu Ala Leu Ala Ala Leu Cys 5 10 15 Ile Ala Gly Gln Ala Gly Ala Lys Pro Ser Gly Ala Glu Ser Ser Lys 20 25 30 Ala Phe Val Ser Lys Gln Glu Gly Ser Glu Val Val Lys Arg Pro Arg 35 40 45 Arg Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro 50 55 60 Leu Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys Asp Glu 65 70 75 80 Leu Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly 85 90 95 Pro Val

SEQ ID NO: 6 Sequence length: 310 Sequence type: Nucleic acid Number of strands: Double-stranded tropology: Linear Sequence type: cDNA Sequence characteristics 11..76 E sig peptide 77..142 E pro peptide 143..154 E cleavage-site 155..300 E mat peptide Sequence: GAATTCCGAT CGT GCT CTG ACC CTG CTA GCC CTG CTG GCA CTG GCT GCA CTA 54 Arg Ala Leu Thr Leu Leu Ala Leu Leu Ala Leu Ala Ala Leu- 45 -40 -35 TGC ATC GCA GGT CAG GCA GGT GCT AAA CCG TCT GGC GCC GAA TCT TCC 100 Cys Ile Ala Gly Gln Ala Gly Ala Lys Pro Ser Gly Ala Glu Ser Ser -30 -25 -20 AAA GCA TTC GTT TCT AAG CAG GAG GGA TCC GAA GTT GTA AAA CTG GTT 148 Lys Ala Phe Val Ser Lys Gln Glu Gly Ser Glu Val Val Lys Leu Val -15 -10 -5 CCG CGG TAC CTG TAT CAG TGG CTG GGC GCT CCG GTT CCA TAC CCG GAC 196 Pro Arg Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp 1 5 10 CCT CTC GAG CCG CGC CGT GAG GTT TGT GAA CTG AAC CCG GAC TGC GAT 244 Pro Leu Glu Pro Arg Arg Glu Val Cys Glu Leu Asn Pro Asp Cys Asp 15 20 25 30 GAG CTG GCT GAT CAC ATC GGT TTT CAG GAA GCA TAC CGT CGC TTC TAC 292 Glu Leu Ala Asp His Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr 35 40 45 GGT CCG GTA TAATCTAGA 310 Gly Pro Val

SEQ ID NO: 7 Sequence length: 21 Sequence type: Amino acid Tropoposity: Linear Sequence type: Synthetic peptide Sequence: Tyr Leu Tyr Gln Trp Leu Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu 5 10 15 Glu Pro Arg Arg Cys 20

SEQ ID NO: 8 Sequence length: 7 Sequence type: Amino acid Tropoposity: Linear Sequence type: Synthetic peptide

SEQ ID NO: 9 Sequence length: 15 Sequence type: Amino acid tropology: Linear Sequence type: Synthetic peptide Sequence: Cys Ile Gly Phe Gln Glu Ala Tyr Arg Arg Phe Tyr Gly Pro Val 5 10 15

[Brief description of drawings]

1 shows the DNA sequence for expressing human osteocalcin of the present invention in Example 1. FIG. In the figure, the solid line is synthetic DNA.
The boundary of the fragment and the dotted line indicate the restriction enzyme cleavage plane. Further, a to l respectively represent the oligonucleotides synthesized in Example 1 when they were separated at the boundary position of the synthetic DNA fragment.

FIG. 2 Cloning vector pHS in Example 1
A method for preparing a modified p399 of G399 is shown.

FIG. 3 The plasmid pBPR-PRE in Example 1
The construction design of is shown.

FIG. 4 Plasmid pBPR-MOC in Example 1
The construction design of is shown.

FIG. 5 shows a construction design of plasmid pBPR-OC in Example 2.

FIG. 6 shows the construction design of plasmid pKK-OC in Example 3.

FIG. 7 shows the synthetic DNA fragment prepared in Example 6. In the figure, the solid line indicates the boundary of the synthetic DNA fragment, and the dotted line indicates the restriction enzyme cleavage surface. m and n represent synthetic oligonucleotides when they are separated at the boundary position of the synthetic DNA fragment.

FIG. 8 shows the construction design of plasmid pOC28 in Example 6.

FIG. 9 shows the construction design of plasmid pKOC28 in Example 6.

FIG. 10: Human osteocalcin E in Example 8
The result by IA is shown.

FIG. 11 shows the measurement results of human osteocalcin-containing polypeptide concentration in Example 9.

FIG. 12 shows the results of activity evaluation of antibody-immobilized beads in Reference Example 4.

13 shows a calibration curve of the fully human osteocalcin measurement system in Reference Example 4. FIG.

FIG. 14 shows a construction design of a DNA sequence for expressing human growth hormone-human osteocalcin fusion protein in Example 10. In the figure, (1) is a DNA sequence encoding human growth hormone, (2) is a DNA sequence encoding an FXa cleavage sequence, and (3) to (5) are B sequence, C sequence, and D sequence in order.

FIG. 15 shows the design of the ligated DNA portion in Example 10.

FIG. 16: Plasmid pGH-PO in Example 11
3 shows a CI construction design.

FIG. 17: Plasmid pGH-PO in Example 11
The construction design of CII is shown.

FIG. 18: Plasmid pGH-PO in Example 12
The result of SDS-PAGE of CI is shown.

FIG. 19: Plasmid pGH-PO in Example 12
The result of SDS-PAGE of CII is shown.

FIG. 20 shows the result of SDS-PAGE in Example 14.

Front page continuation (72) Inventor Kenji Hosoda 4-3-2 Asahigaoka, Hino City, Tokyo Teijin Limited Tokyo Research Center

Claims (17)

[Claims] 1. The following formula [I] ABCD ... [I] wherein D is a DNA sequence (D sequence) encoding human osteocalcin or a protein physiologically equivalent thereto, and C is 1 shows a DNA sequence (C sequence) encoding a cleavage sequence peptide, where B represents B-1 and / or B-2, where B-1
Is a DNA sequence (B-1 sequence) encoding an additional polypeptide for expressing human osteocalcin in a large amount in a host cell, and B-2 is a Gla-enzyme-recognizing polypeptide of human osteocalcin. DNA to do
The DNA sequence for human osteocalcin expression represented by: (B-2) sequence, A represents a DNA sequence (A sequence) encoding a signal peptide which may be optionally bound. 2. The DN having the D sequence shown in SEQ ID NO: 1.
The DNA sequence for expressing human osteocalcin according to claim 1, which is the A sequence. 3. The DN whose C sequence is shown in SEQ ID NO: 3.
The DNA sequence for expressing human osteocalcin according to claim 1, which is the A sequence. 4. The DNA sequence for human osteocalcin expression according to claim 1, wherein B comprises B-1 and B-2 (B-1 sequence and B-2 sequence). 5. The DNA sequence for human osteocalcin expression according to claim 1, wherein the B-2 sequence is the DNA sequence shown in SEQ ID NO: 2. 6. The DNA sequence for human osteocalcin expression according to claim 1, wherein the B-1 sequence is a DNA sequence encoding human growth hormone. 7. A vector for expressing human osteocalcin, which comprises the DNA sequence for expressing human osteocalcin according to claim 1 and can be expressed in a host cell. 8. A vector for human osteocalcin expression according to claim 7, which comprises the DNA sequence for expressing human osteocalcin according to claim 1 and a DNA sequence having an expression regulation function for human osteocalcin (expression regulation sequence). 9. The human osteocalcin expression vector according to claim 8, wherein the expression control sequence is an expression control sequence capable of functioning in E. coli. 10. The human osteocalcin expression vector according to claim 7, which is a plasmid capable of self-replication in Escherichia coli. 11. A recombinant host cell transformed with the human osteocalcin expression vector according to claim 7. 12. The host cell is E. coli.
A recombinant host cell as described. 13. A recombinant host cell according to claim 11 is cultivated, and in the culture, the following formula [II] B′-C′-D ′ ... [II] wherein D ′ is human osteocalcin or physiology thereof. C'indicates a cleavage sequence peptide, B'indicates B'-1 and / or B'-2, wherein B'-1 is a human osteocalcin Gla-recognizing enzyme recognition poly. B'-2 represents a peptide, and B'-2 represents an additional polypeptide for expressing human osteocalcin in a large amount in a host cell, which produces a polypeptide-containing protein represented by, and then cleaves this protein, A method for producing human osteocalcin or a protein physiologically equivalent thereto, which comprises isolating osteocalcin or a protein physiologically equivalent thereto (D '). 14. The recombinant host cell according to claim 11 is cultured, and in the culture, the following formula [II] B′-C′-D ′ ... [II] wherein D ′ is human osteocalcin or physiology thereof. C'indicates a cleavage sequence peptide, B'indicates B'-1 and / or B'-2, wherein B'-1 is a human osteocalcin Gla-recognizing enzyme recognition poly. A peptide, B'-2 is an additional polypeptide for the large expression of human osteocalcin in the host cell, producing a polypeptide-containing protein of Glu to G
A method for producing human osteocalcin or a protein physiologically equivalent thereto, which comprises isolating and further cleaving human osteocalcin or a protein physiologically equivalent thereto. 15. Human osteocalcin obtained by the method according to claim 13 or a protein physiologically equivalent thereto. 16. Recombinant Escherichia coli K-12 strain XL1-Blue, which has been submitted to the Institute of Microconducting as Accession No. BP-2950.
F ^- [pKOC28]. 17. A recombinant Escherichia coli K-12 strain HB101 [pG, which has been submitted to the Institute of Microorganisms as Accession No. BP-3175.
H-POCI].