JPH07173187A - Hpct gene - Google Patents

Abstract

PURPOSE:To obtain a HPCT gene coding for a human partially amino acid- substituted calcitonin precursor having a specific amino acid sequence, and capable of giving a precursor for C-terminated amidated peptides to be used for e.g. treating hypercalcemia, osteoporosis, Paget's disease etc. CONSTITUTION:This gene has an amino acid sequence of formula I and consists of a double-stranded polynucleotide of formula II coding for a calcitonin precursor (HPCT) with the Met at 8-site of human calcitonin replaced by Val. This gene can be obtained by chemical synthesis through such processes that plural oligodeoxyribonucleotides are synthesized by the solid phase method using a solid phase carrier such as aminomethylated polystyrene resin and separated from the resin and then mutually linked successively. The HPCT gene represented by the formula II is bound to a vector into a recombinant plasmid which is then incorporated into a host cell to make a transformation, and the transformant produced is cultured to obtain a calcitonin presenting powerful physiological activity which consists of a precursor for C-terminated amidated peptides.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a gene which can be used for producing a precursor of a peptide having a C-terminal amidated group to give a -CONH ₂ group, a plasmid having the gene inserted therein and a plasmid thereof. It relates to a transformed unicellular organism or animal cell.

[0002]

2. Description of the Prior Art Some C-terminal amidated peptides are pharmacologically active. For example, calcitonin consists of 32 amino acid residues whose C-terminal is amide type. It is used as a peptide for the treatment of hypercalcemia, osteoporosis, bone-Bezette's disease and promotion of bone formation. The amide type C-terminal structure is said to be essential for activity expression.

To date, calcitonin has been isolated from pigs, cows, sheep, humans, rats, salmon, and eels, and the structure of each has been clarified. Among them, humans, eels, pigs,
Salmon calcitonin is commercially available for therapeutic use. These are extracted from the living body or chemically synthesized, but the calcitonin content in the living body is low, and the synthesis is difficult.

The present inventors intended to produce calcitonin in large quantities and at low cost by utilizing gene manipulation techniques, but C-terminal amide type peptides cannot be produced by the current gene manipulation techniques.

[0005]

[Means for Solving the Problems] However, a precursor obtained by adding two or more basic amino acids such as glycine (Gly) and then lysine (Lys) or arginine (Arg) after the amino acid to be amidated is I knew it was pharmacologically active. It is considered that the reason is probably that the precursor is cleaved and modified by an enzymatic action in vivo to generate a C-terminal amidated peptide.

The present invention is based on this new finding, part of which is expressed by the following formula:

[Chemical 2]

The above precursor can be produced by the method described below.

[0008] It is preferable to add a methionine codon at the beginning of the nucleotide sequence of this gene and a translation stop codon at the end, and more preferably, a sequence to be cleaved with a restriction enzyme at both ends thereof, for example, upstream (5 'side). EcoR
I, BamHI cleavage site is added downstream (3 'side).

In addition, one or more restriction enzyme cleavage sites such as RsaI, KpnI, Sm in the nucleotide sequence.
You may provide an aI and XmaI cutting point. A preferred example of double-stranded DNA design for a gene is shown in FIG. For the chemical synthesis of the gene, first, the 20 DNA fragments F1 to F20 shown in FIG. 3 were subjected to the solid phase method (Miyosh).
i, K. Nucl Acid. Res. , 8 , 550
7, 1980) and obtain double-stranded DNA from these fragments by a ligase reaction.

Part of the present invention is a DN containing the above gene.
A is a plasmid or hybrid plasmid derived from a unicellular organism or animal cell in which A is inserted.

As an embodiment, a structural gene of an operon (eg, APase or β-galactosidase) containing a promoter region derived from a unicellular organism, for example, a microorganism such as bacteria, yeast, filamentous fungi, or an animal cell, for example, monkey kidney cells. And a plasmid in which the above genes are read and ligated so that the frames match each other. A preferred example is FIG.
PAHPCT38 shown in FIG. Alternatively, the pHPCT4 shown in FIG. 6 may be used. pHPCT4 is PKO1
3 (see Japanese Patent Application No. 56-163303)
RI, BamHI was obtained by inserting the above-mentioned chemically synthesized gene at the breakpoint, and pAHPCT38 was an alkaline phosphatase (APas) of Escherichia coli.
e) PBR322-derived plasmid pAα carrying the gene
Eco of NE1 (see Japanese Patent Laid-Open No. 58-71894)
It can be obtained by inserting the above-mentioned chemically synthesized gene at RI and BamHI breakpoints.

Part of the present invention is a unicellular or animal cell transformed with the above plasmid.

In one embodiment, the plasmid pHPCT
4 into Escherichia coli K12WA802 strain to obtain a transformed strain WA802 / pHPCT.
HPCT38 was transformed into the Escherichia coli K12 / E15 strain to obtain the transformed strain E15 / pAHPCT.

E15 / pAHPCT38 strain is SBMC1
It has been deposited at the Institute for Microbial Industry, Institute of Industrial Science and Technology, with the identification number of 38, and is given the deposit number, Micromachine Research Institute, No. 6523.

Transformation with a plasmid incorporating a gene having a nucleotide sequence encoding the amino acid sequence of the peptide of the above formula (I) having a code for Met added to the 5'-terminal side, and subsequently, a nucleotide sequence encoding a translation termination. The above-described peptide can be produced as a protein component in the culture by culturing the host cell.

An example of transformed host cells is E1 described above.
5 / pAHPCT. To explain the culture in this example, for example, when the APase is induced by first culturing in a high-phosphate-containing medium and then transferring to a low-phosphate medium, a desired peptide precursor is produced in the cells as a protein component.

The preferable operation and conditions for culture are selected depending on the type of plasmid and host cell. Then, the cells were separated from the culture, treated with formate cyanogen monobromide, and then subjected to concentration gradient elution fractionation using a pyridine-acetic acid buffer on an SP-Sephadex column to obtain a fraction containing the desired peptide. Can be collected and purified by HPLC.

The C-terminal amidated peptide precursor thus obtained exhibited the same activity as the C-terminal amidated peptide. For example, the precursor shown below in FIG.
Abbreviated as), strongly reduced calcium concentration in rat blood and exhibited calcitonin activity.

In the following, human calcitonin Met ⁸ is Va
The calcitonin precursor replaced by 1 is abbreviated as HPCT.

[0020]

【Example】

Example Chemical Synthesis of HPCT Gene Fragment The chemical synthesis of 20 oligodeoxyribonucleotides, designated F1 to F20, respectively, shown in FIG. 3, is basically Ken-ichi except for the improvements described below.
Solid-phase method [Ke reported by Miyashi et al.
n-ichi Miyashi et al (1980)
NuCl Acids, Res. 8 5507] was used.

The outline of the chemical synthesis of oligodeoxyribonucleotides by this improved technique is as follows. a) First, as shown in FIG. 4, a 3'-terminal mononucleoside was added to an aminomethylated polystyrene resin. We started by fixing the unit. That is, as the aminomethylated polystyrene resin shown in FIG. 4, a resin crosslinked with 1% divinylbenzene was used, and a resin in which two β-alanines were linked to each other was synthesized.

By reacting three kinds of compounds (B is thymine, N-benzoylcytosine or N-isobutyrylguanine) with this resin, a polystyrene resin having three kinds of 3'-terminal nucleosides immobilized on the resin is obtained. It was

An example of the method of synthesizing this resin is as follows when B in FIG. 4 is shown for thymine.

Aminomethylated polystyrene. HCl (di
Vinylbenzene 1%, 100-200 mesh, N
H₂: 0.63 mmole / g) CH resin 15 g₂C
l₂After swelling well with 75 ml, Nt-BOC-β
-Add alanine (1.01 g, 11.34 mmole)
Well, use DCC (2.34 g, 11.34 mmole)
And allowed to condense at room temperature for 3 hours. Filter the resin through a glass filter
After separating, CH₂Cl₂Four times with 150 ml, DMF15
Wash 4 times with 0 ml, then 4 times with 150 ml pyridine
It was The resin is then treated with 75 ml of 25% acetic anhydride-pyridine.
Acetylation of unreacted amino groups by treatment for 30 minutes
did. Next, add 20% CF resin₃COOH-CH₂Cl₂
By treating with 90 ml at room temperature for 25 minutes, t-BOC group
Removed, then 5% triethylamine-CH₂Cl₂1
Wash the resin with 50 ml and then CH ₂Cl₂150m
The resin was washed 4 times with 1. Repeat the same operation again
Therefore, the resin of FIG.
A resin in which two β-alanines are linked to a styrene resin is obtained.
It was 5 g of the resin (Fig. 4) obtained here was added to 30 ml of DMF.
Kendakushi, Figure 4 compound (B is N-thymine) 5mmo
le and 500 mg of triethylamine are added, and room temperature is 12 hours.
Shaken. After the reaction, filter the resin using a glass filter.
Separated and washed with DMF followed by pyridine. Then resin
3 hours with 75 ml of 10% phenyl isocyanate pyridine
The unreacted amino group is protected by
After washing the resin with pyridine and methanol,₂O_FiveExistence
Figure 4 resin (B is the
Got).

A part of the resin obtained here (FIG. 4, B is thymine) is a part of [M. J. Gait et al. (19
80) NuCl. A cids Res. 8 108
1] was used to quantify the amount of the 3'-terminal nucleoside immobilized on 1 g of the resin.
It was e. A resin (B is N-benzoylcytomine or N-isobutylguanine) was also produced by the same method as the above-mentioned synthetic method. The amount of 3'-terminal nucleoside immobilized on 1 g of the resin was 0.150 mmole for resin 4 (B is N-benzoylcytomine) and 0.147 mmole for resin 4 (B is N-isobutylguanine).

B) The three kinds of resins obtained here and the 3'phosphoric acid diester dimer block shown in FIG. 5 (FIG. 5)
n is 0) and 3 ′ phosphodiester trimer block (1 in FIG. 5n) is used, and F1 to F2 having a constant base sequence are used.
20 oligodeoxyribonucleotides consisting of 0 were synthesized. That is, the dimethoxytrityl group of the resin (abbreviated as DMTr above) was replaced with 2% benzenesulfonic acid CHCl ₃ −.
After removing the DMTr group by treatment with MeOH7 = 3 v / v, 4 equivalents of 3'phosphodiester dimer (0 in FIG. 5n) or 3'to 1 equivalent of 3'nucleoside unit immobilized on the resin. 3 equivalents of phosphoric acid diester trimer (FIG. 5, n is 1) were sequentially condensed using 20 to 50 equivalents of the condensing agent mesitylenesulfonyl tetrazolide (MsTe). Excess 3'phosphate diester block and condensing agent can be easily removed by filtering the reaction mixture through a glass filter.

Next, in order to protect the unreacted 5'hydroxyl group, the resin was treated with 10% acetic anhydride-pyridine at room temperature for 1 hour to acetylate the unreacted 5'hydroxyl group. The resin was then treated with 2% benzene sulfonic acid (CHCl ₃ -MeOH,
7: 3 v / v) to treat the 5'end DMTr
The group was removed and the following condensation reaction was performed. This operation was repeated until the desired length was obtained.

An example of this series of operations is as follows for the synthesis of the F3 fragment. Fig. 4 Resin (B-
Resin in which DMTr group of resin was removed by treating 200 mg of N-benzoylcytosine) with 20 ml of 2% benzenesulfonic acid (CHCl ₃ -MeOH 7: 3 v / v) (hereinafter abbreviated as 2% BSA) at room temperature for 1 minute twice. Got
To this resin was added 90 μmole of 3'phosphate trimer block, GAG, (FIG. 5, n = 1, B ₁ is N-isobutyrylguanine, B ₂ is N-benzoyladenine, B ₃ is N-isobutyrylguanine). After repeating azeotropic distillation with 3 ml of pyridine 3 times, 2 ml of pyridine was added, and the condensing agent MsT was added.
A condensation reaction was performed at room temperature for 2 hours using e 1 mmole. After the reaction, the excess 3'phosphate trimer and the condensing agent can be easily removed by filtering the reaction mixture with a glass filter and washing the resulting resin well with pyridine. Next, to protect the unreacted 5'hydroxyl group, 10
% Acetic anhydride-Pyridine 20 ml at room temperature
Shake for hours. After the reaction, the resin is filtered off with a glass filter and washed well with pyridine and then with CHCl ₃ . The resin was then loaded with 2% BSA (CHCl ₃ : MeOH, 7: 3 v
/ V) The DMTr group was removed from the resin by treatment with 20 ml for 1 minute and twice. The resin was then washed well with CHCl _{3 and} then pyridine, and block condensation was repeated in the same manner until the desired length was obtained. That is, in order to synthesize the oligodeoxyribonucleotide of F _3, the following 3'phosphate block, CCT, AA, GGT,
Was successively used to repeat the condensation reaction to obtain a completely protected oligodeoxyribonucleotide resin having the base sequence of F3. Similarly, other fragments F1 and F2
And F4 to F20 fragments were also prepared by the same method as above except that the nucleotide combination was changed.

C) Cleavage of the oligodeoxyribonucleotide completely protected with a certain base sequence thus obtained from the resin and the complete deprotection reaction, the resin was treated with concentrated aqueous ammonia-pyridine (2: 1 v / v) to 55%. It was obtained by treating at 10 ° C. for 10 hours and then at room temperature for 10 minutes with 80% acetic acid.

The deprotection reaction conditions for the fragment F ₃ are as follows. 50 mg of the resin of fragment F3 was shaken in 1 ml of pyridine and 2 ml of concentrated aqueous ammonia in a sealed tube at 55 ° C. for 10 hours, the resin was filtered off, and the filtrate was concentrated under reduced pressure. Toluene is added to the obtained residue and azeotropic distillation is repeated to remove pyridine. Next, 2% of 80% acetic acid was added to the residue, and the mixture was treated at room temperature for 10 minutes to obtain a completely deprotected oligodeoxyribonucleotide.

D) Separation and purification of the final product was carried out using high performance liquid chromatography (HPLC). This HPLC uses ALTEX model 110A liquid chromatography and uses solvent A (0.005M, KH ₂ P
O ₄ , pH 4.0 and solvent B (0.05M, KH ₂ PO)
_The compound was separated and purified on a Permaphase AAX (DuPont) column (0.21 × 50 cm) using a linear concentration gradient method with 4-1.0 M KCl, pH 4.0). A gradient was applied starting with solvent A and adding 3% solvent B every minute. Elution is 2 ml per minute at 58 ° C
At a flow rate of The peaks of interest were collected, desalted by dialysis, and lyophilized. Each fragment thus obtained was further linearized with solvent A (0.01 M ethylenediamine acetic acid / 2.5% acetonitrile pH 7.0) and solvent B (0.01 M ethylenedimine acetic acid / 25% acetonitrile pH 7.0). Using the concentration gradient method, μ
-Bondapak C18 column (0.4 x 25 cm)
The compound was further purified by. A gradient was applied starting with solvent A and adding 3% solvent B every minute. Elution was performed at room temperature with a flow rate of 2 ml per minute. By collecting the peaks of interest and freeze-drying,
₂₀ DNA fragments of _{1 to} F ₂₀ were obtained.

E) 2 of F1 to F20 thus obtained
The homogeneity of 0 fully deprotected oligodeoxyribonucleotides was checked by electrophoresis on a 20% polyacrylamide gel after labeling the 5'end with [γ- ³² P] ATP using T4 polynucleotide kinase. . The base sequence of each oligodeoxyribonucleotide was T4 polynucleotide kinase, 5 ′ end was labeled with [γ- ³² P] ATP, and then partially hydrolyzed with nuclease to obtain pH3 using cellulose acetate. .5 electrophoresis and D
It was confirmed by a two-dimensional mapping method obtained by performing homochromatography with EAE-cellulose. [Ba
mbara, J, E, et al. (1974) NuC
l. Acids. Res. 1 331].

Ligation of HPCT gene fragment After chemically phosphorylating the 5'end of 18 chemically synthesized oligonucleotide fragments (F2 to F19, see FIG. 3), the fragments of F1 and F20 were added and annealed to obtain T4 DNA ligase. 122 and
A gene corresponding to HPCT consisting of base pairs was synthesized.
Details are shown below.

4.2 μg of each of the 18 fragments except F1 and F20 was dissolved in 22 μl of distilled water, heated at 90 ° C. for 2 minutes, and immediately cooled to 0 ° C. 1 in this aqueous solution
0 μCi of r [ ³² P] ATP (5100 Ci / mmo
l) was added and the solution was added to 50 mM Tris-HCl, pH 9.6.
10 mM MgCl ₂ , 2 mM spermine, 100 mM
After adjusting to KCl, 10 mM DTT (oligonucleotide kinase buffer), 3 units of polynucleotide kinase was added to make the total volume 30 μl. 15 at 37 ° C
By reacting for 5 minutes, the 5 terminal was labeled with ³² P. Then 4 nmol of AT to phosphorylate all 5 ends
Add P and 3 units of polynucleotide kinase at 37 ℃
At 45 ° C. for 45 minutes, and the reaction was stopped by heating at 90 ° C. for 5 minutes. 18 fragments of the above phosphorylated F2 to F19 and unphosphorylated F1 and F2
2.1 μg of each of 0 was mixed and placed in a dialysis tube and dialyzed against distilled water at 4 ° C. for 16 hours to obtain unreacted AT.
P and kinase buffer components were removed. This dialyzed DN
The solution A was concentrated to dryness, and 14.5 μl of ligase buffer (20 mM Tris-HCl, pH 7.6, 10 mM MgC was added.
I ₂ ). This solution was placed in a 0.5 ml volume Eppendorf tube and heated at 95 ° C. for 2 minutes, and then the temperature was slowly lowered (reduced to room temperature in 30 minutes). 2
The 0 fragment was annealed and then placed at 0 ° C. This DNA solution was used as a ligase buffer solution containing 0.4 mM ATP and 10 mM DTT, 30 units of T4 DNA ligase was added to make the total volume 80 μl, and the reaction was carried out at 11 ° C. for 16 hours. A part of the reaction solution was taken out and examined by 8% polyacrylamide gel electrophoresis. Two types of gel were used, one containing 7 M urea and the other not containing 7 M urea. After electrophoresis, the reaction products were examined by radio autograph. As a result 1
DNA corresponding to 22 base pairs was bound at a rate of about 20% (see FIGS. 11 and 12). Next, the total amount of the above reaction solution was separated by 8% polyacrylamide gel electrophoresis. After confirming 122 base pair DNA by radio autograph, a gel piece of the portion was cut out and placed in a dialysis tube, and 9 mM tris borate (pH 8.3), 0.
DNA in the gel was eluted by filling with 25 mM EDTA buffer and sealing, followed by electrophoresis in the same buffer at a constant voltage of 200 V for 3 hours. Next, the DNA solution was taken out from the dialysis tube and freeze-dried. 10 dry matter
After dissolving in 0 μl of 0.3 M sodium acetate (pH 7.0), 2.5 times amount of ethanol was added, the mixture was left at −80 ° C. for 30 minutes, and the DNA precipitate was collected by centrifugation. The obtained DNA was dissolved in 30 μl of kinase buffer, 6 units of polynucleotide kinase and 0.8 nmol ATP were added, and the reaction was carried out at 37 ° C. for 1 hour. 3 μl after reaction
After the addition of 3 M sodium acetate, the DNA was precipitated by adding 2.5 times the amount of ethanol to obtain about 2 μg of HPCT gene corresponding to 122 base pairs.

Construction of Recombinant Plasmid FIG. 6 shows a method for constructing a recombinant plasmid containing the HPCT gene, which will be described in detail below.

(A) Construction of plasmid pHPCT The construction method of plasmid pKO13 is described in Japanese Patent Application No. 56-163.
As specified in No. 303 specification. The plasmid pKO13 is a plasmid having most of the β-galactosidase gene from the promoter region of the Escherichia coli lactose operon, possesses one EcoRI and BamHI restriction enzyme cleavage site, respectively, and has the EcoRI and BamHI of chemically or naturally obtained. Adhesive site (coh
The gene carrying the active end) is transformed into the plasmid pKO
13 can be easily introduced. Furthermore, under the control of the lactose promoter, a gene incorporated downstream of the EcoRI restriction enzyme cleavage site can be expressed as a hybrid protein with β-galactosidase, and after decomposition with cyanogen bromide, the desired peptide can be separated and purified.

Then, 5 μg of pKO13 was added to 40 μl of 1
XTA reaction solution (33 mM Tris acetate buffer pH 7.6,
Restriction enzyme Ec in 66 mM potassium acetate, 10 mM magnesium acetate and 0.5 mM dithiothreitol)
1 unit each of oRI and BamHI was added, and the reaction was carried out at 37 ° C. for 60 minutes. After the reaction solution was heated at 65 ° C. for 30 minutes to inactivate the enzyme, 0.7% agarose gel electrophoresis was performed, and a large DNA fragment was recovered by the electrophoresis method.
EcoRI and BamH of pKO13 thus obtained
I fragment DNA / μg and 0.6 μg of the previously obtained 5′-OH-phosphorylated HPCT gene were added to 40 μl of T4 DNA ligation reaction solution (20 mM Tris-HCl buffer, p
H7.6, 10 mM MgCl ₂ , 10 mM DTT,
It was dissolved in 0.4 mM ATP) and reacted with 2 units of T4 DNA ligase at 5 ° C. for 16 hours. 2.5 volumes of cold ethanol was added to the reaction solution to obtain DNA as a precipitate. This D
The NA precipitate was dissolved in 15 μl of distilled water, and 10 μl of which was dissolved in E. E. coli strain WA802 was transformed. The transformant was 10 μg / m 2 of a colony formed after overnight culture at 37 ° C. on a nutrient agar medium containing 40 μg / ml of ampicillin.
A tetracycline-sensitive colony was selected by replicating on a nutrient agar medium containing 1 tetracycline. Transformants resistant to ampimiline and sensitive to tetracycline WA802 / pHPCT / ~ WA802 / pHP
It was named CT5. Of these, strain WA802 / pHPC
As a result of analyzing the DNA base sequence of the small EcoRI, BamHI fragment obtained from T4 as described below, it was confirmed that the pHPCT4 plasmid had the DNA base sequence of the desired HPCT gene. This WA
In 802 / pHPCT4 bacteria, HPCT is expressed as a β-galactosidase-HPCT hybrid protein under the control of the lactose promoter, and HPCT is separated from the resulting hybrid protein by the methionine-decomposing reaction of cyanogen bromide as a complete target peptide. it can. However, since there are 23 residues of methionine in the β-galactosidase protein, a large number of unwanted contaminating peptides are produced during the decomposition of cyanogen bromide, which makes it difficult to separate and purify the desired peptide. Therefore, we have developed a plasmid using the Escherichia coli alkaline phosphatase gene as a vector to improve the above-mentioned drawbacks and proved its usefulness (Japanese Patent Application No. 56-170543). Therefore, in order to isolate and purify the HPCT peptide from Escherichia coli, it was considered preferable to construct a plasmid in which the HPCT gene was recombined with a plasmid using the alkaline phosphatase gene, and the following plasmid was constructed.

H in the plasmid of the above transformant strain
The PCT base sequence was analyzed as follows. HPCT base sequence pHPCT1, pHPCT2, pHPCT3, pHPC
150 μg of each T4 plasmid DNA was digested with 200 units of restriction enzyme EcoRI and 200 units of restriction enzyme SalI to separate and purify a DNA fragment of about 400 base pairs containing the PHCT gene. Next, after dephosphorylating the 5'end with bacterial alkaline phosphatase, γ- [
^The 5'end was ³² P-labeled with ³² P] ATP and polynucleokease. Next, 100 units of restriction enzyme BamH
The 122 base pair DNA fragment was separated and purified after degradation using I, and the base sequence was determined by the Maxam-Gilbert method (Proc. Nat. Acad. Sci. USA. 7
4 , 560-564, 1977). As a result pHC
All had the same nucleotide sequence as originally designed except T1. In pHCT1, the third codon (AAC) corresponding to asparagine, which is the 17th amino acid of HPCT, was converted from C to T. However, the codon AAT also corresponds to asparagine.

(B) Construction of plasmid pAHPCT38 The construction method of plasmid pAαNE1 is described in Japanese Patent Application No. 56-170.
543. pAαNE1 is EcoRI of Escherichia coli alkaline phosphatase structural gene
It is a plasmid that incorporates the alpha neo-endorphin gene downstream of the restriction enzyme cleavage site, and is a vector that expresses the alkaline phosphatase-alpha neo neoendorphin hybrid protein under the control of expression control of alkaline phosphatase. The method for constructing the plasmid pAHPCT38 in which the HPCT gene is inserted in place of the alpha neo endorphin gene is described in detail below.

10 μg of plasmid pAαNE1 was added to 10
Restriction enzyme EcoRI 10 in 0 μl of 1 × TA reaction solution
After partially cutting with the unit at 37 ° C for 30 minutes,
Perform 0.7% agarose gel electrophoresis and
The DNA fragment cleaved with EcoRI was recovered from agarose by electrophoresis. 3 DNA fragments collected
After complete digestion with 5 units of the restriction enzyme BamHI in 0 μl of 1 × TA reaction solution at 37 ° C. for 60 minutes, 2.5 volumes of cold ethanol was added to precipitate the DNA. This D
The NA precipitate was dissolved in 30 μl of distilled water. PHPCT4 was prepared by using 0.5 μg of this restriction enzyme EcoRI-BamHI-cut DNA fragment and the restriction enzyme EcoRI-BamHI.
30 μg of 0.5 μg of HPCT gene DNA
Dissolve in T1 DNA ligation reaction solution
A ligation reaction was carried out using 2 units of DNA ligase at 5 ° C. for 16 hours. 2.5 volumes of cold ethanol was added to the reaction solution to obtain DNA as an ethanol precipitate. D
After adding 10 μl of distilled water to the NA precipitate to dissolve it, E. coli E15 (Hayashi et al. 1964 J. Biol. Chem. 239 3091) was transformed. The transformant was cultured overnight at 37 ° C. on a nutrient agar medium containing 40 μg / ml of ampicillin. Regarding the obtained 72 ampicillin-resistant strains, the restriction enzyme S of the plasmid
The presence or absence of the maI cleavage site was examined. As a result, it was found that 3 strains were plasmids having a SmaI cleavage site in the HPCT gene, and one strain among them was E15 /
It was named pAHPCT38 and used for the subsequent separation and purification of the HPCT peptide.

The above Escherichia coli E15 /
pAHPCT38 was attached to the Institute for Microbial Industry, Institute of Industrial Science, labeled SBMC138, and was given the deposit number, Microindustrial Research Institute No. 6523.

The human calcitonin precursor E. ColiE
Purification from E. 15 / pAHPCT38 Eli E1
In 5 / pAHPCT38, the HPCT gene is inserted into the alkaline phosphatase structural gene. Therefore, HPCT should be expressed as a hybrid protein with alkaline phosphatase. But the first HPC
As shown in the T gene design, by inserting methionine at the amino acid terminal site of HPCT, HPCT can be easily obtained by cleaving the hybrid protein of alkaline phosphatase and HPCT with cyanogen bromide. Therefore, the E. coli K
HPCT was separated and purified from 12E15 / pAHPCT38. Grace, E. coli K12E15 / pAHP
The method for culturing the CT38 strain and the method for inducing the alkaline phosphatase-HPCT hybrid protein are described in Japanese Patent Application No. 56-17.
It was carried out according to the method described in the specification of 0543. Strain E. E. coli E15 / pAHPCT38 TG + 20 containing 40 μg / μl of ampicillin (high phosphate medium)
After culturing in 100 ml at 37 ° C for 16 hours, 10 l of TG + 1
(Low-phosphate medium) and inoculated at 37 ° C. for 24 hours. Incubate the cells by continuous centrifugation to 2.36g
Wet cells were obtained. 20 ml of a 70% formic acid solution containing 500 mg of cyanogen bromide was added to the wet cells, and the suspension was suspended and left at room temperature for 24 hours in the dark.

HPCT strain E. coli E15 /
Purification from pAHPCT1 The Bromcian reaction solution of the cells was freeze-dried, 60 ml of distilled water was added, and then ultrasonication was performed at room temperature for about 1 hour. The suspension thus obtained is centrifuged (10,000 r
pm. 15 minutes) 30 ml of distilled water was added to the precipitate, leaving the supernatant.
Was sonicated again and centrifuged to obtain 90 ml of the supernatant together with the previous one. 10 ml of distilled water in this solution
To 100 ml and add SP-Sephadex C-2
5 columns (1.4 x 20 cm) were applied. The column was equilibrated with 0.025M acetic acid in advance, the sample was applied to the column, and then washed with 230 ml of the same acetic acid solution.
Subsequently, 1.5% pyridine-acetic acid buffer-abbreviated as PA buffer below- (pH 4.47) 200 ml, 2.5% pA
It was washed with 160 ml of a buffer solution (pH 4.65). Next, 60 ml of 2.5% pA buffer (pH 4.65) and 3.7
Elution was performed with a linear concentration gradient using 60 ml of 5% pA buffer (pH 4.76), followed by elution with 60 ml of the latter buffer, and then 3.75% pA buffer (pH 4.76).
76) 160 ml and 5.0% pA buffer (pH 4.8)
7) Elution was performed with a linear concentration gradient using 160 ml, and the eluate was collected in 6 ml portions. Every several fractions, 100 μl of each fraction was analyzed using HPLC (HPLC conditions are described later). Fractions corresponding to each main peak obtained from HPLC were dried and dissolved in 150 ml of 6N hydrochloric acid containing 0.1% phenol and hydrolyzed at 110 ° C. for 24 hours. The decomposed products were evaporated to dryness and then analyzed for amino acids (using Hitachi # 835-50 type amino acid analyzer) to monitor the elution status from the HPCT column.

As a result, 2.5% to 3.75% gradient elution fractions (total 120 ml), all 3.75% elution fractions (60 ml), and 3.75% to 5.0%.
The first 10 of the gradient elution fractions (60 m
The presence of HPCT was confirmed in l).

Fractions containing these HPCT were collected, lyophilized and then purified by HPLC. The dried solid was dissolved in 8.0 ml of 0.1N acetic acid, and 10 of
0-300 μl each of μ-Bondapak C18
(Waters) 0.39 × 30 cm column was used for purification. Elution is 0.1% with 10% acetonitrile
Trifluoroacetic acid and 50% acetonitrile in 0.
A linear gradient with 1% trifluoroacetic acid was used and monitored at a UV wavelength of 210 nm. As shown in the typical chromatogram of FIG. 7, the peak corresponding to HPCT can be clearly detected. Fractions containing this peak were collected and further subjected to HPLC under the same conditions (FIG. 8) to obtain 2.15 mg (0.5) of pure HPTC.
6 μmol) could be obtained.

Confirmation of structure of HPCT The amino acid composition of HPCT was found to be 0.
2% at 110 ° C with 6N hydrochloric acid containing 1% phenol
It was determined from the result of amino acid analysis of the product hydrolyzed for 4,48,72 hours, and the result of amino acid analysis after hydrolyzing HPCT according to a conventional method (1) formic acid oxidation of 6N hydrochloric acid at 110 ° C. for 24 hours. 1). When the amino acid sequence analysis of HPCT performance oxide was carried out using the Edman degradation method (2), the sequence from the amino terminus to the 15th position could be determined (FIG. 9). HPC
In order to determine the amino acid sequence of the entire T, we tried tryptic digestion of HPCT to obtain a peptide fragment.

After 160 μg of HPCT was suspended in 300 μl of 0.1 N PA buffer (pH 7.80) and sonicated for 30 minutes, 10 μl of TPCK-treated (3) trypsin solution (1 mg / ml) was added, and the mixture was incubated at 37 ° C. for 2 hours. I left it. The reaction solution was freeze-dried, dissolved in 400 μl of 0.1N acetic acid and subjected to HPLC. HPLC conditions are HP
The chromatogram monitored at a UV wavelength of 210 nm was exactly the same as the conditions used for CT purification, and showed a pattern as shown in FIG. 10. From the amino acid composition of each peak T ₁ , T ₂ , T ₃ , T ₁ Was estimated to be a peptide from the 19th amino acid to the 34th amino acid of HPCT, T ₂ was a peptide from the amino terminus to the 18th, and T ₃ was a peptide from the amino terminus to the 34th. 1).

The documents referred to in the above (1), (2) and (3) are shown below. (1) C.I. H. W. Hirs, "Method in
Enzymology ”, Academic Pres
s, New York, Vol. 11, P.I. 197 (1
967) (2) J. P. Van Eerd et. al. , Bi
Chemistry, 15 , 1171 (1976) (3) S. -S. Wang et. al. J. Bio
l. Chem. , 240 , 1619 (1965)

Subsequently, amino acid sequence analysis was performed using T ₁ and T ₂ which had been subjected to peroxidic acid oxidation (FIG. 9). Further H
Amino acid sequence analysis of the carboxyl terminal portion of PCT or T ₁ and T ₂ was carried out by the carboxypeptidase method (T.I.
sobe et al. Mol. Biol. , 102 , 349
(1976)) to carry out a carboxyl terminal analysis by a hydrazine decomposition method (A. Tsug: ta et al., Pro.
c. Nat. Acad. Sci. , U. S. A. 46 .
1462 (1960)) (FIG. 9). From the above results, the amino acid sequence of HPCT was determined as shown in FIG.

Purification of degradation product HPCT-Cp (1-33) using carboxypeptidase of HPCT 500 μg of HPCT was added to 0.1 N PA buffer (pH).
7.80) Suspended in 300 μl, sonicated for 1 hour and then carboxypeptidase B solution (0.67 mg / m 2
l) was added in an amount of 7.5 μl and left at 37 ° C. for 6 hours. After checking the released amino acids (2 mol of lysine and 1 mol of arginine) by amino acid analysis, the reaction solution was freeze-dried, dissolved in 500 μl of 0.1 acetic acid, and purified using HPLC. The HPLC conditions were the same as those used for HPTC purification, and the chromatogram was monitored at a UV wavelength of 210 nm. By the amino acid analysis, the peptide purified in this way was identified as HPPT-Cp (1-33), a peptide from the amino terminus of HPCT to the 33rd amino acid.
Was confirmed (Table 1). Furthermore, the yield determined from the results of this amino acid analysis was 302 μg, and the yield was 68%.

[0051]

Table 1 Amino acid composition of HPCT, T ₁ , T ₂ and HPCT-Cp (1-33) HPCT T ₁ T ₂ HPCT-Cp (1-33) ─────────────── ──────────────────── CySO ₃ H 1.6 1.8 1.8 Asp 3.1 3.0 3.0 3.0 Thr 4.7 1.8 2 .8 4.7 Ser 1.1 1.1 1.1 Glu 2.0 1.0 1.1 2.1 Pro 2.0 1.9 1.9 Gly 4.8 2.8 2.0 5 Ala 2.2 1.9 2.1 Val 1.9 1.0 1.0 2.0 2.0 Ile 1.1 1.0 1.1 Leu 2 1.9 2.0 Tyr 1.0 1.0 1.0 1.0 Phe 2.7 1.9 1.1 3.0 Lys 2.9 1 1 1.1 His 0.9 0.9 0.9 1.0 Arg 1.0 ─────────────── ────── ──────────── 36 16 18 33 In the table, the composition values of other amino acids were calculated based on the underlined values.

Biological activity of HPCT HPCT and HPCT · Cp purified by the above method
The biological activity of (1-33) was measured using the serum calcium lowering effect in rats as an index.

Experimental method Male Wistar rats weighing 300 to 350 g were fasted overnight, fixed in the dorsal position under ether anesthesia, and the lower leg of the right leg was filled with sodium heparin sodium injection (Japan Upjohn) in advance. polyethylene tubing ^{(INTRAMEDIC R PE-50, Clay} A
dams). Then, the rat was fixed in a Pallman cage, and 30 minutes after the anesthesia was awakened, 0.5 ml of blood was added to 50 ml from a polyethylene tube inserted into the above-mentioned lower leg artery.
It was collected in a polyethylene centrifuge tube containing a unit of sodium heparin. Immediately afterwards, physiological saline solution, HPCT
Solution, HPCT-Cp (1-33) solution or porcine calcitonin solution was subcutaneously administered to the back of 0.2 ml per rat, and 0.5 ml of blood was passed through the polyethylene tube at 1, 2, 4 and 6 hours after the administration. Was collected. The collected blood was immediately collected with an Eppendorf microcentrifuge (Eppendorf 5414) at a rate of 13,
It was centrifuged at 000 rpm for 1 minute, and the supernatant was used as a serum fraction.

Serum calcium concentration is determined by a commercially available calcium measuring reagent RM117-K (CaSET) (Yatron).
Was measured by the o-cresolphthalein complexone (OCPC) method. Ie 50 μl of serum
In a test tube, and a coloring reagent containing R-compound of o-cresolphthalein and 8-hydroxyquinoline (R
Add 0.5 ml of M117-2) and stir. Then monoethanolamine borate buffer (RM117-1)
5.0 ml was added and stirred, and within 90 minutes, the absorbance was measured at a wavelength of 575 mμ using a self-recording spectrophotometer (Hitachi Model 320). The calcium concentration was calculated from the absorbance. Physiological saline solution, HPCT solution,
The method for preparing the HPCT-Cp (1-33) solution and the porcine calcitonin solution will be described below. a) Physiological saline: bovine serum albumin (SIGMA ^R )
Is dissolved in the Japanese Pharmacopoeia physiological saline solution (Otsuka Pharmaceutical Co., Ltd.) at room temperature,
The albumin concentration was adjusted to 0.1%. b) HPCT solution: HPCT was added to the above albumin at 0.1
% Saline solution and add 0.2 ml of H
It was prepared to contain 4.5 μg of PCT. c) HPCT / Cp (1-33) solution: HPCT / Cp
(1-33) was diluted with the physiological saline solution containing 0.1% of albumin described above, and HPCT / Cp was added to 0.2 ml of the solution.
It was prepared so as to contain 20 μg of (1-33). e) Porcine Calcitonin Solution: Calcitar ^R containing 160 international units of porcine-origin calcitonin in 1 vial (Yamanouchi Pharmaceutical Co., Ltd.), purified gelatin 16%,
Dissolution solution containing 0.5% of the Japanese Pharmacopoeia phenol 4.0
It was dissolved in ml, diluted with a physiological saline solution containing 0.1% of the above-mentioned albumin, and 0.2 ml or 1 international unit of porcine calcitonin was prepared in 0.2 ml of the solution.

Experimental Results Table 2 shows the experimental results. The serum calcium concentration of the rat subcutaneously administered with 4.5 μg of HPCT was statistically significantly decreased 1 and 2 hours after the administration. The time course of serum calcium concentration after HPCT administration is very close to the time course of serum calcium concentration in rats subcutaneously administered with 0.2 international units of porcine calcitonin, and under the present experimental conditions, 4.5 μg of HPCT is generally porcine calcitonin. 0.
It was presumed to have activity equivalent to 2 international units.
On the other hand, the serum calcium concentration of rats subcutaneously administered with 20 μg of HPCT-Cp (1-33) decreased statistically significantly 1 hour after the administration, but the serum calcium concentration lowering effect was observed 2 hours after the administration. I couldn't do it. It is known that a peptide having a free C-terminal (desamido peptide) has a very low biological activity as compared with a peptide generally found in nature and having an amidated C-terminal. On the other hand, the HP shown in the embodiment of the present invention
CT (Val ³ human calcitonin precursor) and HPC
As shown in Table 2, T.Cp (1-33) does not allow absolute comparison, but suggests that it has a strong physiological activity. In addition, although much research has been done on amino acid substitutions in the amino acid sequence of calcitonin and partial removal of amino acids or peptides, the fact that the peptides such as those of the present invention show activity was completely unknown. Not not.

[0056]

[Table 2]

[0057]

EFFECTS OF THE INVENTION By using the HPCT gene of the present invention, a plasmid into which it is inserted, and cells transformed with the plasmid, a precursor of a C-terminal amidated peptide can be obtained. Although not amidated, it shows strong physiological activity itself.

[0058]

[Brief description of drawings]

The drawings relate to the embodiments of the invention shown in the examples,
In FIG. 1, the upper row shows the amino acid sequence that was the basis of the design of the synthetic gene, the lower row shows the structure of mature calcitonin, and the lower row shows the parts common to the upper row by lines. Figure 2
Is the design of the HPCT gene, and FIG. 3 is a fragment of the chemically synthesized HPCT (1-36) gene (F1-F
20), FIG. 4 shows 3'on the aminomethylated polystyrene resin.
Step of immobilizing terminal mononucleotide unit, FIG.
Is the structure of the 3'-phosphate diester dimer (or trimer) block, Fig. 6 is a step of constructing a recombinant plasmid containing the HPCT gene, Fig. 7 is an elution pattern by HPLC of HPCT produced by a bacterium transformed with the above plasmid, FIG. 8 shows the H of the fraction showing the peak in FIG.
The elution pattern by PLC is shown. Figure 9 shows the above HPL
It is the amino acid sequence of HPCT isolated by C. Figure 1
0 shows the elution pattern of tryptophan degradation product of HPCT by HPLC, and FIGS. 11 and 12 show the mapping of the gene synthesized from oligonucleotide fragments F1-20 by 8% polyacrylamide gel electrophoresis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location C12N 15/09 ZNA // A61K 38/00 ADP C12P 21/02 C 9282-4B (C12P 21/02 C12R 1:19) A61K 37/02 ADP

Claims (12)

[Claims] 1. The following formula: 2. The gene according to claim 1, wherein a translation termination codon is added to the end of the codon for methionine at the beginning of the nucleotide sequence of the gene. 3. The gene according to claim 2, wherein a sequence that is cleaved with a restriction enzyme is further added to both ends of the nucleotide sequence. 4. The gene according to claim 1, wherein EcoRI is added upstream (5 ′ side) and BamHI cleavage site is added downstream (3 ′ side). 5. RsaI, Kp in the nucleotide sequence
The gene according to claim 1, 2, 3 or 4, which is provided with a site which is cleaved by at least one of nI, SmaI and XmaI. 6. A unicellular or animal cell-derived plasmid or hybrid plasmid into which the DNA containing the gene of claim 1 is inserted. 7. The plasmid according to claim 6, wherein the gene of claim 2, 3 or 4 is ligated to the structural gene of the operon containing a promoter region derived from a unicellular organism or an animal so that the reading frames are matched. 8. The plasmid according to claim 7, wherein the structural gene is Escherichia coli APase or β-galactosidase structural gene. 9. The plasmid according to claim 7, which comprises pAHPCT38 shown in FIG. 10. The plasmid according to claim 7, which comprises pHPCT4 shown in FIG. 11. A unicellular organism or animal cell transformed with the plasmid of claim 7. 12. The plasmid pAHP whose plasmid is shown in FIG.
The unicellular organism or animal cell according to claim 11, which is CT38.