JPS62226998A - Human calcitonin precursor peptide and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I (P is peptide residue having amino acid sequence of human calcitonin). USE:A remedy for hypercalcemia, Behcet's disease, etc. PREPARATION:For example, a microorganism, e.g. Escherichia coli, etc. is transformed with a plasmid containing a DNA containing a gene capable of coding a peptide expressed by formula II (n is a positive integer) and then cultivated in a culture medium to produce a fused protein containing the peptide expressed by formula II in the resultant culture. The above-mentioned fused protein is then treated with an enzyme, e.g. trypsin, etc., to afford the aimed human calcitonin precursor peptide expressed by formula I.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a peptide hormone precursor and a method for producing the same, and more particularly to a human calcitonin precursor peptide and a method for producing the same by genetic engineering.

[Conventional technology]

Calcitonin is a peptide hormone found in mammals, birds, and fish, and consists of 32 amino acids. Although the sequence varies slightly depending on the species, the general characteristics are 1 and 7.
A disulfide bond based on the cystine residue present at the C-terminus and a proline amide present at the C-terminus. Calcitonin is used to treat hypercalcemia, Bartschett's disease, etc., but the products used for these treatments are either extracted from eel, salmon, pigs, etc., or chemically synthesized. It is. However, the content of calcitonin in living organisms is very low, and calcitonin synthesis requires a lot of time and cost. On the other hand, eel and salmon calcitonin has higher biological activity than human calcitonin;
It is known that because some of the amino acids are different from those of humans, when administered to the human body for a long period of time, antibodies are produced, resulting in unfavorable results.

[Problem that the invention seeks to solve]

From this background, we intended to produce human calcitonin. As a genetic engineering method for producing the precursor, Japanese Patent Publication Nos. 58-501120, 58-501121, and 59-501095 are disclosed.

In the above genetic engineering method, it is not easy to convert the obtained precursor to human calcitonin, and the method is not necessarily advantageous.

An object of the present invention is to provide a peptide that can be easily converted into human calcitonin and a method for producing the same using genetic engineering.

[Means for Solving the Problems] To summarize the present invention, the present invention relates to a human calcitonin precursor and a method for producing the same.
The invention is based on the following general formula! :P-Gly-Arg
+++ CD (wherein P is a peptide residue having the amino acid sequence of human calcitonin, Gly is a glycine residue, and Arg is an arginine residue).

Further, the second invention of the present invention is based on the following general formula (■): [Arg-
P-Gly-IHArg-a7B ---rn] (wherein CyS is a cysteine residue, n is a positive integer, and Ar
gXP and Gly are synonymous with the above general formula I).Escherichia coli transformed with a plasmid into which a DNA encoding a peptide represented by the above general formula I has been inserted is cultured, and the peptide represented by the above general formula II is introduced into the culture. The present invention relates to a method for producing a human calcitonin precursor peptide represented by the general formula (2) above, which comprises producing a fusion protein containing the fusion protein, and then treating the fusion protein with an enzyme.

The present invention will be explained in detail below.

Human calcitonin is a peptide consisting of 32 amino acids with an amidated C-terminus, and when such a low-molecular-weight peptide is directly produced in microorganisms using genetic engineering techniques, it is difficult to decompose it by enzymes in the microorganism. Generally, the unnecessary portions are removed by appropriate means after producing the fusion protein as a fusion protein. Bromosyanide, which specifically cleaves methionine residues in peptides, and enzymes (trypsin), which specifically cleave basic amino acid residues, are used to remove unnecessary parts, but there are Due to the presence of methionine and lysine, neither method can be used as is. The present inventors focused on the fact that Arg is not included in the human calcitonin sequence, and Arg is added to the N-terminus, and Pro (proline) is added to the C-terminus.
Next, a gene encoding a peptide having a sequence to which Gly-Arg was added was chemically synthesized. This is the gene encoding the phosphate binding protein of E. coli (ph
o 8 ) containing plasmid vector psN 5182
European Journal of Biochemistry
Biochemistry) Volume 130 No. 427-4
55 (1985)] and succeeded in expressing it in significant quantities as a fusion protein with a phosphate-binding protein.

Furthermore, it has been found that a peptide having the sequence P-Gly-Arg (same as the above general formula I) can be efficiently separated from the fusion protein.

The above P -(ly -Arg is converted to p p -G], y by carboxypeptidase treatment. Then,
P-Gly is converted to cytocalcitonin by an amidating enzyme. Regarding amidation enzymes, see, for example, Nature, Vol. 298, No. 686.
~688 pages (1982) or Fetus Letter (FEE
8 Letter) Volume 152, pages 277-281 (
(1983).

The present invention was completed based on the above findings.

The peptide to be produced by genetic engineering in the present invention is represented by the following formula I.

p-a 1y-Arg
--- [:I] Here, P is a peptide residue having the amino acid sequence of human calcitonin, and its next structure is the following formula % formula % In the formula This is a glycine residue necessary to amidate the C-terminus of P. Furthermore, Arg is an arginine residue added to enzymatically excise a peptide that can be converted into human calcitonin from the produced fusion protein. Since human calcitonin does not contain an Arg residue in its molecule, enzymes that specifically cleave the carboxyl side of Arg, such as mouse mandibular grotease (commercially available from Pierce Chemical Co.) or clostripain (commercially available from Boehringer or Sigma), can be used. A peptide having the sequence P-Gly-Arg is produced from the fusion protein using a commercially available method. Furthermore, a peptide having the sequence P-Gly-Arg can also be obtained by protecting the lysine residue in the fusion protein with citraconic acid and then treating it with trypsin.

Next, construction of a gene for producing the peptide represented by formula (2) will be explained. In the present invention, general formula II
A preferred example of the base sequence for producing the peptide represented by is shown in FIG. When n is 1 in the above formula (2), the base sequence shown in FIG. 2 is chemically synthesized, the 5' end is phosphorylated, and the sticky end is made into a blunt end using DNA polymerase. DNA ligase is then used to attach one end of the Hpa lumini linker. Thereafter, it is cut with the restriction enzyme HpaI. The DNA fragment thus obtained is plasmid p8N 5182
It becomes possible to perform Hpa Lumiecite rowing. psN 5182 is a plasmid that integrates a gene (pho day) encoding a phosphate binding protein of E. coli into the pst knee Kco R site of pBR322, and E. coli introduced with this plasmid can change from a high phosphate medium to a low phosphate medium. Upon transfer to acid medium, significant amounts of phosphate-binding proteins are produced.

pal+5 whose 5' end was dephosphorylated after cutting with Hpa
A plasmid containing a fusion gene of pho8 and the peptide represented by formula II is obtained by ligating 182 with the above synthetic compound IIIA, introducing this into E. coli, and selecting colonies that grow on a high phosphate medium containing tetracycline. can be obtained by introducing E. coli. The introduction method itself is a known technique. Techniques for chemically synthesizing the base sequence shown in FIG. 2 are generally known. That is, the designed DNA base sequence is divided into seven appropriate wood-like fragments, each of which is chemically synthesized, and then the 5' end is phosphorylated as required. Each fragment is mixed and annealing is performed so that the fragments form a correct duplex. Thereafter, a ligation reaction of adjacent fragments is performed using DNA IJ gauze. The desired synthetic gene can be obtained by subjecting the reaction product to polyacrylamide electrophoresis to separate and purify it.

The base sequence shown in FIG. 2 is designed to facilitate the synthesis of a peptide in which n is 2 or more in the formula (2). If such a gene is cloned and expressed, n molecules of calcitonin precursor peptide can be obtained from one molecule of the gene product, and the stability of the gene product can also be improved by polymerizing the peptide. Therefore, an efficient method for producing human calcitonin precursor peptide becomes possible.

In order to synthesize and clone such a gene, the following procedure may be performed. First, we purified the synthetic gene shown in Figure 2, phosphorylated the 5' end, and then performed self-ligation (self-ligation reaction) using DNA ligase, thereby linking n genes in Figure 2. Genes can be synthesized. This was made blunt-ended in the same manner as above, combined with Hpa lumini linker, and restriction enzyme Hp
Hpa of plasmid psN 5182 after cutting with a.
Incorporate lumiesite and introduce into E. coli.

As described above, the gene encoding a peptide that can be converted to human calcitonin was transferred to the Hpa of p81J 5182.
Although it is possible to obtain E. coli into which a plasmid containing lumiesite has been introduced, general methods can be used to examine whether the obtained E. coli retains the desired recombinant DNA. The E. coli bacteria to be investigated are cultured, and plasmids are extracted from the resulting bacterial cells. After cutting this with the restriction enzyme Hpa, it is subjected to polyacrylamide electrophoresis to determine whether a DNA fragment of the desired length is detected by staining with an appropriate method. Furthermore, the DN
The base sequence of the A fragment can be determined using established methods such as Maxam-
Investigate by Gilbert method and dideoxy method (dideo:cy method).

Whether or not E. coli into which the desired recombinant DNA has been introduced is producing a fusion protein containing the peptide sequence represented by formula (2) is determined by the following method. After culturing E. coli overnight in a high phosphate medium, the cells are collected and transferred to a low phosphate medium to induce synthesis of the fusion protein. After several hours, the bacterial cells are collected and subjected to repeated freezing and thawing, and then the whole protein is dissolved by heating in a buffer containing EIDEI and subjected to 8D8-polyacrylamide electrophoresis. After transferring this to nitrocellulose, it is bound with an anti-calcitonin antibody, and further bound with an enzyme-labeled second antibody. The target band is detected by developing the enzymatic activity of the bound second antibody with a chromogenic substrate.

The fusion protein from E. coli can be purified, for example, as follows. The collected bacterial pellet is suspended in a highly concentrated sucrose solution, then dispersed in a solution that does not contain sucrose, and the pellet is collected by centrifugation that leaks out the periplasmic fraction, followed by sonication. Remove the soluble fraction.

The insoluble fraction is solubilized with 7M urea and applied to column K of DEAF cellulose equilibrated with a buffer containing 7M urea to obtain a non-adsorbed fraction. This is applied to a column of 0M cellulose, and the fusion protein is eluted by increasing the concentration of sodium chloride. A purified fusion protein is obtained by desalting and lyophilization.

There are several methods for separating the P-Gly-'Arg from the fusion protein. For example, P-(ly-Arg can be released by using an enzyme that specifically cleaves the carboxyl side of arginine (mouse mandibular protease, clostripain, etc.). However, the enzyme used in this case It is necessary that there is no contamination with other proteases in the fusion protein.For example, by protecting the lysine residue in the fusion protein with citraconic acid anhydride and then treating it with trypsin, it is possible to protect the lysine residue with citraconic acid. P-G containing a taridine residue
ly-Arg is obtained.

Citraconic acid can be easily removed under acidic conditions.

In this way, P-Gly-Arg, which can be converted into human calcitonin from the fusion protein, is produced as a peptide mixture, and the target product can be further separated and purified by appropriate means, such as high-performance liquid chromatography. P-G17-Arg is converted to P-01,y by carboxypeptidase, and P-G17-Arg is converted to P-01,y by carboxypeptidase.
Gly is converted to human calcitonin by an amidating enzyme derived from animal tissue.

〔Example〕

Examples of the present invention will be described below, but these are not intended to limit the present invention.

Example 1 (A) Chemical synthesis of single-stranded oligonucleotides The base sequence of the gene shown in Figure 2 was divided into 12 single-stranded oligonucleotides (fragments) shown in the figure, and each was isolated using the solid-phase triester method. We synthesized 7 fragments of . Since the synthesis method is common to each fragment, an example will be shown for the synthesis of fragment 1. The sequence of fragment 1 is: 5'CGC! TGOGGTAACOT' 3 in a known manner
Weigh out 5 μmole of a protected nucleoside (T in this case) with '-OR bound to a polystyrene solid support,
Small column with glass filter (15x100a)
After adding methylene chloride and leaving it for a while, filter it with suction. Methylene chloride containing 1% trichloroacetic acid (TOA) was added to the column to protect the 5'-OH protecting group D
Remove MTr group (dimethoxytrityl group) (1.5
111/X2 times). After washing the column with anhydrous pyridine, 25 μmole of a protected nucleotide monomer (C in this case) from which one of the protecting groups (cyanethyl group) of 3'-phosphoric acid has been removed was added as an anhydrous pyridine solution, and the column was washed with anhydrous pyridine. Connect to a vacuum pump to completely dehydrate. Next K
After adding 25 μm of the mixture MSNT (mesitylenesulfonyl nidrotriazole) and 0.25 μm of anhydrous pyridine, the column was tightly stoppered and kept at 50 to 55° C. for 20 minutes. Return the column to room temperature and wash the column with anhydrous pyridine. Next, the unreacted 5'-OH group of T is acetylated by adding a solution of 0.1 M acetic anhydride in pyridine. After 5 minutes, the column is washed with pyridine and methylene chloride in this order, and the 5'-OH protecting group (DMTr group) of the bonded C is removed by the method described above. Hereinafter, protected nucleotide dimers were prepared using the same method as AC, %TA, and GG.

Go, OT, and CG are sequentially combined in this order. When the last CG dimer is completed, a 5 M TMG-PAO (
5 of tetramethylguanidine-pyridine aldoxime)
By adding 1.5 d of 0% dioxane solution and incubating at 30°C for 24 hours, the synthesized oligonucleotide is separated from the carrier and the protective group (0-chlorophenyl group) of the internucleotide is removed.

The reaction solution is concentrated to dryness and subjected to reverse phase column chromatography on silica gel. Elute with increasing concentrations of acetonitrile in 50 mM TEAA (triethylamine acetate buffer, pH 7.0) and collect the desired fraction from absorbance at 256 nm.

After deprotecting DMTr with 80% acetic acid, purified oligonucleotide is obtained by reverse phase high performance liquid chromatography. Yield was approximately 100 μ2. The base sequence was confirmed by two-dimensional homochromatography.

The other 11 fragments were synthesized in exactly the same manner, and their nucleotide sequences were confirmed.

03) - Construction of human calcitonin precursor peptide gene from wood-like oligonucleotides Among the seven chemically synthesized fragments, the 5' ends of each of the seven fragments, excluding 1 and 7 shown in Figure 2, were phosphorylated by the following method. did. Dissolve 7 fragments of 25 μm in 10 μt of distilled water and add polynucleotide kinase (PNX) buffer (50 mM) Lis-HO2, pH 7, 6, 10.
mM g O12, 10mM mercaptoethanol)
It was incubated for 60 minutes at 37°C in 2 μt of a solution containing 1 mM ATP and 5 units of PNK.

After heat treatment, 2.5.
10. Mix 11 and 12 with phosphorylated fragment 1 and add DNA ligase buffer (66mM).
Lis-HCl, pH 7,66, 6mM MgCl2
), 150 μt solution containing 10 mM DTT (dithiothreitol).

Similarly, fragment 4.5.6.8.9 and non-phosphorylated fragment 7 were mixed to form the same solution. An annealing operation was performed for each of these two solutions. That is, each solution was heated to 70°C for 10 minutes, then gradually returned to room temperature over 3 hours, and further maintained at 15°C. To each solution, 1mM ATP, a ligase bag, and 250 units of DNA ligase were added to make a total volume of 200 μt, and the mixture was reacted at 15° C. for 6 hours. Both reaction solutions were combined, 150 units of DNA ligase was added, and the reaction was continued at 15°C overnight. After the reaction solution was extracted with phenol and then with chloroform, twice the amount of cold ethanol was added to the aqueous layer, DNA was precipitated at -70°C, and 1) Dnh was recovered by centrifugation.

This was separated by 10% polyacrylamide electrophoresis, the target band was detected under ultraviolet irradiation, and the cut out gel pieces were collected using gel elution buffer (Q, 5M ammonium acetate, [l
LOIM magnesium acetate, 1mM EDTA),
DNA was extracted by incubation at 7°C overnight. This was precipitated with twice the amount of ethanol to obtain about 20 μ2 of DNA.

(0) Cleavage and dephosphorylation of vector DNA by Hpa engineering 7μ of plasmid vector psN 5182? , Hpa
Buffer for I (10mM) Lisu・HO4spH15,
7mM MgO1l, 100mM KOt, 7
(mM mercaptoethanol), restriction enzyme Hpa 1
40 fits of the reaction solution containing 0 units were incubated overnight at 37°C, subjected to phenol extraction, chloroform extraction, and ethanol precipitation to obtain DNA cut with HPA.
was collected and dissolved in 20 μt of distilled water. This is 100
65 mM) Lis-Hct (pHaO) in a 40 μt reaction solution containing 1.2 units of alkaline phosphatase.
℃, and incubated for 60 minutes. fiDNA was recovered by phenol extraction, chloroform extraction, and ethanol precipitation, and dissolved in 14 μt of distilled water. This was subjected to 1% agarose gel electrophoresis and separated.

Cut out the band of interest and prepare the gel piece with 50mM) Lis-HO.
ICpHan), 400μ containing 1mM BDTA
Extracted with t buffer. The extract was subjected to phenol extraction, chloroform extraction, and ethanol precipitation.
2 DNA was recovered.

(D) Binding of synthetic gene and vector ■ Example when n in formula ■ is 1 Synthetic gene obtained in (B) above N A 0.3 μy (4
, 5 pmole) in 5 pt of distilled water and 10
1J101J, PNK buffer and 5 units of P
The reaction was carried out at 37° C. for 1 hour in a 5 μt reaction solution containing IJK. 5μt of this reaction solution was mixed with 5μM dNTP, DNA polymerase buffer (67mM potassium phosphate,
p, H7,4,6,7mM Mg, 012.1mM
mercaptoethanol) and 10 units of DNA polymerase 1 (Klenow fragment) and 10 units of DNA polymerase 1 (Klenow fragment).
fragment)] in a reaction solution of 20 pl at 25°C for 60 minutes. The reaction solution was extracted with phenol, then extracted with chloroform, and precipitated with ethanol to obtain blunt-ended DNA with a phosphorylated 5' end.
was recovered. This was dissolved in 8 μt of distilled water. on the other hand,
Commercially available Hpa Lumini Linker GTTAA (:!) 2
00 pmole was phosphorylated at the 5' end as previously described. 5 μt of the phosphorylated linker and 8 μt of the blunt-ended, 5′-end phosphorylated DNA were added to I) NA sorgase buffer, 10 mM DTT.

[I], 5mM ATP, and 200 units of DNA ligase in a 20 μL reaction solution at 15° C. overnight. 100 units of DNA ligase and 1o qmol
A'l'P of e was added and the reaction was further continued at 15°C for 5 hours. After the reaction solution was extracted with phenol and chloroform, 1) Dyh was recovered by ethanol precipitation, and 10 μt
dissolved in distilled water. Add this to 20μ of a reaction solution containing HpaI buffer and 10 units of restriction enzyme HpaI.
The reaction was carried out overnight at 37°C. The reaction solution was subjected to 10% polyacrylamide gel electrophoresis, a gel piece containing the target DNA was cut out, and the DNA was extracted and collected by the method described above.

Recovered DNA and vector 0.2 prepared in (0) above
μm, ligase buffer, 10mM DTT, α5m
2 containing MATP and 200 units of DMA ligase
The reaction was carried out in a 0 μt reaction solution at 15° C. overnight. 10 μt of this reaction solution was used for transformation of E. coli.

■ Example when n is 2 or more when formula (■) is satisfied. Dissolve 5 μf of the DMA fragment obtained in (■) above in 20 μt of distilled water, and phosphorylate the 5' end in the same manner as in (g)) above. did. Phosphorylated DNA solution 50 liters
, 10mM DTT.

Reaction was carried out at 15° C. for 2 hours in a 100 μt reaction solution containing α5mM ATP, DNA ligase buffer, and 250 units of DNA ligase. A portion was subjected to electrophoresis and it was confirmed that self-ligation (self-binding) of the synthesized genes had occurred. The reaction solution was subjected to phenol extraction, chloroform extraction, and ethanol precipitation.
A was collected. Thereafter, the sticky ends were made into blunt ends in the same manner as in (9)-(1), further combined with an Hpa lumini linker, cut with the restriction enzyme Epa, and subjected to 10-way polyacrylamide gel electrophoresis. A band with a length corresponding to n = 2 or more was cut out, extracted from the gel, and collected.

Recovered DNA and vector prepared in (C) 0.2 μm
Ligase buffer, 10mM DTT
, 20μ of a reaction solution containing 0.5mM ATP and 200 units of DNA ligase was reacted overnight at 15°C. 10 μt of this reaction solution was used for transformation of E. coli.

(E) Transformation of Escherichia coli Escherichia coli AN] K 10 (stored at the Institute of Microbial Diseases, Osaka University) was mixed with 5-L medium (Bactodrypton 1oy, "z,
Yeast extract 5 f/! ,, 1Jact5 f/l
, pH 72) at 37°C overnight with shaking. The [1L05-] was inoculated into a new L medium of 5- and cultured with shaking under the same conditions until the optical density at 600 nm reached 0.6.

The 2.5 d was cooled on ice, centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed. To this, 2.5 d of Q, 1M MgO/4, which had been ice-cooled in advance, was added, mixed, and then centrifuged to remove the supernatant. Further ice-cooled αI M
OaC! 1.25 of 4 was added, mixed gently and homogeneously, kept in ice water for 30 minutes, and then the supernatant was removed by centrifugation. To this, 10 μt of the reaction solution obtained in (D)-〇 above
was added and kept in ice water for 30 minutes. Next, the mixture was heated at 40°C for 5 minutes, and L medium 2-, which had been previously warmed to 37°C, was added thereto and kept at 37°C for 30 minutes. The culture was further incubated at 37°C for 1 hour with shaking. The culture solution was treated with tetracycline 1
It was spread on an agar medium containing 5 μt/−. After overnight culture at 37°C, the grown colonies were isolated and treated with tetracycline 1.
0 μ? After confirming growth in the medium containing /-, this was subjected to plasmid analysis.

(9) Analysis of plasmid The transformed E. coli obtained in (E) above was grown in 250-L medium containing 10 μf/- of tetracycline at 37°C for 2 hours.
After culturing for 4 hours, the bacteria were collected. Add to this a solution of 10-1 (
50mM glucose, 10mMRDTA, lysozyme 2 xq/vt, 25mM Tris wCt%
pHaO) and kept in ice water for 50 minutes, then
A solution of It ((L2M NaOH 11% BD day) was added. After 5 minutes, a solution of Ill of 15- (5M sodium acetate, p)!4B) was added and kept at 0°C for 30-60 minutes, then 100d Add cold ethanol and incubate at -20℃ for 30 minutes.
Hold for minutes. Collect the precipitate by centrifugation, add a solution of 25- (0.1M sodium acetate, Q, 05M Tris-HO
After adding 50-mL cold ethanol and keeping it at -20°C for 10 minutes, it was removed by centrifugation.
) A DNA precipitate was obtained. This was added to a 4-TE solution (50 m
M) Lis HCl, pHNo, 1mM KDTA
), transfer to a centrifuge tube containing 4,75 t of cesium chloride, and add 0.42 q/- of ethidium bromide.
After adding m and mixing, the mixture was kept in ice water for 30 minutes, and the supernatant was obtained by centrifugation. Transfer the supernatant to an ultracentrifuge tube (
Put it in a quick seal tube (Manufactured by Beckman) 55
．． 00 Orpm, - night ultracentrifugation was performed. The plasmid band was detected under ultraviolet irradiation and extracted using a syringe. 5MNa0t, 10mM) Lis-H
C1% pHa 5.1mM]! Extraction was repeated 7 to 8 times with Inglovir alcohol saturated with a solution containing 1DTA to remove ethidium bromide, and the plasmid was recovered by ethanol precipitation (yield: about 20 μ?).

After dissolving this in 200μt TFi and cutting a part with Hpa, polyacrylamide electrophoresis was performed.
Expected DNA fragment, (102n-1-12)'hp
(identical to nld formula II) was confirmed. In addition, the insertion direction of the synthetic gene was determined from the size of the DNA fragment generated by double digestion with the restriction enzymes Ace and O1a (141 bp fragment in the positive direction, 215 bp fragment in the negative direction). ).

In this way, a recombinant was obtained into which a plasmid into which a gene in which 1 to n synthetic genes were connected in a straight chain was inserted in the forward direction was introduced. The results are shown in Table 1. The nucleotide sequence of the inserted DNA was determined by the dideoxy method after it was incorporated into an M13mp phage, and the correctness of the sequence was confirmed.

Table 1: Number of recombinants obtained, n = 1 of 1 of the plasmids thus obtained, where n has the same meaning as formula II.
The plasmid was named psOT 815, and the Escherichia coli into which the plasmid was introduced was called Escherichia coli ANOK 1.
It was named pSOT 1315 and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology.
No. 00 (FEBM P-8700)].

An outline of the construction of psOT 815 is shown in FIG.

That is, FIG. 3 is a schematic diagram showing the construction process of a plasmid into which the human calcitonin precursor peptide gene is integrated.

(G) Production and purification of human calcitonin precursor fusion protein Culture and induction of recombinant E. coli are described by A. Nakata et al. European Journal of Biochemistry Vol. 130, No. 427.
It was carried out according to the method described on page 435 (1985).

Escherichia coli ANCK 10/pBOT 815 (FE
RM'P-8700) and tetracycline 10μ2/-
Tris/glucose high phosphate medium (0,2% glucose Q, IM) containing Tris/HCl5 pH 7,2 and 0.6
The inorganic salt containing 4 mM Kn and PO4) was inoculated into three test tubes containing 5Tnt, and cultured with shaking at 67°C for 20 hours.

Three Erlenmeyer flasks were inoculated with 2 containing 500 ml of the same medium, and cultured at 37° C. overnight. Bacteria were collected by centrifugation, transferred to a medium with the same composition except for KH2PO4, and cultured for an additional 6 hours. Bacteria were collected by centrifugation and treated with 1 omM) Lis-Hat (pH 7,5) containing 2 μM paraaminophenylmethanesulfonyl fluoride (p-APMSF), 5 mM KDT.
A, Bacterial cells were suspended in 5 mM mercaptoethanol and disrupted by sonication.

The solution thus obtained was heated to 12,000 rpm.
The mixture was centrifuged for 30 minutes and the precipitate was collected. This precipitate:
2 pM p-APMSF, 1 mM Mg
12. Dissolve in 1omu) IJS・HCt buffer (pH 75) containing SO4 and 7M urea. OOOrpm
The mixture was centrifuged for 30 minutes. D] whose supernatant was equilibrated in advance with the same buffer solution! ! AF! Cellulose power ram (1
, 3tMφX 12 cm ) and the same buffer solution 5
It was eluted at 0d. 2-/Vial and gallipart sorting,
The fusion protein was confirmed by 5DS-polyacrylamide electrophoresis, and the target fraction was collected. This was equilibrated in advance with the same buffer and applied to a CM-cellulose column (1.5 mφ x 30 pieces). Then in the same buffer,
From 0 M NaOt (50 m) to 0.35 M NaOt (
After elution with a linear concentration gradient of 50WLt), both fractions containing the fusion protein were confirmed and collected, dialyzed against water and freeze-dried to obtain human calcitonin precursor fusion protein (2[11rIg). .

(6) Human calcitonin precursor peptide (P-Gly-Ar
g) Separation and Purification Excision of the human calcitonin precursor peptide (P-Gly-Arg, ) from the fusion protein was performed by T. D. Boxtra (J.D.
) et al [Nature Vol. 285, pp. 456-461 (19
[80]].

[Phase] The combined protein (2) was suspended in 7-100 mM ammonium bicarbonate aqueous solution, and while stirring, 14 μt of citraconic anhydride was added 5 times at 15 minute intervals for reaction. At that time, the pH was adjusted to aO using 2N aqueous sodium hydroxide solution.
Do this while maintaining the

As the reaction progresses, the fusion protein dissolves, yielding a protein in which only lysine residues are selectively citraconylated. This reaction solution was dialyzed against 100 mM ammonium bicarbonate. In the solution after dialysis (7rytt), 42
μ2 of trypsin was dissolved in 70 μt of I M QaOt2 aqueous solution and added, the reaction was carried out at 37°C, and after 1 hour p
-A 1N hydrochloric acid solution containing 200 μm of APMSF was added to stop the reaction. This solution was left at 40° C. for 6 hours to remove the citraconyl group bonded to the shirizine residue. The reaction solution was desalted and concentrated using a sup-PAK (co, ) cartridge (manufactured by Waters).

300 μt of the above concentrated solution was added to TSK-80TM (1.5×1
It was purified by HPLC using a Toyo Soda Co., Ltd. column (No. 5). 0.1%) IJ fluoroacetic acid aqueous solution (liquid A)
and a 0.1 aqueous solution of trifluoroacetic acid (solution B) containing 90% acetonitrile.
Monitored by ultraviolet absorption at 210 nm. Each peak was measured by enzyme immunoassay using an anti-calcitonin antibody (DAKO), and the peak of human calcitonin precursor peptide (p-G177Arg) was identified. This peak completely matched the peak of a standard product chemically synthesized using a peptide synthesizer manufactured by Abride Biosystems, Inc. (ABI), USA. This peak was fractionated to obtain 100 μm of pure human calcitonin precursor peptide.

Figure 1 shows the H of the obtained human calcitonin precursor peptide.
This is a graph showing a PLO pattern, and the analysis conditions are the same as above. That is, FIG. 1 is a graph showing the HPL (!) elution pattern of human calcitonin precursor peptide in terms of the relationship between elution time (minutes, horizontal axis) and absorbance at 21 nm (vertical axis).

After hydrolyzing 4 μV of the obtained peptide with hydrochloric acid, the amino acid composition was examined using an amino acid analyzer (manufactured by Hitachi Seisakusho), and the result was almost identical to the theoretical value.

Furthermore, the results of amino acid sequence analysis using a gas phase protein sequencer (manufactured by ABI) were as expected. Furthermore,
The obtained peptide was placed in a 96-well microplate) (NUN (
After blocking with bovine serum albumin, rabbit anti-human calcitonin antibody (Dako) was applied. After washing, peroxidase-labeled donkey anti-rabbit second antibody (Amajam) was bound, and after washing, ABTS
(chromogenic substrate), color was developed with a sensitivity of about 10 times that of human calcitonin.

〔Effect of the invention〕

As explained in detail above, the human calcitonin precursor peptide newly provided by the present invention can be easily converted into human calcitonin, and the precursor can be efficiently produced by genetic engineering techniques. The present invention has a great effect in that a method has been developed.

[Brief explanation of drawings]

Figure 1 shows the H of the human calcitonin precursor peptide of the present invention.
PL(!) A graph showing the elution pattern, Figure 2 is a diagram showing an example of the base sequence for producing the raw material peptide of the method of the present invention, and Figure 3 is the construction process of a plasmid into which the human calcitonin precursor peptide gene has been integrated. FIG.

Claims (1)

[Claims] 1. The following general formula I: P-Gly-Arg...[I] (wherein, P is a peptide residue having the amino acid sequence of human calcitonin, Gly is a glycine residue, and Arg is arginine) Human calcitonin precursor peptide represented by (residues shown). 2. General formula II below: [Arg-P-Gly]-_nArg-Cys...[
II] (wherein, Arg is an arginine residue, P is a peptide residue having the amino acid sequence of human calcitonin, Gly is a glycine residue, n is a positive integer, and Cys is a cysteine residue). A fusion protein containing the peptide represented by the general formula II is produced in the culture by culturing Escherichia coli transformed with a plasmid into which a DNA containing the encoding gene has been inserted, and then the fusion protein is treated with an enzyme. A method for producing a human calcitonin precursor peptide represented by the following general formula I: P-Gly-Arg... [I] (wherein P, Gly and Arg have the same meanings as in the above formula II).