JPS62205795A - Production of physiologically active peptide amide - Google Patents

Abstract

PURPOSE:To obtain a physiologically active peptide amide useful as pharmaceuticals, etc., by treating a specific precursor in an aqueous medium with a C-terminal amidation enzyme originated from mammal. CONSTITUTION:A fish calcitonin derivative (precursor) having amino acid sequence of formula (X is Asn or Asp; Y is Thr or Val; Z is Sr or Ala) is mixed with a C-terminal amidation enzyme extracted from swine pituitary gland, etc. The obtained aqueous medium is added with 20-100muM of copper ion, 100-500muM of ascorbic acid, 50-200mug/ml of catalase and 50-1,000muM of serine protease inhibitor (e.g. phenylmethylsufonyl fluoride) and/or thiol protease inhibitor (e.g. N-ethylmaleimide) and made to react at 20-42 deg.C and 6-9pH. The reaction medium is purified and the objective physiologically active peptide amide is collected from the medium.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more specifically, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more specifically, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more particularly, the present invention relates to a method for producing a bioactive peptide amide, and more specifically, a precursor of a bioactive peptide having an additional glycine at the C-terminus. The present invention relates to a method for producing a physiologically active peptide amide by converting it into a bioactive peptide amide.

[Conventional technology]

Various peptides having physiological activity are known and are used as medicines.

For example, calcitonin can be used to treat hypercalcemia, Page
A therapeutic effect has been found for t (Beget's) disease. This substance has also been confirmed to have a bone resorption inhibiting effect and a bone neogenesis promoting effect, and is also expected to have a therapeutic effect on senile osteoporosis.

Calcitonin from pigs, salmon, and eel is currently commercially available as therapeutic calcitonin, and these calcitonins are obtained by extraction from living organisms or chemical synthesis. However, its production quantity is small and it is expensive. Therefore, there is a strong desire to develop a new method that can produce calcitonin economically and in large quantities.

In order to achieve this purpose, methods for producing calcitonin precursors using genetic engineering methods have been proposed, for example, in Japanese Patent Application Laid-Open No. 58-203953 and pcτ Publication Patent Publication No. 58-50.
1121. PCT Publication Patent Publication Sho 59-501095
, PCT Publication No. 59-501243. In addition, Japanese Patent Application No. 59-170492, Japanese Patent Application No. 60-98891, filed by the same applicant as this patent application,
Patent application 1986-113254 and patent application 1988-1308
No. 15 describes a novel fish calcitonin derivative having an additional glycine at the C-terminus and a method for its preparation.

By the way, some peptide physiologically active substances require a peptide amide with an amidated C-terminus for their activity; for example, for the activity of calcitonin, the 32nd position It is known that it is important that proline be amidated. For example, in the above-mentioned Japanese Patent Application No. 60-130815, the proline at position 32 of the C-terminus is not amidated, but instead it is added. It has been described that the activity of a derivative of fish calcitonin with glycine is about one-seventh of the activity of the corresponding natural calcitonin.

However, it is extremely difficult to directly produce a peptide whose C-terminal amino acid is amidated using genetic engineering methods. It often has to be converted to an amide.

Nature, 298.686 (19B) describes D-Tyr-
Conversion of Vat-Gly to 0-↑yr-Val-NHz has been described, BBRC.

112372 (1983) describes the conversion of R-Tyr-Val-Gly to D-Tyr-Val-NH3 using a preparation of a C-terminal amidation enzyme derived from pig brain.

921 (1983), D-Tyr-Val-Gly using a C-terminal amidation enzyme preparation derived from bovine brain.
Conversion to Tyr-Val-NHZ is described and F
EBS Lett, 167.160 (1984) describes thyrotropin-releasing hormone precursor (TRI-Gly, pGlu-His-Pro-Gl) whose C-terminus is glycylated using a mouse brain-derived C-terminal amidation enzyme preparation.
y) to pGIu-11is-Pro-NH, and in the 1985 Chemical Society, Ac-Tyr-Phe-
The conversion of Gly to octc-Tyr-Phe-NHz has been described. However, in these reports, peptides consisting of 3 to 4 amino acid residues including glycine are used as substrates, and their N-termini contain D-Tyr (D-tyrosine) and pGIu (pyroglutamic acid). , Ac-Tyr (acetyltyrosine) are present, which protect them from undesired degradation by proteases. Therefore, these reports do not suggest at all the possibility of amidation of a physiologically active peptide such as calcitonin, which is composed of many amino acids and does not have a protected amino acid at the N-terminus.

P t, ProcJur, P t, S m, 18
th, 1984 contains sequences of 1, 6, 8, 10, and 12 amino acids on the C-terminal side of human calcitonin, and further contains the protected amino acid D-T at the N-terminus.
Amidation using an enzyme preparation derived from pig pituitary gland using a synthetic peptide with yr added and Gly added to the C-terminus as a substrate has been reported. However, in this case as well, the number of amino acid residues in the substrate peptide is relatively small, and concrete data show that the reactivity worsens as the number of amino acid residues increases. In addition, the N-terminus of all substrates is protected by D-Tyr, an unnatural amino acid, and this report also shows that the N-terminus of a peptide consisting of many amino acids, such as complete calcitonin, is not protected at the N-terminus. Please do not suggest anything about the possibility of change.

The enzyme called C-terminal amidation enzyme is present in extremely small amounts in the pituitary gland, etc., and has 1. Its enzymatic properties have not been investigated in detail, and a purification method has not been established. Therefore, enzyme preparations containing this enzyme contain large amounts of various proteases, and therefore, such enzyme preparations can be used to perform reactions without undesirable side reactions, such as undesired cleavage of the substrate. It is thought that it is impossible to perform only the desired amidation reaction, and in fact, it is difficult to carry out only the desired amidation reaction, and in fact, it is difficult to carry out only the desired amidation reaction.
It was not possible to amidate peptides whose -termini were not protected from protease degradation with C-terminal amidation enzyme preparations.

As mentioned above, this is why prior art methods using C-terminal amidation enzymes used only peptides consisting of a relatively small number of amino acids as substrates and whose N-terminus was protected against protease degradation. This is because it is based on the level of technology.

[Problem that the invention seeks to solve]

The present invention utilizes a partially purified C-terminal amidation enzyme derived from mammals to synthesize bioactive peptide precursors consisting of a relatively large number of amino acids with an additional amino acid at the C-terminus. - It is an object of the present invention to provide a method capable of converting bioactive peptide amides without requiring terminal protection.

[Means for solving problems]

The present inventors have discovered that C-
As a result of various methods for purifying terminal amidation enzymes and research on methods for amidation of calcitonin derivatives using these crude enzyme preparations, partial purification was achieved. By carrying out the reaction using a C-terminal amidation enzyme, the product has an additional glycine at the C-terminus, and the N-terminus is not protected from protease degradation by D-Tyr, acetylated amino acids, pGlu, etc. , and that a bioactive peptide precursor comprising a relatively large number of amino acids can be converted into a bioactive peptide amide with almost no undesired side reactions, such as cleavage in the middle portion of the peptide. In other words, we obtained completely new knowledge, and based on this we completed this invention.

Therefore, the present invention provides a precursor having an additional glycine at the C-terminus of a physiologically active peptide by treating the precursor with a C-terminal amidation enzyme derived from a mammal in an aqueous medium. The object of the present invention is to provide a method for producing a bioactive peptide with an amidated C-terminus, which is characterized by converting the bioactive peptide into an amidated bioactive peptide and collecting the bioactive peptide.

[Specific explanation]

Preparation of Enzyme The enzyme preparation used in the present invention can be prepared from any mammal,
For example, it can be prepared from the pituitary gland of livestock such as pigs and cows, or experimental animals such as rats. Preferably, it is prepared from the pituitary gland of a pig because it is relatively easy to obtain and can be obtained in large quantities. In the preparation of enzyme preparations, that is, the partial purification of enzymes, various proteases and other impurities are removed and the relative concentration and cotton concentration of the target C-terminal amidation enzyme are increased. Any enzyme purification method can be used.

For example, the pituitary gland is removed from a pig, collected in large numbers, and homogenized in an appropriate buffer, such as 20 mM Tris-HCl buffer (pH 7.5) containing 250 mM sucrose. This homogenate is subjected to centrifugation, filtration, etc. to remove solids and obtain a supernatant or filtrate. The C-terminal amidation enzyme is extracted and solubilized by freezing and thawing this solution, and then subjecting it to ultrasonication. Next, a solution in which the C-terminal amidation enzyme is dissolved is obtained by centrifugation, filtration, etc. Next, if desired, solvent precipitation, such as acetone precipitation, is performed, and after dissolving the precipitate, salting out, for example, with ammonium sulfate, is performed to obtain a 10 to 40% saturated fraction. These two fractions are subjected to column chromatography,
For example, chromatographic purification is performed using a DEAE-1-Yopard column, Sephadex G-150 column, etc., and then further purification is performed using a 5F4 (ff) chelate affinity column to obtain an active fraction.

The titer of the C-terminal amidation enzyme is determined as follows. The enzyme preparation was mixed with copper sulfate 50μM and ascorbic acid 200μM.
M, 1. in the presence of catalase I001tg/ml. c.
+M' ”1-D-Tyr-Val-Gly (
(Reaction volume: 50 μl) Incubate at 37° C. for 4 hours, and stop the reaction by adding 10 μl of IN hydrochloric acid to 50 μl of the reaction solution. The reaction solution is centrifuged, and 3 μβ of the supernatant is spotted on an aluminum sheet silica gel thin layer plate. This was mixed with chloroform:methanol:water:ammonia (6:4).
: 0.5 : 0.5) and the remaining substrate on the separated thin layer plate "'I-D-T
yr-Val-Gly and generated ttsl-04yr-
Val-N)lz is detected by autoradiography. A portion of the thin layer plate corresponding to each spot is cut out, and the radioactivity of ItJ is counted with a gamma counter to determine the proportion of amidated substrate. When the reaction was carried out under these conditions, 20% of the substrate (10 pmol
) is defined as one position.

Ball-1 The method of the present invention can be applied to, for example, thyrotropin-releasing hormone (TRH) (3 amino acids in the active peptide, C-terminal structure Pro-NL), oxytocin, vasopressin (9 amino acids, GlyJHz), luteinizing hormone. Release hormone (LH-RH (10 pieces, Gly-N)
), caerulein (10 pieces, Phe-NHz), secretin (27 pieces, Val-NHz), calcitonin (32 pieces, Pro-N) lx), avian pancreatic polypeptide (36 pieces, Tyr-NHt), vasoactivity ( vasoactive
) Intestinal peptide (28 pieces, Asn-NHz eight cell stimulating hormone α-MSI ((13 pieces, Val-NHz
), mast cell granule-reducing peptide (Mast-ce
ll degranu-fating peptide
(22 pieces, Gin-NHz), Melitchin (26 pieces,
Gin-NHg), Peptide 401-Be, Venom (P
eptide 401-Beevenom) (22 pieces, Asn-NHl) -'-Aerotoxin ■-Scorpion (64 pieces, His-NHt), etc. That is, the above-mentioned peptide in which the amidic NH, group at the C-terminus is replaced with Guy can be used as a substrate.

As a substrate for producing calcitonin, for example fish calcitonin, for example the following amino acid sequence: Cys S
er Asn Leu Ser Thr C
ys Val LeIj GlyLys Leu
Ser Gin Glu Leu His Lys L
eu GlnThr Tyr Pro Arg
Thr X Y Gly Z G
lyThr Pro Gly (In the sequence, X represents Asn or Asp, Y represents Thr
or Val, and Z represents Ser or ^la) can be used, and a method for producing this derivative is described, for example, in Japanese Patent Application No. 6
0-130815 in detail.

In the enzymatic amidation reaction, the C-terminal amidation enzyme preparation prepared as described above and a precursor or derivative having glycine at the C-terminus of a physiologically active peptide are allowed to coexist in an aqueous medium. To do this.

The concentrations of substrate and enzyme are interrelated and can be determined experimentally depending on the type of substrate. Copper ions such as copper sulfate, ascorbic acid or its salts, and catalase are present in the reaction medium. These concentrations vary depending on the type of substance, substrate concentration, reaction conditions, etc., but for example, copper ions are 20 to 100 μM, for example 50 μM.
M, ascorbic acid is 100-500 a M,
For example, about 200 μM, and catalase is 50-
200 tt g/mg.

For example, about 100 μg/m1. Furthermore, it is preferable to add a serine protease inhibitor, such as phenylmethylsulfonyl fluoride, and a thiol protease inhibitor, such as N-ethylmaleimide, to prevent undesired p-reactions caused by contaminant proteases contained in the enzyme preparation. . These are, for example, 50 to 1,000
It is added at a concentration of 0 μM, preferably about 250 μM.

The reaction temperature is about 20'C to 42°C, preferably about 37°C.
and the pH of the reaction medium is 6-9, preferably about 7-8. The reaction time can be arbitrarily selected such that the reaction is substantially complete. As the reaction medium, for example, Tris-HCl buffer, sodium phosphate buffer, etc. can be used.

Recovery and purification of the product from the reaction medium can be carried out by any conventional method depending on the type of desired product.

Next, the method of the present invention will be explained in more detail with reference to Examples.

On the left plate C-O''' Amidohye, 2,.8.ff
Pituitary glands were collected from 50 pigs per pig, and were added to a 20mM Tris-HCl buffer containing 250mM sucrose (
pH 7.5) and homogenized using a Botter homogenizer. This homosyne-1.
, Centrifuge for 15 minutes at OOOxg, and remove the supernatant at 80 m
I got 1. Add sodium deoxycholate (final concentration 0.1%) and potassium chloride (final concentration 0.5M) to this supernatant.
) was added, and the target enzyme was solubilized by ultrasonication for 15 minutes.

30. A supernatant was obtained by centrifugation at OOOxg for 30 minutes, and this supernatant was diluted with 20mM Tris-
Sodium deoxycholate and potassium chloride were removed by dialysis against hydrochloric acid buffer (pH 7.5), and 9 times the volume of cold acetone was added to form a precipitate. The precipitate was dissolved in 20 mM Lys-Salt #buffer (ρ) containing 0.1% sodium deoxycholate and 0.5 M potassium chloride and centrifuged to remove insoluble material. Removed. The centrifugation supernatant was fractionated by ammonium sulfate salting out to obtain a 10-40% saturated ammonium sulfate fraction.

Next, this ammonium sulfate fraction was added to D[! Add to AE-1-Yopal column and add 20mM Hlys-HCl buffer (CpH 1,5)
Medium 0.05-0.2M sodium chloride dry%
? Elution was performed with an X degree gradient. C- thus obtained
The terminal amidation enzyme active fraction was further applied to a Copper(II)-chelating Sepharose 6B column, and the column was washed with 20mM #acid/acetic acid buffer (pH 4) containing 0.5M sodium chloride. ,0)) and then 20mM containing 50mM imidazole and 0.5M sodium chloride.
A C-terminal amidation enzyme active fraction was obtained by elution with Tris-HCl buffer (pH 7.5). The active fraction thus obtained was dissolved in 20mM Tris-HCl buffer containing 0.15M sodium chloride (pl+7.5
) and then used for amidation of salmon calcitonin derivatives.

Recovery of the ``man'' 1-death reaction in 20mM Tris-HCl buffer containing 0.15M sodium chloride (pH
7,5), 1 mg of a salmon calcitonin derivative (a peptide having an additional glycine at the C-terminus of the proline at position 32 of salmon calcitonin), and iJI! as described above.
C-terminal amidation enzyme fraction (18,000 units of enzyme)
was added, and further copper sulfate 50μM, ascorbic acid 200μM
1. μM, catalase 100 μg/ml, phenylmethylsulfonyl fluoride 250 μM, and N-ethylmaleimide 250 μM. A reaction mixture of Qrnl was prepared and the reaction was carried out by shaking at 37°C for 8 hours.

Next, this reaction mixture was subjected to gel filtration using a Sephadex G-50 column to obtain a fraction containing amidated natural salmon calcitonin. In this case, the calcitonin mixture in the Sephadex elution fraction is ODS-
Detection was performed by reverse phase high performance liquid chromatography (IIPLc) using an 80TM column. Both fractions in which calcitonin was detected were collected and lyophilized, the residue was dissolved in 1 rrl of pure water, and then subjected to ion exchange HPLC using an ES-502C column (elution conditions: 90 mM ammonium formate, pH
3, 5), a peak having the same retention time (Fig. 1) as standard natural salmon calcitonin was fractionated.

After freeze-drying both of these fractions, they were further purified by HPLC using an ODS-80TM column (elution conditions: 20-80% acetonitrile, 0.1% trifluoroacetic acid) (Figure 2), yielding about 280 μg of amidated product. I got it.

The product obtained according to Example 2 was identified as follows.

(1) As a result of amino acid analysis, the amino acid composition was as follows.

Amine 7″ ・A sp 2.12 2T h
r 4.52 5S e r
3.70 4G l u 3.1
9 3G ly 3.47
3Ala-Val 1.06 11/2Cys
1.91 2Met
- 11e - L eu 5.0 5Tyr
1.01 1Phe
-Lys 2.06
2) (is 1.07
1A r g 1.06
1P r o 2.16
2 (2) Next, the product was subjected to amino acid sequence analysis without decomposition, and 80 μg of the product was separately collected at 0.2 μg.
Digested using trypsin in 2M ammonium acetate (PIT 8.0), amino acid sequence analysis was performed on the four types of digested fragments obtained, and by arranging the obtained sequences, the product was determined to be the amino acid of natural salmon calcitonin. It was confirmed that the sequence was correct. The results are shown in FIG.

(3) Next, in order to confirm the structure of the C-terminus, the following three types of peptides: (A) H-Thr25-Pro"-Gly-O
H(B) H-Thr”-Pro”-NHt(C
)H-Thr"-Pro"-OH was chemically synthesized,
These and the trypsin-digested fragment IV were combined with C4-reverse phase 11P.
Comparison was made in LC. Solution A (0.1% T
FA) and B solution (20% acetonitrile 10.1% TF
A) was used to elute with a concentration gradient of solution B from 2% to 20% over 10 minutes, and the peak positions were compared. As a result, trypsin-digested fragment IV has the same retention time as the synthetic peptide CB), and as a result, it was confirmed that trypsin-digested fragment IV has a structure in which proline is amidated at its C-terminus. It was done. This elution pattern is shown in FIG.

Furthermore, the molecular weight of trypsin-digested fragment IV was determined by mass spectroscopy, and the molecular weight MH' = 733 (S
II'lS), which was in good agreement with the molecular weight of the target substance.

Furthermore, the trypsin-digested fragment (1) was compared with the trypsin-digested product of standard salmon calcitonin in C4-reverse phase HPLC, and it was confirmed that it had the same retention time (Figure 5).

The molecular weight of the trypsin-digested fragment (2) was determined by mass spectrometry, and the molecular weight was MH"-1124 (SIMS), which was in good agreement with the result obtained for the trypsin-digested fragment (2) of standard salmon calcitonin.

From the above results, it was confirmed that the reaction product had the same structure as natural salmon calcitonin.

140 Wistar rats (Japan Charles River Co., Ltd.), male only, were purchased at 3 weeks of age, and after being reared for 1 week, healthy animals were raised to 4 weeks of age. 70 fish were used.

The animals were housed in an automatically controlled breeding room with a temperature of 22±2° C., 55±10%, ventilation 13 times per hour, and lighting 12 hours a day (7 a.m. to 7 p.m.). Animals were kept in metal mesh cages (
The animals were housed in pairs (250 x 350 x 200 mm (Nippon Cage Co., Ltd.)), and were given free access to the same type of feed (CMF: Oriental Yeast Co., Ltd.) and tap water.

As test compounds, salmon calcitonin (lyophilized product) prepared by the method of the present invention (referred to as GC-3) and commercially available salmon calcitonin I (lyophilized product) (referred to as CT) were used.

These test substances were dissolved in the following solvent, and 10,000
A test solution of ng/ml was prepared.

Sodium citrate 1.3mM citric acid
22mM sodium chloride
120mM gelatin 0.16%p
For H6,0 GC-3, the maximum dose was 80 ng/kg, and the following common ratios were 57, 40, 28, 20, 14, and L.
7 doses of ng/kg, CT the highest dose of 40 n
g/kg, and the following common ratio is 20.10 and 5 ng.
Four doses were established: /kg. The test solution is administered in an amount equal to 1 body weight.
Each dose was diluted with a solvent to give 1 ml per 00 g.

The animals were fasted from 6 pm on the day before administration of the test method, and 70 animals were randomly divided into 7 groups with 10 animals per group, and their body weights were measured. Based on this body weight, apply the test solution at 1% per 100g of body weight.
．． 0m7! was administered intravenously in the tail vein. Exactly 1 after administration
After a certain period of time, blood was collected from the abdominal aorta of the animal under ether anesthesia. Blood was left at room temperature for approximately 1 hour, then 30
The serum was collected by centrifugation at 00 rpm for 10 minutes. The obtained serum was 0-Cresolphthaiein C
It was used to measure calcium concentration using the onplexone method. Clinical test automatic analyzer JCA is used to measure calcium concentration.
-VX100O (JEOL Ltd.) was used, and the average value of duplicate measurements was taken as the calcium concentration measurement value.

The activity per protein of the reaction product was equivalent to that of a commercial salmon calcitonin preparation.

[Brief explanation of drawings]

Figure 1 shows the ion exchange HPLC (E
The elution pattern of S-502C) is shown. Figure 2 shows the reverse phase HP of the product obtained by the method of the present invention.
The elution pattern in LC (ODS-807M) is shown. FIG. 3 shows the results of amino acid sequence analysis of the products obtained by the method of the invention. FIG. 4 shows a comparison of the tryptic fragment (C-terminal fragment) of the product obtained by the method of the present invention and a chemically synthesized peptide by reverse phase HPLC. Figure 5 shows the reverse phase HP reaction of the tryptic fragment I (N-terminal fragment) of the product obtained by the method of the present invention and the tryptic fragment (N-terminal fragment) of standard salmon calcitonin.
A comparison by LC is shown.

Claims (1)

[Scope of Claims] 1. A precursor having an additional glycine at the C-terminus of a physiologically active peptide is reacted with a mammalian-derived C-terminal amidation enzyme in an aqueous medium to convert the precursor to C-terminus. −
C, characterized in that the terminal is converted into a physiologically active peptide with amidation, and the physiologically active peptide is collected.
- A method for producing a physiologically active peptide having an amidated terminal. 2. The method according to claim 1, wherein the mammalian-derived C-terminal amidation enzyme is a pig pituitary gland-derived C-terminal amidation enzyme. 3. The method according to claim 1, wherein the C-terminally amidated physiologically active peptide is calcitonin. 4. The method according to claim 3, wherein the calcitonin is fish calcitonin. 5. The method according to claim 4, wherein the fish calcitonin is salmon calcitonin. 6. The method according to claim 1, wherein a protease inhibitor is present in the aqueous medium. 7. The method according to claim 6, wherein the protease inhibitor is a serine protease inhibitor and/or a thiol protease inhibitor. 8. The method of claim 7, wherein the serine protease inhibitor is phenylmethylsulfonyl fluoride and the thiol protease inhibitor is N-ethylmaleimide. 9. The method according to claim 1, wherein copper ions, ascorbic acid, and/or catalase are present in the reaction medium.