JPH01215296A - Human secretin precursor and production thereof and use thereof - Google Patents

Abstract

NEW MATERIAL:A compound consisting of amino acid sequence expressed by the formula (X is -Gly-Lys-Arg-COOH, -Gly-Lys-COOH, -Gly-Arg-COOH, -Gly-Arg-Lys-COOH or -Gly-COOH). USE:An antiulcer agent. PREPARATION:For example, a gene coding human secretin precursor substance having amino acid sequence expressed by the formula is prepared by ordinary DNA synthesis, etc. and then integrated to a vector capable of propagating in a host to provide recombinant vector capable of propagating in host cell. Then the host cell is transformed using the resultant vector and then the product is recovered by culturing the transformant to provide the human secretin precursor substance expressed by the formula.

Description

[Detailed description of the invention] 1 Rigomi The present invention relates to human secretin precursor substances.

More specifically, the present invention relates to human secretin precursor substances, their genetic engineering production methods, and their uses.

Secretin is a compound known as a gastrointestinal hormone, and is known to promote the secretion of water, bicarbonate, etc. from the pancreas.

Clinically, porcine secretin represented by the following formula is used for pancreatic function tests and as an ulcer treatment agent.

His-8er-Asp Gly Thr-Phe
-Thr-5er-Glu-Leu-5er-Arg-
Leu-Arg-Asp-5er-Ala-Arg-
Leu-Gin-Ar g-Leu-Le u-G
1 n-G ly-Leu-Vat NH2 Usually, to obtain the above secretin, natural secretin (usually futasecretin) is used, but it has been found that the amino acid sequence of bovine secretin is also identical to that of futasecretin. ) extraction and chemical synthesis have been implemented.

However, all of these methods still have problems in terms of cost, rapid production, production volume, etc.

In addition, the products extracted and purified from pig small intestine contain many gastrointestinal hormones (motilin, VIP, CCK-PZ).
There is also the difficulty that accurate bioassays or radioimmunoassays for secretin cannot be performed using methods such as GIP, GLI, GLI, etc.).

By the way, with the recent development of genetic engineering technology, it is becoming possible to produce various physiologically active polypeptides using this technology.However, in the case of secretin produced by genetic engineering, the C-terminus of the secretin polypeptide is It has been reported that it does not have the expected physiological activity because it does not have the amide structure of valine.

Subsequently, amidating enzymes that amidate the C-termini of these peptides were discovered in higher animals, and a method for obtaining natural secretin by using this amidating enzyme with secretin-Gly-COOH as a substrate has been disclosed. (Refer to Japanese Patent Application Laid-Open No. 60-69098.) However, this method has not been put into practical use because the manufacturing process is complicated and the yield is low.

By the way, recently, -Gly- was added to the C-terminus of porcine secretin.
A porcine secretin precursor substance with three amino acids added, Lys-Arg-COO
FEBSLetters, 184 (2), 34
7 (1985)).

However, the current situation is that even the existence of a precursor substance for human secretin has not been reported.

m (7) [! 10i The present invention is to chemically synthesize a structural gene of a human secretin precursor substance, to produce a human secretin precursor substance by genetic engineering using this gene, and to recover this as a homogeneous human secretin precursor substance. This was done after confirming that these human secretin precursor substances had extremely high biological activity.

Therefore, the secretin precursor substance according to the present invention is characterized by consisting of the following amino acid sequence.

H2N-Hi s-5e r-As p-G I
y-Thr-Phe-Thr-Ser-Glu
-Leu-5er-Arg-Leu-Arg -Crl
u-Gly-Ala-Arg-Leu-G 1
n-Ar g-Le u-Leu-G 1 n
-Gly-Leu-Va l -XIn the formula, X is -Gly-Lys-ArB-COOH, -G
1y-Lys-COOH.

-Gly-Arg-qoOH, -Gly-Arg-Ly
Indicates s-COOH or -Gly-COOH.

Furthermore, the method for producing the human secretin precursor substance represented by the above formula (1) has seven characteristics, including the following steps a) to 2).

b) Prepare a gene encoding a human secretin precursor substance.

(1) Introducing this gene into PETAR, which can proliferate within the intended host, to create a recombinant vector that can proliferate outside of this cell.

-8) Transforming host cells using this vector.

2) Cultivating the resulting transformant and collecting the produced human secretin precursor substance.

Further, the anti-ulcer agent according to the present invention is characterized in that it contains as an active ingredient a human secretin precursor substance consisting of the amino acid sequence represented by the above formula (1).

The human secretin precursor substance according to the present invention is a new substance, and is expected to have various physiological activities in addition to the medicinal effects of secretin itself. In addition, drugs containing this as an active ingredient have stronger biological activity than natural secretin, and are therefore extremely useful as anti-ulcer agents. Furthermore, according to the production method of the present invention, human secretin precursor substances can be produced in large quantities and purified and recovered rapidly, making industrial production possible. Alternatively, it is possible to change several species, and this can be an extremely important method for studying the structure-activity relationship of human secretin.

The human secretin precursor substance as used in the present invention is a peptide consisting of the following amino acid sequence, and may be in the form of any inorganic or organic salt as required.

H2N-HI s-5e r-As p-G ly
-Th r-Ph e-Th r-5e r-G l
u-Leu-8er-Arg-Leu-Arg-Gl
u-Gly-Ala-Arg-Leu-G l n-A
r g-Le u-Leu-G l n -Gly-
Leu -Va l -X where X Lt-Gly-Lys-Arg-COOH,
-Gly-Lys-COOH.

-Gly-Arg-COOH, -Gly-Arg-Ly
Indicates s-COOH or -Gly-COOH.

jLM-Friend The manufacturing method according to the present invention consists of the following steps a) to 2).

b) Prepare a gene encoding a human secretin precursor substance.

(1) Incorporating this gene into a vector that can proliferate within the intended host to create a recombinant vector that can proliferate outside of this cell.

c) Transforming host cells using this vector.

2) Cultivating the resulting transformant and collecting the produced human secretin precursor substance.

Hereinafter, these steps will be explained in detail.

(1) Gene The gene encoding the human secretin precursor substance in the present invention may be any gene as long as it encodes the amino acid represented by the above formula (1). There is something indicated by .

(1) Vector The vector used in the present invention is a vector that can be replicated in the intended host, has a marker, for example, a drug resistance gene, and has the function of expressing a desired structural gene. It refers to the body's DNA.

Functions for gene expression include promoters, operators,
The preparation of vectors, which are realized by providing transcription initiation regions, ribosome binding sites (RBS), etc., is now well known.

In the embodiments of the present invention, pBR322 is used as a standard, and a fusion type trp promoter (Japanese Patent Laid-Open No. 62
285788), but the invention is not limited thereto.

(1 Summer I) Recombinant vector The recombinant vector according to the present invention consists of a vector containing at least a promoter and a so-called parasanger consisting of a structural gene encoding a secretin precursor substance or a fusion protein thereof. This fusion protein is composed of a human secretin precursor substance and other proteins bound together via a linkage consisting of a cleavable amino acid sequence. The protein other than the human secretin precursor substance mentioned here may be any protein that is used in normal fusion methods, such as protein A.
5β-galactosidase or growth hormone can be used. Preferably the 'frettin precursor substance is
Through the linkage, it may be bound to the C-terminus of a protein other than the human secretin precursor, that is, to the downstream side in terms of the corresponding gene sequence, or to the N-terminus, that is, similarly to the upstream side. You may. It goes without saying that in this recombinant vector, the gene encoding the fusion protein is located downstream of the promoter.

As a specific example of the cleavable amino acid sequence referred to herein, methionine shown in Examples below can be used. That is, methionine is inserted into the connecting part of the fusion protein, and the resulting fusion protein is treated with cyanogen bromide (
When treated with CNBr), this portion is specifically cleaved and the human secretin precursor substance can be recovered.

In addition, recently, in cutting out the desired protein,
A method using an enzyme has been proposed (Jiatsu et al., 9th Molecular Biology Society of Japan, 3A-35, Nagoya, 1986), but by creating an amino acid sequence recognized by the enzyme at the linkage site, it is possible to If there is also a cleavable amino acid sequence, it will be cleaved into several fragments. Therefore, on the condition that the stabilizing effect of protein parts other than the human secretin precursor substance is not excessively lost, by deleting, converting, or newly inserting the cleavable amino acid sequence within the protein, ,
Purification of the human secretin precursor substance after the cleavage operation becomes easy. That is, it is possible to greatly change the molecular weight or physical properties between the obtained human secretin precursor substance and other proteins or fragments thereof, which facilitates the purification of the desired protein (details can be found in Japanese Patent Application No. 1983-1999).
304937).

(IV) Transformant (Host Bacteria) The host bacterium can be any microorganism as long as the above-mentioned recombinant vector can proliferate within its body.A specific example of such a host bacterium is Escherichia
There is an E. coli strain HBIOI belonging to the genus Hia) [Pr
oc, Natl, Acad, Sci, USA, , 8
1, 5958-5960 (1984)].

(Transformation) The transformation operation itself can be carried out using conventional methods known in the field of molecular biology [for example, when transforming Escherichia coli, the Kushner method (Genetic Engineering) is used.

1978.17 (1978))], and when transforming Bacillus subtilis, genetic techniques for transformation of Escherichia coli can be applied. Specifically, the book “Practical Gene Recombination Technology 3”
Published by Science Forum, p. 33 (1982), published by R.
Molecular cloning a 1abol
atory manual J p247-268, C
o1d Spring Harbor Laborat
ory (1982), etc.

For the specific transformation method for E. coli, please refer to the experimental examples below.

(Transformant) As a specific example of a recombinant transformant created by transferring a recombinant vector, a strain transformed by a transformation operation is a strain transformed by transforming E. coli HBIOI into pAM425, pAM425, pAM425,
Transformants can also be selected using the properties [ampicillin resistance (Amp')] of transformants obtained by transformation with AM426, pAM427, and pAM428 as an indicator.

(Production and Recovery) The human secretin precursor substance is produced by culturing the above transformant according to a conventional method.

For specific methods, please refer to the experimental examples below.

To recover the human secretin precursor substance, the culture is treated by centrifugation or the like to collect bacteria, and then the bacteria are destroyed by any means such as a sonicator. Thereafter, after the human secretin precursor substance is released from the fusion protein containing the human secretin precursor substance, a homogeneous human secretin precursor substance can be recovered by subjecting it to the following steps.

C) Subjecting to purification by reverse phase chromatography.

That is, the fusion protein containing the human secretin precursor obtained in the above step is subjected to enzymatic or chemical treatment to release the human secretin precursor, and then extracted with acid to collect the supernatant. Preferably, the mixture is treated with cyanogen bromide (BrCN) and then extracted with an acetic acid solution. Impurities can be precipitated and removed by gradually adding alkali to the resulting acid solution. First, unnecessary fusion proteins or fragments thereof other than the human secretin precursor substance, peptides and proteins derived from E. coli begin to become insolubilized in the vicinity of weak acidity, and are removed by centrifugation as necessary.

Furthermore, in order to purify it, it can be purified by raising it to a weak alkalinity and performing the same operation. Both fractions obtained in this way contain highly pure human secretin precursor substances (normally, the fractions obtained in this way are purified by reverse phase chromatography to produce human secretin precursor substances). can be obtained as a single product (see Examples below for details).

Note that the reversed phase chromatography referred to here is a conventional method in which elution is performed with a water-containing organic solvent using a carrier, such as silica, as a stationary phase to which a hydrophobic group such as an alkyl group (C8 or 018) is chemically bonded as a ligand. In such systems, the desired protein is usually eluted using acetonitrile or alcohol. Moreover, it is preferable to carry out the reaction by a linear concentration gradient method in which the concentrations thereof are increased linearly.

As a preferred embodiment of the present invention, it is preferable to use reversed phase high performance liquid chromatography as shown in the experimental examples below.

(11M) The anti-ulcer agent of the present invention comprises the above-mentioned secretin premedication and others. Depending on the type of adjuvant, it can be administered orally or parenterally in the form of powder, granules, tablets, capsules, syrups, liquids, injections, or the like.

Further, if necessary, special dosage forms such as transmucosal drugs may be used (see Japanese Patent Application No. 62-29334).

Furthermore, other ulcer agents may be formulated.

The dosage can be adjusted appropriately depending on age and body weight, but as an injection, it is usually about 10 to 1000 secretin units, more preferably about 50 to 500 secretin units, as a secretin precursor substance for adults.

A preferred embodiment of the present invention is a unit dosage form in which the daily dose is divided and administered two to four times a day.

The anti-ulcer agent of the present invention is characterized in that the human secretin precursor substance as an active ingredient is natural human secretin.The anti-ulcer effect of the human secretin precursor substance in the present invention refers to both preventive and therapeutic effects on gastrointestinal ulcers This shows that.

NU'1 Takumi1, vector creation pAM-41 with hGH connected downstream of the trp promoter
0 plasmid vector (see Figure 2) can be made more efficient by adding IAA as an inducer.
can be expressed.

Therefore, around 120 amino acids from the N-terminus of hGH,
In order to obtain the target substance from the fusion protein, Net (ATG) was added in front of the old S at the N-terminus of the human secretin precursor substance. Furthermore, the 14th Net of hGH was
changed to °This made it possible to easily purify the fusion protein even if it was treated with cyanogen bromide.

A chemically synthesized DNA (ATG changed to TTG to make Leu at position 14) was inserted between the former nfl and Hha I restriction enzyme recognition sites upstream of hGH, and this vector was designated as pAM-420 ( (See Figure 2).

2. Creation of recombinant vectors Genes encoding four types of human secretin precursor substances (A-D) were inserted into the large NA fragment obtained by cutting the above pAM-420 with BamHI%SalI to create recombinant vectors. (See Figure 3).

Among these vectors, the vector encoded by the A gene (human secretin-Gly-Lys-Arg) was pAM426, and the vector encoded by the B3tl gene (t:)t'7f-Gly-Lys)@ pAM4
25. What is encoded by the C3g gene (human secretin-Gly-Arg) was added to pAM427 and D
I! The gene encoded by one gene (human t-creten-Gly-Arg-Lys) was named pAM428 (see Figure 3).

These four vectors are based on the sea-fence method (M
methods inEnzymno1ology*65t
499 (1980)), the structural gene encoding the junction and human secretin precursor substance was identified.

Then, these vectors pAM425, pAM426
, pAM427 and pAM428 t-
E. coli HBIOI was transformed by the Iz method.

3. Expression of fusion protein After culturing E. coli carrying each vector in L-broth containing 40 μg/ml ampicillin, the bacteria were harvested and transformed into M9.
- IAA, an inducer of the TRP promoter, was transferred to a medium containing 1% casamino acids (containing 40 μg/ml ampicillin).
(3-β-1ndoleaerylic acid) was added and cultured at 37°C overnight. In addition, the amount of glucose in the M9 medium was 1%, and the concentration of IAA was 8 μg/ml.

4. The purified and cultured bacterial cells were collected by centrifugation and placed in 20ml Tris-H.
C.I.

Dispersed in pH 8.0. The cells were broken with an ultrasonicator (sonicator) at 8000 rpm, 4℃, 1
The mixture was centrifuged for 0 minutes to collect precipitated proteins (insoluble proteins).

Insoluble proteins were purified with 7M urea in 20mM Tris-H.
C.I.

Dissolve at pH 8.0 and centrifuge at 18,000 rpm, 4°C, for 20 minutes to obtain a supernatant. This was dialyzed against 20mM acetic acid, and the white precipitate that came out during the dialysis was collected by centrifugation.

The centrifuged material (white precipitate) was dissolved in 70% formic acid, cyanogen bromide was added, and the reaction was carried out at room temperature overnight with shaking.

Thereafter, acetic acid extraction was performed to remove the insolubilized precipitate to obtain a supernatant. Next, aqueous ammonia was gradually added to the supernatant to adjust the pH.
set to 6.8. Centrifuge to remove the supernatant, add Trizma base (manufactured by Sigma) to raise the pH to 7.8, and centrifuge to obtain the supernatant.

Next, this was subjected to reverse phase C-18HPL (C-18HPL(
Y column). Purification was performed at 4°C except for HPLC.
Do it below.

All purified products could be recovered as single products. - As an example, human secretin-Gly-Lys-
The liquid chromatography and polyacrylamide gel electrophoresis patterns of Arg are shown in FIGS. 4 and 5.

5. Amino acid analysis (1) Amino acid composition analysis After hydrolyzing by a conventional method, it was quantified by the ninhydrin method. It was confirmed that the results were in line with the theoretical values.

(2) N-terminal analysis For N-terminal analysis, HPLC analysis was performed after Edman degradation. The device is a 470A Budane Sequenchy (
Applied Biosystem) and an isocratic method using an HPLC system LC-4A (Shimadzu Corporation) ODS-80TM column. As a result, the water was as designed (Hjs-Ser-A
sp-Gly-Thr-Phe-Thr-3er-Gl
u-Leu-Ser-ArB-Leu-ArB-Glu
).

(3) C-terminal analysis The results of the investigation were as follows.

Peptide derived from Danhyeongsing 1pAM426 Lys-Arg-CoolpA
M425-derived peptide Lys-Coo
lpAM427 derived peptide /lrg
-coo pAM428-derived peptide Ar
Human secretin precursor substances were identified at the protein level of g-Lys-COOH (1) to (3).

6. Biological activity Biological activity was measured using pancreatic juice secretion promoting effect in rats as an indicator.

(1) Method S and D rats that had been fasted from the previous day were treated with urethane (0%
The animal was anesthetized with −75 g/kg (i-p, ) and fixed in the anterior position.

After laparotomy, the bile duct was cannulated to secrete bile, and the bile duct was immediately ligated.

Next, pancreatic duct cannulation was performed distally from the ligated site, and a ligation was performed just before the duodenal opening to secrete pancreatic juice. Bile was administered using a cannulation tube into the femoral vein, and the amount of pancreatic juice secreted was measured by marking the tube at regular intervals (20 minute intervals). The distance traveled by the fluid was measured, and the amount of secretion was calculated. Chemically synthesized human secretin and pig secretin were also used as controls.

(2) Results The results are shown in FIG.

Human secretin-Gly-Lys-Arg showed approximately the same biological activity as natural human secretin.

Furthermore, linearity of 0, 2 μg/rat (i, v,)
The results obtained using the same solid were human secretin-Gly-Lys-Arg, human secretin-Gly-Lys, and human secretin-Gly-Ar when expressed in relative values with human secretin as a control (100%).
g and human secretin-Gly-Ar8-Lys showed a strong activity of 120-130%.

This shows the base sequence of the gene.

Figure 2 shows plasmids pAM-410 and pAM42.
This shows the structure of 0.

Figure 3 shows plasmids pAM-425, pAM-426,
The structure of pAM-427 and pAM-428 is shown.

Figure 4 shows the H of human secretin-Gly-Lys-Arg.
This shows a PLC pattern.

FIG. 5 shows the pattern of polyacrylamide gel electrophoresis of human secretin-Gly-Lys-Ar8.

FIG. 6 shows the amount of pancreatic juice secreted in H) 1 Cretene-Gly-Lys-Arg.

Claims (1)

[Claims] 1. A human secretin precursor substance consisting of the following amino acid sequence. [There is a gene sequence] In the formula, X is -Gly-Lys-Arg-COOH, -G
ly-Lys-COOH, -Gly-Arg-COOH
, -Gly-Arg-Lys-COOH or -Gly
- indicates COOH. 2. A method for producing a human secretin precursor substance comprising the following steps a) to d). b) Prepare a gene encoding a human secretin precursor substance. b) Incorporate this gene into a vector that can proliferate within the intended host to create a recombinant vector that can proliferate within this cell. c) Transforming host cells using this vector. d) Cultivating the resulting transformant and collecting the human secretin precursor substance produced. 3. The host cell is Escherichia
) The manufacturing method according to claim 2, which belongs to the genus A. 4. Recovery of human secretin precursor substance is performed in the following step a)
A manufacturing method according to any one of claims 2 and 3 consisting of -c). a) Subjecting to extraction with an acid solution. b) Adding alkali to remove impurities. c) Subjecting to purification by reverse phase chromatography. 5. An anti-ulcer agent containing a human secretin precursor substance having the amino acid sequence shown below as an active ingredient. [There is a gene sequence] In the formula, X is -Gly-Lys-Arg-COOH, -G
ly-Lys-COOH, -Gly-Arg-COOH
, -Gly-Arg-Lys-COOH or -Gly
- indicates COOH.