JPH04364199A - Synthetic gene of human type glicentin - Google Patents

Abstract

PURPOSE:To obtain a new synthetic gene efficiently producing a human type glicentin, comprising a DNA sequence encoding a human type glicentin having a specific amino acid sequence, by transforming Escherichia coli by introducing the gene to a plasmid. CONSTITUTION:A DNA which contains a DNA sequence shown by formula II encoding a human type glicentin having an amino acid sequence shown by formula I and contains a bond part to a vector, is synthesized by division of said DNA to 5 fragments and respectively by using cyanoethylphosphoamidate method, then purified by using a strong anion exchange chromatography column, 5' ends are phosphorylated with T4 polynucleotide kinase, ligated with T4 ligase, the fragments are bonded, linked to a proper vector to prepare a recombinant plasmid, which is inserted into Escherichia coli to give a transformant. The transformant is cultured to efficiently give a human type glicentin shown by formula I.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a synthetic gene having a novel DNA sequence encoding the amino acid sequence of human glicentin.

0002

[Prior Art] A polypeptide hormone composed of 29 amino acid residues that is secreted from Langerhans islet A cells in the pancreas of animals and is known to activate the adenylate cyclase system in cell membranes of the liver and other tissues. From the study of glucagon, Sundby, F. isolated a large molecular weight polypeptide with antigenicity against glucagon antibodies from a pig small intestine extract and named it glicentin [Sundby, F. et al. et al. , Hor
m. Metab. Res. , Vol. 8, 36
6-371 (1976)]. Next, Thim, L.
and Moody, A. J. determined its structure and found that glicentin is composed of 69 amino acid residues, and that the amino acid sequence from 33rd to 61st is the same as glucagon [Regulator
y Peptides, Vol. 2:139 (19
81)]. In this way, porcine glicentin was purified and the structure was elucidated, but since porcine glicentin exists in the small intestine of pigs, human glicentin, which is naturally expected to exist in humans, was not extracted. Due to the difficulty in obtaining the raw material, human intestinal tract material, it has never been isolated as a purified product, and therefore its amino acid sequence has not been determined. Due to difficulties in isolation, the physiological activity of human glicentin has not yet been elucidated.

By the way, Bell, G. I. et al. deduced the sequence of the human preproglucagon gene by hybridizing the hamster preproglucagon cDNA with the EcoR I degradation product of the human gene, and elucidated the amino acid sequence of human glicentin from the structure of the exon portion of the gene [ Nature, Vol. 304, 36
8-370 (1983)]. Although the amino acid sequence of human glicentin was thus deduced from the corresponding DNA sequence of human preproglucagon, no recombinant DNA vector was created from the DNA encoding this human glicentin and used for transformation. No attempt has yet been made to produce human glicentin from host cells.

0004

[Problems to be Solved by the Invention] As mentioned above, human glicentin has not been obtained as a purified product so far because it is difficult to extract from biological samples. Therefore,
Although the physiological activities and pharmacological effects of human glicentin are still unknown, it is a peptide substance produced in living organisms, and porcine glicentin is known to suppress gastric acid secretion in rats [P ．． Kirk
egaard et al., Nature, Vol. 297,
156-157 (1982)], some kind of medical use is expected for human glicentin.

[0005] Therefore, it is thought that human glicentin can be produced in large quantities by synthesizing DNA encoding the amino acid sequence of human glicentin, ligating it to a vector, and culturing host cells transformed with this. Based on this idea, the present inventors attempted to establish a method for producing human glicentin using so-called genetic engineering techniques.

[0006] Therefore, the problems to be solved by the present invention are as follows:
The aim is to elucidate the synthetic gene that allows human glicentin to be expressed most efficiently by host cells.

[0007]

[Means for Solving the Problem] According to the present invention, the following amino acid sequence

A synthetic gene of a DNA sequence encoding human glicentin having [Chemical formula 3], a plasmid recombinant in which this synthetic gene is inserted into a plasmid vector, a host cell transformed with this plasmid recombinant, and a host cell transformed with this plasmid recombinant, and A method for producing human glicentin by culturing to express desired human glicentin is provided.

[0008] Various DNA sequences encoding the above-mentioned amino acid sequence of human glicentin can be established based on the amino acid sequence, and the DNA sequences shown in the above-mentioned prior documents also indicate that this sequence is present in human cells. This is a DNA sequence for the biosynthesis of human glicentin, but when it is intended to express human glicentin in a transformed host cell, the following points should be considered when setting the DNA sequence. That is, (1) Select codons that are frequently used in host cells (eg, E. coli) and that are familiar to the host cells. (2) Provide specific restriction enzyme recognition sites within the set DNA sequence and at both ends thereof to facilitate insertion and ligation into other DNA sequences or plasmid vectors, and To facilitate verification of whether a set DNA sequence is correctly maintained. (3) Target DNA by linking DNA fragments
When synthesizing a DNA fragment, select a DNA fragment that will not cause erroneous ligation. (4) Design the set DNA sequence so that no DNA sequence that is inconvenient for peptide synthesis, such as a stop codon, occurs.

Based on such considerations, the present inventors have discovered a synthetic gene having the following DNA sequence as a preferable gene for producing human glicentin in Escherichia coli.

0010

[C4]

[0011] The above synthetic gene is shown as a mutually complementary double-stranded DNA sequence encoding human glicentin, but each of the single-stranded DNA sequences constituting this is also a synthetic gene of the present invention. It constitutes.

[0012] When using E. coli as a host cell, this DNA sequence selects codons that are frequently used in E. coli.
Restriction enzyme recognition sites such as t I and Sac I are present, thereby making it easier to test whether this DNA sequence is correctly maintained in the recombinant.

[0013] Considering the connection to the vector, the synthetic gene having this DNA sequence is divided into five fragments, namely fragments G1, G3, and the DNA sequence including the connecting part to the vector, as shown in Table 1 below. These oligonucleotide fragments were divided into G5, G7, and G9, along with complementary fragments G2, G4, G6, G8, and G10, that is, a total of 10 oligonucleotides were produced by synthetic means.

[0014]

[Table 1]

[0015] Each of the oligonucleotide fragments can be synthesized using DNA synthesis means known in the art, and for this purpose a DNA synthesizer is used, such as the one manufactured by Pharmacia. There is a gene assembler.

Among the oligonucleotide fragments obtained, fragments G2 to G9 were phosphorylated at their 5' ends, annealed with non-phosphorylated fragments G1 and G10, and subsequently treated with ligase to obtain the following sequence. Double-stranded DNA was obtained.

[0017]

[C5]

[0018] This double-stranded DNA has in its structure a gene portion for causing Escherichia coli to produce the human glicentin of the present invention as described above, and the following vector DNA at both ends.
Nco I and Hi as cohesive ends when connecting to A
It has an end capable of binding to the nd III site.

The DNA sequence containing the human glicentin gene thus obtained is then ligated to a suitable plasmid vector. In this case, as mentioned above, this D
The NA sequence has Nco I and Hind I at both ends.
Plasmid vectors have cohesive ends capable of binding to the Nco I and Hind III sites.
Foods that can be digested by humans are used. However, this plasmid vector must contain a promoter site necessary for subsequent expression of genetic information and a gene that serves as a marker for transformed host cells, such as an antibiotic resistance gene site. is preferred.

The present inventors have found that it is suitable to use plasmid pKK233-2 for this purpose.

[0021] The plasmid recombinant in which the human glicentin gene has been linked is used to transform host cells. It is also possible to produce the desired human glicentin using this transformed host cell.

In a specific example of carrying out the present invention by the present inventors, plasmid pKK233-2 was cut with Nco I and Hind III, and a synthetic gene having the DNA sequence encoding the human glicentin gene described above according to the present invention was obtained. was inserted into this cut plasmid, thus obtaining a plasmid named pGL3, which was used to transform E. coli, and pGL3 was cloned by culturing this E. coli.

Plasmid pGL thus obtained
When 3 was digested with EcoR I and Hind III, tr was generated upstream of the DNA sequence of the human glicentin gene.
A fragment with the c promoter was obtained, and this and EcoR
Plasmid p digested with I and Hind III
Upon ligation with UC18, a plasmid named pGL5 was obtained. Plasmid pUC18 is a plasmid with multicopy properties, and pGL5 has the glicentin gene connected to the lac promoter and trc promoter in tandem, and a plasmid recombinant with a high plasmid copy number was obtained. Become.

[0024] By transforming Escherichia coli using this pGL5 plasmid, human glicentin can be produced extremely efficiently.

[0025] In order to confirm that the glicentin produced in this way has the desired structure, we performed assays using antigen-antibody reactions, analyzed the amino acid composition by chemical analysis, and determined the amino acid sequence. The results of these tests all indicated that the product produced was the desired human glicentin.

The present invention has been explained above using an example in which Escherichia coli is used as a host cell, but it goes without saying that other than Escherichia coli, microorganisms such as Bacillus subtilis and yeast, and cells of mammals including humans can also be used. .

[0027]

[Example] Next, in order to explain the present invention in more detail,
According to Examples, a glicentin gene having a novel DNA sequence encoding human glicentin of the present invention, a recombinant DNA containing the glicentin gene having this new DNA sequence, and a host transformed with this recombinant DNA, Expressing human glicentin from this host,
A method for assaying that the expressed product is the desired human glicentin will be explained in detail.

Preparation of a fragment of the human glicentin gene As described by Bell, G. et al. I. et al, Natu
re, Vol. 304, 368-370 (198
Based on the amino acid sequence of human glicentin elucidated by 3), we studied the DNA sequence for E. coli to most efficiently produce human glicentin. That is, when E. coli is transformed with recombinant DNA containing DNA encoding human glicentin, transformation of E. coli with recombinant DNA containing DNA containing frequently used codons in this host E. coli is planned. Ta. For this purpose,
In order to construct a DNA that encodes human glicentin and has codons that are frequently used by E. coli,
The DNA containing the human glicentin gene and the connecting portion to the vector was divided into 5 fragments as shown in Table 1 above, and 10 oligonucleotides were prepared by synthetic means along with fragments complementary to these 5 fragments. .

[0029] The above oligonucleotide fragments G1 to G1
0 was synthesized by the β-cyanoethyl phosphoramidite method and subsequently purified using a strong anion exchange chromatography column.

Preparation of human glicentin gene Among the oligonucleotide fragments synthesized and purified as described above, the 5' ends of fragments G2 to G9 were phosphorylated with T4 polynucleotide kinase (manufactured by Takara Shuzo Co., Ltd.). i.e. 50mM Tris-HCl pH 7.5, 10
5 μg of each of the above oligonucleotide fragments was added to 50 μl of a solution containing mM dithiothreitol, 10 mM MgCl2, and 5 mM ATP, and 4 units of T4 polynucleotide kinase was added thereto. After reacting at 37°C for 1 hour, The enzyme was inactivated by holding at ℃ for 5 minutes. In this way, eight fragments, fragments G2 to G9, were phosphorylated at their 5' ends.

Non-phosphorylated fragments G1 and G10
and 2 μg of each of the phosphorylated fragments G2 to G9 were mixed and ethanol precipitated according to a conventional method (Perba
l, B. , A Practical Guide
to Molecular Cloning, 198
8, 2nd edition, p. 78, JOH
N WILEY & SONS). The precipitate thus obtained was dissolved in 40 μl of distilled water, and
5 μl of a solution containing M Tris-HCl and 0.1 M MgCl2 was added, heated to 90°C, and then cooled to 30°C over about 3 hours. Add 2.5μl of 0.2M dithiothreitol and 0.5μl of 0.1M ATP to this solution.
l, 0.5μl of 20mM spermidine and 2μl of distilled water.
To this add T4 ligase (manufactured by Takara Shuzo Co., Ltd.) 35
Ligation was performed by adding 0 units and reacting at 14° C. for 16 hours to bind the oligonucleotide fragments. The reaction product was developed by 5% polyacrylamide gel electrophoresis, and a band at a position corresponding to 230 bp was excised. Add 0.5M ammonium acetate and 1mM to the cut out gel.
1 ml of a solution containing EDTA was added and extracted overnight at 37°C. The extract was centrifuged at 12,000 rpm for 5 minutes to separate into a supernatant and a precipitate, and the supernatant was subjected to phenol extraction, followed by ethanol precipitation according to a conventional method. The DNA thus precipitated and separated was phosphorylated again with T4 polynucleotide kinase.

[0032] In this way, the oligonucleotide fragments described above are linked to form a 228 base pair nucleotide having 4 base cohesive ends at both ends as shown in the formula 5 above, and human glicentin is present therein. D to code
A double-stranded DNA containing NA and complementary DNA to this DNA was obtained, which had cohesive ends at both ends for ligation to the next vector DNA.

Cleavage of plasmid pKK233-2 with restriction enzymes Plasmid p used as a vector in this example
KK233-2 (manufactured by Pharmacia) with restriction enzyme Nco
I (manufactured by Takara Shuzo Co., Ltd.) and Hind III (manufactured by Takara Shuzo Co., Ltd.)
Co., Ltd.). Nco I and Hind II
The plasmid DNA cleaved at the I cleavage site was then treated with phenol and precipitated with ethanol. 8 μg of precipitated DNA was mixed with 1 unit of calf intestine alkaline phosphatase (CIP) (manufactured by Boehringer) at 37 μg.
℃ for 30 minutes to dephosphorylate the 5' end. After phenol extraction of the dephosphorylated DNA,
Ethanol precipitation was performed.

[0034] Linkage of glicentin gene and vector DNA phosphorylated double-stranded human glicentin gene 2
0ng and the above dephosphorylated plasmid pKK23
400 ng of DNA, which is the cleavage product of 3-2 with Nco I and Hind III, was mixed and ligated using T4 ligase. Using this ligated DNA, Escherichia coli
HB101 was transformed (Prebal, B., A.
Practical Guide to Molec
ular Cloning, 1988, 2nd e
dition, pp. 414-416, JOHN
WILEY & SONS), each of the obtained transformed strains was added to 5 ml of LB medium containing 50 μg/ml ampicillin.
Hattor was cultured at 37°C for 16 hours.
i, M. and Sakaki, Y. The plasmid was extracted and purified by the method of (Anal. Bioc
hem. Vol. 152, 232-238 (19
86)).

[0035] The obtained plasmid was subjected to BamH I, Hi
The plasmid was reacted with restriction enzymes nd III and Sac I to select a plasmid containing each restriction enzyme cleavage site in the glicentin gene. The base sequences of these plasmids were confirmed by the dideoxy method. Dideo
For xy method base sequencing, 7-Deaza manufactured by Takara Shuzo Co., Ltd.
This was carried out using a Sequencing Kit according to the company's manual. A strain containing a plasmid in which the glicentin gene portion was arranged as designed according to base sequencing was saved, and this plasmid was named pGL3. Plasmid pGL3 can be easily replicated by culturing this strain (Feikoken Bibori No. 11030).

[0036] Furthermore, using this plasmid pGL3, E
A strain obtained by transforming S. scherichiacoli JM105 (Feikoken Bibori No. 11029) was similarly prepared.

[0037] The above oligonucleotide fragments G1 to G1
By annealing and ligation of 0, a sticky end capable of binding to the Nco I site was created on the upstream side, and a Hi
Generation of a synthetic DNA duplex encoding human glicentin with cohesive ends capable of binding to the ndIII site and cleavage sites for BamH I and Sac I in the sequence, and the restriction enzyme Nc of plasmid pKK233-2.
o Cleavage of the same plasmid with I and Hind III and synthetic DNA encoding the human glicentin
The production of plasmid pGL3 by ligation of the double strand of and the cut plasmid pKK233-2 is shown in Figure 1.
is schematically shown.

Vector conversion The pGL3 vector was converted into a multicopy plasmid pUC.
lac promoter and trc by converting to 18
A new plasmid was constructed in which the promoters are linked in tandem and have a high copy number.

That is, as shown in FIG. 2, plasmid pGL3 was digested with restriction enzymes EcoR I and Hind I.
II, and a fragment containing the glicentin gene portion corresponding to approximately 500 bp was separated by 1.2% agarose gel electrophoresis, using the method described in the literature (Perbal, B.
．． , A Practical Guide to M
Olecular Cloning, 1988, 2
nd edition, pp. 372-373,
JOHN WILEY & SONS)
The NA fragment was extracted and purified.

[0040] Vector pUC18 was digested with restriction enzyme EcoR.
Digested with I and Hind III, extracted with phenol and precipitated with isopropyl alcohol. For precipitation with isopropyl alcohol, add sodium acetate (pH
4.8) to a concentration of 0.3M, and isopropyl alcohol to a final concentration of 40%.
This was done by leaving it for 5 minutes. The resulting precipitate was washed with 40% isopropyl alcohol-water and then with 70% ethanol-water. The vector fragment thus obtained was dephosphorylated by CIP, and ligated with T4-ligase at a ratio of vector fragment 2 to fragment 1 containing the glicentin gene previously obtained from pGL3. Using the DNA obtained from this ligation, Esc
Herichia coli JM105 was transformed. The obtained transformant was treated with ampicillin at 50 μg/m.
The plasmid was extracted by culturing in LB medium containing a concentration of
Restriction enzymes EcoR I, Hind III and Ba
This was digested with an appropriate combination of mH I to confirm whether the fragment containing the glicentin gene was inserted in the correct direction and at the correct length. In this way, a plasmid in which the glicentin gene was correctly inserted was confirmed, and this was named pGL5.

[0041] Furthermore, using this plasmid pGL5, E
A strain obtained by transforming S. scherichiacoli HB101 (Feikoken Bibori No. 11031) was similarly prepared.

The production amount of glicentin by this strain was 0.08 mg per liter of culture solution.

[0043] Further, using this pGL5, Escherichia coli S, which is a non-protease producing strain, was grown.
A strain transformed from G21059 (Feikoken Bacterium No. 122
No. 83) was also produced. The production amount of glicentin in this strain was 0.4 mg per liter.

EcoR I and H of pGL3 described above
Removal of the glicentin gene fragment carrying the trc promoter by digestion with indIII and pU
Digestion of C18 with EcoR I and Hind III and the thus cut plasmid pUC18
The purification of plasmid pGL5 by ligation of the glicentin gene fragment obtained above is schematically shown in FIG.

Preparation of anti-glicentin rabbit antiserum In order to assay glicentin produced by the transformed E. coli of the present invention, anti-glicentin rabbit antiserum was prepared.

That is, a peptide from the N-terminus to the 32nd amino acid sequence of glicentin having the following amino acid sequence,

embedded image was synthesized using a peptide synthesizer (manufactured by Applied Bio) using the BOC method. This synthesis followed the company's manual.

Using the above synthetic polypeptide as an antigen and keyhole limpet hemocyanin as an antigen carrier, a rabbit was immunized by sensitizing it six times at 14-day intervals to form antibodies. The antibody titer of the obtained anti-glicentin rabbit antiserum was evaluated by enzyme antibody method. That is, 5 to 500 ng of the antigen was adsorbed onto a nitrocellulose membrane, and the membrane was assayed using an enzyme immunoassay kit (manufactured by BioRad) according to the company's manual. The antiserum was diluted 200 times, and the secondary antibody was diluted 2000 times.

[0048] As a result, a 5 ng sample showed sufficient color development compared to the control, indicating that detection from 5 ng is possible using this antibody.

Expression and purification of glicentin Escherichia coli harboring plasmid pGL5
50μ of JM105 strain (Feikoken Bacteria No. 11028)
6 hours at 37°C in LB medium containing g/ml ampicillin.
It was cultured in The culture solution was centrifuged at 6500 rpm for 15 minutes to separate and precipitate the bacterial cells.

[0050] Microbial cells equivalent to 20 liters of culture solution were added to 300 m
20mM Tris-HC containing 1 of 6M guanidine hydrochloride
The suspension was suspended in a pH 7.5 solution and sonicated five times for 3 minutes each. Centrifuge at 15,000 rpm for 10 minutes and collect the supernatant, which is diluted to 3 liters with 0.1% trifluoroacetic acid solution.
Next, insoluble matter was removed by centrifugation at 10,000 rpm for 20 minutes. The supernatant was transferred to a reversed-phase silica gel column [Preparative C18100Å manufactured by Waters Co., Ltd.] that had been equilibrated with 0.1% trifluoroacetic acid in advance.
(inner diameter 16mm x 100mm)] and 200m
After washing with 1 ml of 0.1% trifluoroacetic acid, linear gradient elution was performed from 0.1% trifluoroacetic acid to 90% acetonitrile to 0.1% trifluoroacetic acid over 30 minutes; at a flow rate of 5 ml per minute. The eluate was fractionated into 10 ml portions, 2 .mu.l of each fraction was adsorbed onto a nitrocellulose membrane, and a glicentin-containing fraction was selected using the enzyme-antibody method described above. Selected fractions were collected and concentrated to dryness using a rotary evaporator. Add this to 20mM sodium acetate pH 5
．． 6 Suspend in 100 ml, remove insoluble matter by centrifugation at 15,000 rpm for 10 minutes, and
It was adsorbed onto a cation exchange chromatography column (MonoS 5-5 for FPLC manufactured by Pharmacia) that had been equilibrated with 0 mM sodium acetate pH 5.6.
After washing with 30 ml of mM sodium acetate pH 5.6, elution was performed using a linear gradient from the same solution to 20 mM sodium acetate pH 5.6 containing 0.5 M NaCl at a flow rate of 1 ml per minute for 30 minutes. A glicentin-containing fraction was collected by the enzyme antibody method described above using 1 μl of each fraction.

[0051] This was carried out using a high performance liquid chromatography device (
655 series (manufactured by Hitachi) and a reverse phase column (Inertsil ODS 5 μm, manufactured by Gascro Kogyo Co., Ltd.). The separation conditions were a flow rate of 1 ml per minute and a linear gradient from 24% acetonitrile 0.05% trifluoroacetic acid to 40% acetonitrile 0.05% trifluoroacetic acid over 20 minutes.

Peak detection was performed by ultraviolet absorption at 280 nm. The column temperature was 40°C. The obtained main peak fraction was collected and dried under reduced pressure.

[0053] The amount of glicentin produced by this JM105 strain was found to be 0.08 mg per liter of culture solution.

Confirmation of Glicentin Whether the sample obtained as described above was pure human glicentin was confirmed by Western blotting, amino acid analysis, and amino acid sequencing.

[0055] Western blotting was performed in the following manner. That is, electrophoresis was performed using a ready-made gel-based electrophoresis device (Fast System manufactured by Pharmacia) according to the company's manual. The gel used was a 20% polyacrylamide homogeneous gel, and the sample was adjusted to pH 6.8 by SDS polyacrylamide gel electrophoresis.
of 5mM Tris-HCl, 2% SDS, 5% 2-mercaptoethanol, 7.5% glycerol, and 1
0.1 in a solution containing 0 μg/ml bromophenol blue.
Dissolve to a concentration of mg/ml and incubate at 100°C.
was heated for 3 minutes and electrophoresed. For electrophoresis, the same sample was run in two lanes at the same time, and one of the lanes was stained with Kumashi brilliant blue according to the company's method. On the other hand, the gel was separated from the plastic membrane, and the gel was used to electrophoretically transfer the proteins in the gel onto a nitrocellulose membrane using a transfer device (semi-dry blotter manufactured by Sartorius). The method was carried out according to the company's manual. After the transfer, the nitrocellulose membrane was treated with anti-glicentin antiserum in the same manner as used for assaying the glicentin-containing fraction after the chromatography, and the position of the band containing glicentin was identified. As a result, the sample showed a single band when stained with Coomassie brilliant blue, and was also strongly colored when stained with antibodies, indicating that it was a single band.

[0056] Amino acid analysis was carried out by the following method. That is, 80 μg (10 nmol) of a sample of a single peak obtained by the above high-performance liquid chromatography was placed in a hydrolysis tube, 200 μl of 6N hydrochloric acid was added thereto, the tube was sealed under reduced pressure, and then heated at 110° C. for 24 hours. The contents were dried under reduced pressure, and the residue was dissolved in 0.01N hydrochloric acid and analyzed using an automatic amino acid analyzer (Model 835, manufactured by Hitachi). The results are shown in Table 2.

[0057]

[Table 2]

Determination of the amino acid sequence 240 μg (30 nmol) of the sample that showed a single peak in the above high-performance liquid chromatography was diluted with 50 μg of the sample at pH 8.8.
5 with mM Tris-HCl and 3 μg of lysyl endopeptidase (4.5 AU/mg, manufactured by Wako Pure Chemical Industries, Ltd.)
The reaction was carried out in 00 μl at 37° C. for 4 hours. The reaction solution was transferred to a high performance liquid chromatography device (Hitachi 655 series).
Reversed phase column (Inertsil ODS manufactured by Gascro Industries)
It was separated using The separation conditions were a flow rate of 1 ml per minute, and a gradient from 0.1% trifluoroacetic acid to 72% acetonitrile to 0.1% trifluoroacetic acid over 60 minutes. Detection of fractions was performed by ultraviolet absorption at 220 nm. The five peaks thus obtained were separated, and the amino acid sequence of each peak was determined using a gas phase amino acid sequencer (PSQ-1 manufactured by Shimadzu Corporation). The operating method followed the company's manual. The amino acid sequences of the peptide fragments of peaks 1 to 5 were revealed to be as shown in Table 3 below, and were consistent with the amino acid sequences of peptide fragments thought to be produced by the decomposition of human glicentin.

[0059]

[Table 3]

[0060] From the above results, it was confirmed that glicentin obtained by the method of the present invention is human glicentin that is thought to exist in the human intestinal tract.

[Brief explanation of drawings]

[Figure 1] A double strand of synthetic DNA encoding human glicentin and plasmid pKK233-2 were transformed into EcoR I
FIG. 2 is a diagram schematically showing the production of plasmid pGL3 by ligation with that obtained by digestion with and Hind III.

FIG. 2: EcoR I and Hi of plasmid pGL3
The glicentin gene fragment carrying the promoter site obtained by digestion with Hind III and plasmid pUC18 were digested with EcoR I and Hind III.
FIG. 2 is a diagram schematically showing the production of plasmid pGL5 by ligation with that obtained by cutting with pGL5.

Claims (2)

[Claims] [Claim 1] The following amino acid sequence [Chemical formula 1] [Claim 2] DN encoding human glicentin
The synthetic gene according to claim 1, wherein the A sequence is: