JPH04346790A - New production method of peptide - Google Patents

Abstract

PURPOSE:To provide a method for massively producing a peptide not having L-methionine in the molecule and having a mol.wt. of 2000-10000. CONSTITUTION:A new method for producing a peptide not containing L- methionine in the molecule and having a mol.wt. of 2000-10000 is characterized by employing a plasmid containing a DNA coding a fused protein prepared by bonding a sequence of a galK protein corresponding to the 200th to 300th amino acid residues from the N-terminal of the galK protein, L-methionine, and the peptide not containing the L-methionine and having a mol.wt. of 2000-10000 to the downstream side end of trp promotor.

Description

[Detailed description of the invention]

0001

TECHNICAL FIELD The present invention relates to a novel method for producing peptides.

0002

BACKGROUND OF THE INVENTION At present, physiologically active peptides are generally obtained by extracting and separating them from nature, by organic chemical synthesis, or by production methods that utilize genetic recombination.

0003

[Problem to be solved by the invention] However, the molecular weight is 2,
000 to 10,000 physiologically active peptides by extraction from animal organs or organic chemical synthesis is not profitable from a cost perspective, and production by genetic recombination is also possible. When expressed in cells, it degrades and is difficult to obtain. Therefore, it is necessary to produce a fusion protein with β-galactosidase, etc. in advance, and then obtain it by an inefficient production method that involves decomposing and purifying it. I didn't get it.

The present inventor has already discovered VIP, which is a peptide hormone derived from the intestinal tract and is known to have useful pharmacological effects such as vasodilatory, hypotensive, and bronchodilatory effects.
In order to obtain a peptide with natural human VIP-like activity, we aim to obtain a peptide with natural human VIP-like activity, without impairing the activity of the protein, and without making production by genetic recombination, which is highly economical, possible by introducing special amino acids. Regarding arrays,
After various studies, the following formula H-His-Ser-Asp-
Ala-Val-Phe-Thr-Asp-Asn-T
yr-Thr-Arg-Leu-Arg-Lys-Gl
n-Leu-Ala-Val-Lys-Lys-Tyr
-Leu-Asn-Ser-Ile-Leu-Asn-
Gly-Pro-R (where R is OH or
Represents Glu-OH or Glu-Ala-OH. )
It has been found that the peptide expressed by However, the example used was based on an organic chemical synthesis method and was not suitable for low-cost mass production.

[0005]

[Means for Solving the Problems] As a result of extensive research into economically efficient production methods through genetic recombination, the present inventors have discovered that L-methionine has a molecular weight of 2,000 L-methionine in the molecule.
In the production of peptides of 0 to 10,000, tr
200 minutes from the N-terminus of the galK protein using the p promoter.
At the C-terminus of the peptide, which corresponds to 300 amino acid residues,
L-methionine and the above molecular weight 2,000 to 10,000
A fusion protein having a peptide with a molecular weight of 2,000 to 1 is expressed in prokaryotic cells, purified, and bromocyanically digested to obtain a peptide mixture.
They discovered that mass production is possible by purifying 0,000 peptides, and completed the present invention.

That is, the present invention is a novel method for producing a peptide with a molecular weight of 2,000 to 10,000 that does not contain L-methionine in the molecule, and uses the trp promoter to cleave 200 to 300 peptides from the N-terminus of galK protein. A fusion protein in which L-methionine and the above-mentioned peptide having a molecular weight of 2,000 to 10,000 are arranged at the C-terminus of a peptide equivalent to amino acid residues is expressed in prokaryotic cells, and the fusion protein is purified,
This method further produces peptides having a molecular weight of 2,000 to 10,000 from a peptide mixture obtained by decomposing bromocyanide.

[0007] In the method of the present invention described above and the following explanation, His is L-histidine and Ser is L-histidine.
- Serine, Asp is L-aspartic acid, Ala is L-
Alanine, Val is L-valine, Phe is L-phenylalanine, Thr is L-threonine, Asn is
L-asparagine, Tyr is L-tyrosine, Arg is L-arginine, Leu is L-leucine, Lys is L-lysine, Gln is L-glutamine, He is
L-isoleucine and Pro each mean proline.

[0008] The novel method for producing the peptide of the present invention will be explained below. The method of the present invention is a novel method for producing peptides, and although it may require the synthesis of structural genes that do not exist in nature, the present inventor has already synthesized the DNA encoding the amino acids constituting the desired peptide.
Among the sequences, we selected sequences that are highly utilized in E. coli, and also ensured that the regions rich in A-T base pairs are not consecutive, and that the synthetic fragments that are the parts for synthesizing this structural gene are intramolecularly self-complementary. We have established a method for obtaining a desired structural gene by designing the gene by taking into account that it does not have any specific base sequences, does not have repetitive sequences, and has a size of about 30 bases.

The structural gene thus obtained is tr
A desired plasmid can be obtained by constructing a gene corresponding to a part of the galK protein with the p promoter upstream and downstream thereof on an existing plasmid that is expressed in prokaryotic cells. As the existing plasmid, it is preferable to use a pBR-based plasmid or a pUC-based plasmid, but it is more preferable to use a pUC-based plasmid since there is no need to use indole acrylic acid (IAA).

For transformation, E. coli strain RR1Δ
M15 (ATCC35102), JM101, JM10
Derivatives of E. coli K12 strain such as E. coli K12 may be used, but especially when considering fed-batch culture, it is more preferable to use a strain that does not require amino acids, such as W3110 (IF012713).

[0011] In order to produce peptides by culturing,
L-medium, M9 medium, M9-casamino acid medium, high phosphate medium, high phosphate-casamino acid medium, etc. can be used in the usual culture method, and when pBR-based plasmids are used,
Although the fusion protein can be mass-produced by adding indole acrylic acid (IAA) and further culturing, it is more preferable to use a fed-batch culture method that allows culturing at high density in a small amount of medium.

[0012] The fusion protein expressed in E. coli can be obtained as a water-insoluble fraction by ultrasonic disruption of E. coli and then centrifugation, and by this procedure, it can be separated from many water-soluble proteins in E. coli. is possible. In order to decompose the fusion protein and excise the desired peptide, the Met bond of the fusion protein purified as described above is decomposed using brom cyanide.

The compound of the present invention can be purified from the peptide mixture obtained by decomposing the fusion protein as described above by subjecting it to cation exchange chromatography and reversed phase chromatography. The thus obtained peptide of the present invention can be confirmed by amino acid composition analysis, amino acid sequence analysis by Edman degradation, and immunoassay using anti-VIP antibodies. EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples.

[0014]

[Example] i) Preparation of recombinant ■ Preparation of pGMVG1/RR1ΔM15 Plasmid pV
3 μg of IPGP1 (described in Japanese Patent Application No. 2-81029) was added to a high salt concentration buffer (10 mM Tris-HCl (pH 7.5), 1
Dissolve in 0mM MgCl2, 1mM DTT (dithiothreitol), 100mM NaCl) and add 40 units of restriction enzyme EcoRI and 4 units of restriction enzyme SspI.
0 unit was added and the reaction was carried out at 37°C for 2 hours. The reaction solution is 1
% agarose electrophoresis, and a band of approximately 0.6 kb was adsorbed onto NA-45 paper (manufactured by S&S). This paper was added to DNA extraction buffer (1M NaCl,
20mM Tris-HCl (pH 8.0), 0.1mM E
DTA) by heating at 65°C for 30 minutes.
Fragments were extracted. This solution was stirred with phenol and then chloroform, and the upper layer was separated with Sephadex G.
Desalted at −25°C.

The obtained DNA fragment was kleno
w buffer (50mM Tris-HCl (pH 7.2), 1
0mM MgSO4, 0.1mM DTT, 50μ
kle in g/ml BSA, 0.2mM dNTPs)
One unit of now fragment was added, and the mixture was reacted at 37°C for 40 minutes. This reaction solution was heated at 65°C for 15 minutes to inactivate the enzyme, then desalted with Sephadex G-50, and pVI
A PGP1 (E-Ss-klenow) fragment was obtained. Plasmid pDR540 (manufactured by Pharmacia) 2μ
g of high salt concentration buffer (10mM Tris-HCl (pH 7)
．． 5), 10mM MgCl2, 1mM DTT,
Dissolve in 100mM NaCl) and add restriction enzyme M
50 units of luI were added and the mixture was reacted at 37°C for 2 hours. This reaction solution was heated at 65° C. for 15 minutes to inactivate the enzyme, and then desalted using Sephadex G-50. Obtained DNA
Fragments were prepared in k1enow buffer (50mM Tris-HCl (pH 7.2), 10mM MgSO4, 0.5mM Tris-HCl (pH 7.2), 10mM MgSO4,
One unit of the Klenow fragment was added in 1mM DTT, 50μg/ml BSA (baby serum), 0.2mM dNTP, and reacted at 30°C for 30 minutes. This DNA fragment was desalted with Sephadex G-50.

The obtained DNA fragment was dissolved in alkaline phosphatase buffer (50mM Tris-HCl (
One unit of alkaline phosphatase was added to the mixture (pH 8.0), 0.1 mM EDTA), and the mixture was reacted at 37°C for 1 hour. The enzyme was inactivated by adding phenol to the reaction solution and stirring. The upper layer after centrifugation was further stirred with chloroform and centrifuged to remove phenol. 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol were added to the upper layer, and pDR540 (
M-klenow-Alp) fragment was purified.

Two types of DNA fragments prepared,
pVIPGP1 (E-Ss-klenow) fragment 36ng and pDR540 (M-klenow-A
90 ng of lp) fragments were mixed in ligation buffer (66 mM Tris-HCl (pH 7.6),
6.6mM MgCl2, 10mM DTT, 0.
The reaction was carried out at 16°C for 20 hours using 350 units of T4-DNA ligase in 1mM ATP). Using this reaction solution, Escherichia coli strain RR1ΔM15 was transformed by the Hanahan method according to a conventional method, and the desired transformant pGMVG1/RR1ΔM15 was obtained on an L-plate containing 50 μg/ml ampicillin. The insertion direction of the inserted gene was confirmed by the cleavage pattern with the restriction enzyme HindIII.

■pGMVG1-trp/C600gal
Preparation of K- Plasmid pGMVG (12 μg) was added to a high salt concentration buffer (10 mM Tris-HCl (pH 7.5), 10 m
M MgCl2, 1mM DTT, 100mM
Dissolve in NaCl) and add restriction enzyme BamHI 5 to this.
Add 0 units and 100 units of restriction enzyme ScaI,
The reaction was carried out at 7°C for 75 minutes. The reaction solution was separated by 0.7% low melting point agarose electrophoresis, and a band of approximately 3.5 kb was excised. This gel fragment was dissolved by heating at 65°C for 5 minutes, phenol was added, stirred, and the upper layer after centrifugation was further stirred and centrifuged with phenol-chloroform and chloroform. Sodium acetate and 2.5 times the amount of ethanol were added, and the target pGMVG1 (E-Sc) fragment was obtained by ethanol precipitation.

[0019] 2 μg of plasmid pDR720 (manufactured by Pharmacia) was mixed with high salt concentration buffer (10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM
DTT, 100mM NaCl), 40 units of restriction enzyme BamHI and 80 units of restriction enzyme ScaI.
The unit was added and reacted at 37°C for 70 minutes. The reaction solution was 0.
Separated by 7% low melting point agarose electrophoresis, approximately 0.9 kb
I cut out the band. This gel fragment was dissolved by heating at 65°C for 5 minutes, phenol was added, stirred, and the upper layer after centrifugation was further stirred and centrifuged with phenol-chloroform and chloroform. The target pDR720 (E
-Sc) fragment was obtained.

Two kinds of DNA fragments prepared, pG
20 ng of MVG1 (E-Sc) fragment and pDR7
100 ng of the 20(E-Sc) fragment were mixed in ligation buffer (66 mM Tris-HCl (pH 7).
．． 6), 6.6mM MgCl2, 10mM DTT, 0.
The reaction was carried out at 16°C for 24 hours using 700 units of T4-DNA ligase in 1mM ATP). Using this reaction solution, E. coli strain C600galK was transformed by the Hanahan method according to a conventional method, and the desired transformation was carried out on an L-plate containing 50 μg/ml ampicillin and 2.5 μg/ml tryptophan. body pGMV
G1-trp/C600galK- was obtained. Gene insertion was confirmed by digestion with restriction enzymes HindIII and PstI.

■pGMVGH1-trp/C600ga
Preparation of lK- (1) Preparation of pUCSTP1 Plasmid pSTP1 (described in Japanese Patent Application No. 2-81029)
5 μg was added to a high salt concentration buffer (10 mM Tris-HCl (pH 7).
．． 5), 10mM MgCl2, 1mM DTT,
Dissolve in 100mM NaCl) and add restriction enzyme E to this.
coRI 100 units and restriction enzyme ScaI 20
0 unit was added and the reaction was carried out at 37°C for 2 hours. The reaction solution is 1
% low melting point agarose electrophoresis, and a band of approximately 0.9 kb was excised. This gel fragment was dissolved by heating at 65°C for 5 minutes, phenol was added, stirred, and the upper layer after centrifugation was further stirred and centrifuged with phenol-chloroform and chloroform. Add sodium acetate and 2.5 times the amount of ethanol,
The desired DNA fragment was obtained by ethanol precipitation. The obtained DNA fragment is kleno
w buffer (50mM Tris-HCl (pH 7.2), 10mM
M MgSO4, 0.1mM DTT, 50μg/
kle in ml BSA, 0.2mM dNTPs)
Four units of now fragment were added and incubated at 37°C for 30 minutes. After adding phenol to the reaction solution and stirring to inactivate the enzyme, the upper layer was further treated with chloroform to remove phenol, and finally desalted with Sephadex G-50 to obtain the desired pSTP1 (E- Sc-
klenow) fragment was obtained.

[0022] 20 μg of plasmid pUC18 was added to a high salt concentration buffer (10 mM Tris-HCl (pH 7.5),
10mM MgCl2, 1mM DTT, 100m
Dissolve in M NaCl) and add restriction enzyme PvuII to this.
100 units and 100 units of restriction enzyme ScaI were added and reacted at 37°C for 2 hours. The reaction solution was separated by 1% low melting point agarose electrophoresis, and a band of approximately 1.5 kb was excised. This gel fragment was dissolved by heating at 65°C for 5 minutes, phenol was added, stirred, and the upper layer after centrifugation was further stirred and centrifuged with phenol-chloroform and chloroform. Sodium acetate and 2.5 times the amount of ethanol were added, and the target pUC18 (P-Sc) fragment was obtained by ethanol precipitation. Two types of DNA fragments prepared,
pSTP1 (E-Sc-klenoW) fragment 1
00ng and pUC18 (P-Sc) fragment 500
Both ng are ligation buffer (66mM Tris-HCl (pH 7.6), 6.6mM MgCl2,
T4 in 10mM DTT, 0.1mM ATP)
- 16°C using 350 units of DNA ligase, 21.5
Allowed time to react. Using this reaction solution, h
E. coli strain RR1ΔM15 was transformed by the anahan method, and the desired transformant pUCSTP1/RR was transformed on an L-plate containing 50 μg/ml ampicillin.
1ΔM15 was obtained. The insertion direction of the inserted gene is determined by restriction enzymes EcoRI and E
Confirmed by coRI-ScaI cleavage pattern

(2) Preparation of pGMVGH1-trp 13 μg of plasmid pUCSTP was added to medium salt concentration buffer (10 m
M Tris-HCl (pH 7.5), 10mM MgC
12, 1mM DTT, 50mM NaCl), 50 units of restriction enzyme HindIII was added thereto, and the mixture was reacted at 37°C for 2 hours. The reaction solution was separated by 0.7% low melting point agarose electrophoresis, and a band of approximately 2.4 kb was excised. The gel fragments are heated at 65°C for 5 minutes to dissolve the gel, phenol is added and stirred, and the upper layer after centrifugation is further stirred and centrifuged with phenol-chloroform and chloroform, and the amount is 1/10 of the upper layer. Add 3M sodium acetate and 2.5 times the amount of ethanol,
The desired DNA fragment was obtained by ethanol precipitation. The obtained DNA fragments were added to alkaline phosphatase buffer (50 mM Tris-HCl (pH 8.
2 units of alkaline phosphatase were added in 0.0) and 0.1 mM EDTA), and the mixture was reacted at 37°C for 1 hour. After inactivating the enzyme by adding phenol to the reaction solution and stirring, the upper layer is further stirred with phenol-chloroform and chloroform, centrifuged, and the upper layer is desalted with Sephadex G-50 to obtain the desired pUCSTP.
1(H-Alp) fragment was obtained. plasmid pG
5 μg of MVG1-trp was added to medium salt concentration buffer (10 m
M Tris-HCl (pH 7.5), 10mM MgC
12, 1mM DTT, 50mM NaCl), 150 units of restriction enzyme HindIII was added thereto, and the mixture was reacted at 37°C for 2 hours. The reaction solution was separated by 1% low melting point agarose electrophoresis, and a band of approximately 1.2 kb was excised. This gel fragment was dissolved by heating at 65°C for 5 minutes, phenol was added, stirred, and the upper layer after centrifugation was further stirred and centrifuged with phenol-chloroform and chloroform. Add sodium acetate and 2.5 times the amount of ethanol, and perform ethanol precipitation to obtain the desired pGMVG1-trp(H).
I got the fragment.

Two kinds of DNA fragments prepared, pG
MVG1-trp(H) fragment 150ng and pU
80 ng of CSTP1 (H-Alp) fragments were mixed together in ligation buffer (66 mM Tris-HCl (pH
7.6), 6.6mM MgCl2, 10mM D
T4-DNA ligase in TT, 0.1mM ATP)
The reaction was carried out using 700 units at 16° C. for 20.5 hours. Using this reaction solution, Escherichia coli strain C600galK- was transformed by the Hanahan method according to a conventional method, and 50μ
The desired transformant pGMVGH1-trp/C600galK was obtained on an L-plate containing g/ml ampicillin and 25 μg/ml tryptophan. Insertion of the gene and its direction are determined using the restriction enzyme HindII.
This was confirmed by cleavage with I and BamHI-ScaI.

■ Preparation of pGMVGH1-trp/W3110 Transformant pGMVGH1-trp/C600galK
Using the plasmid pGMVGH1-trp extracted from
3110, and transform the desired transformant pGMVGH1-t onto an L-plate containing 50 μg/ml ampicillin and 100 μg/ml tryptophan.
rp/W3110 was obtained. Plasmids were confirmed by restriction enzyme digestion.

ii) Expression and confirmation of fusion protein ■Fusion protein (
Confirmation of pGMVG1/RR1ΔM15) Recombinant pG
MVG1/RR1ΔM15 with ampicillin 50μg/m
The cells were cultured overnight at 37°C in L-medium containing l. 50 μl of this culture solution was freshly cultured at 37° C. in 10 ml of L-medium containing 50 μg/ml of ampicillin. O. D.
When 550 = 0.5 to 0.7, add IPTG to a final concentration of 1mM, and then add 5.
After culturing for 5 and 22 hours, 500 μl of this culture solution was centrifuged to collect bacterial cells. Add SDS-PAGE buffer to the cells, heat at 95°C for 5 minutes, and then add 12.5% SDS-PAGE.
I did AGE.

When this gel was stained with CBB (Coomassie Brilliant Blue-R-250) and decolorized, a new band with a molecular weight of about 33,000 was detected. On the other hand, a gel separated from the same sample was electroblotted onto nitrocellulose paper, and mouse anti-VIP antiserum and anti-mouse IgG conjugated with alkaline phosphatase were used.
When Western blotting was carried out using CBB staining, only the same band as that detected by CBB staining was colored. By the method of the present invention, the desired peptide can be converted to gal K
It was confirmed that 10 to 15% of the protein was expressed as a fusion protein.

■Fusion protein (pGMVG1-trp/C6
Confirmation of recombinant pGMVG1-trp/C600galK-
ampicillin 50μg/ml, tryptophan 2.5
The cells were cultured overnight at 37°C in L-medium containing μg/ml and M9 Casamino Acid medium. 50-100 μl of this culture solution
was freshly added to 10 ml of each medium containing 50 μg/ml ampicillin and 2.5 μg/ml tryptophan at 37°C.
It was cultured in O. D. When 550 = 0.5 to 0.6, IAA (indole acrylic acid) was added to a final concentration of 20 μg/ml, and further 5.
After culturing for 23.5 hours, 500 μl of this culture solution was centrifuged to collect bacterial cells. Add SDS-PAGE buffer to the cells, heat at 95°C for 5 minutes, and then add 12.5% SDS-PA.
I did GE. When this gel was stained with CBB and then decolored, a band with a molecular weight of about 33,000, which was the same as the band detected with pGMVG1/RR1ΔM15 in the previous section, was detected. The fusion protein accounted for 50 to 70% of the total protein in E. coli. It was confirmed that the desired peptide was expressed in large amounts as a fusion protein with galK protein by the method of the present invention.

■Fusion protein (pGMVGH1-trp/C
Confirmation of recombinant pGMVGH1-trp/C600galK-)
- ampicillin 50 μg/ml, tryptophan 10
3 in high concentration phosphocasamino acid medium containing 0 μg/ml.
Cultured overnight at 0°C. Add 50 μl of this culture solution to 50 μg/ml of ampicillin and 100 μg of tryptophan.
The cells were cultured at 30° C. in 10 ml of high-concentration phosphocasamino acid medium containing /ml. O. D. When the temperature reached 550=about 0.5, the culture temperature was increased to 37° C., and the culture was further continued for 20 hours. 500 μl of this culture solution was centrifuged to collect the bacterial cells. Add SDS-PAGE buffer to this bacterial cell and
After heating at ℃ for 5 minutes, 12.5% SDS-PAGE was performed. When this gel was stained with CBB and then decolored, a band with a molecular weight of approximately 33,000, which was the same as the band detected with pGMVG1/RR1ΔM15 in the previous section, was detected. The fusion protein accounted for 50 to 70% of the total protein in E. coli. It was confirmed that the desired peptide was expressed in large amounts as a fusion protein with galK protein by the method of the present invention, and without using IAA to induce protein expression, by a temperature shift of 30 to 37°C.

■Fusion protein (pGMVGH1-trp/W
3110) confirmed recombinant pGMVGH1-trp/W3110 was mixed with ampicillin 50 μg/ml and tryptophan 500 μg/ml.
The cells were cultured overnight at 30°C in FB-B medium containing ml. Add 50 μl of this culture solution to 50 μg/ml of ampicillin.
, and cultured at 30°C in FB-B medium containing 500 μg/ml tryptophan. O. D. When 550=approximately 0.5, the culture temperature was increased to 37°C and cultured for 23 hours. 500 μl of this culture solution was centrifuged to collect cells, and SDS-PAGE buffer was added to the cells, and the cells were heated at 95°C for 50 minutes.
After heating for 1 minute, 12.5% SDS-PAGE was performed. After staining this gel with CBB and decolorizing it, the pG
A band with a molecular weight of about 33,000, which is the same as the band detected in MVG1/RR1ΔM15, was detected. The fusion protein accounts for 50% of the total protein in E. coli.
It was ~70%. By the method of the present invention, C600ga
Even in Escherichia coli strain W3110, which is different from lK-, it was confirmed that the desired peptide was expressed in large amounts as a fusion protein with galK protein, and without using IAA to induce protein expression, by a temperature shift of 30 to 37°C. Ta.

iii) Peptide VIPGPEA (17-
Production of VIPGPEA) Purification of recombinant pGMVG1-trp/C600
gaK- was cultured overnight at 37°C in a high concentration phosphocasamino acid medium containing 50 μg/ml ampicillin and 25 μg/ml tryptophan. Add 70 ml of this culture solution to a new 10 liter fermenter with 50 μl of ampicillin.
The cells were cultured at 37° C. in 7 liters of high concentration phosphocasamino acid medium containing g/ml. O. D. When 550 = approximately 0.5, IAA (indole acrylic acid) was added to the final concentration of 20 μg/ml, and the mixture was further cultured overnight. This culture solution was centrifuged (×8000g
, 4° C., 15 minutes) and collected bacterial cells (wet weight: 48.3 g).

[0032] The collected cells were mixed with about 250 ml of a cell disruption buffer (10mM Tris-HCl (pH 8.0), 1
(mM MgCl2) and disrupted the bacterial cells using a high-pressure homogenizer minilab (6,000 psi, x 5
)did. The cell suspension was centrifuged (×15,400g,
(4°C, 30 minutes) and separated into a soluble fraction and an insoluble fraction. By analyzing both fractions by SDS-PAGE, it was confirmed that the target fusion protein was present in the insoluble fraction. 19.4 g (wet weight, from 6 liter culture) of this insoluble fraction was dissolved in 400 ml of 70% formic acid, 12.4 g of cyanogen bromide was added and
Stirred for 4 hours. The reaction solution was concentrated under reduced pressure using an evaporator, diluted with water, and freeze-dried. This freeze-dried powder8.
3g to 400ml of 6M urea/100mM phosphoric acid (
pH 6.5), diluted 4 times with water, and the soluble fraction was collected by centrifugation (×5,000 g, 4° C., 10 minutes).

The soluble fraction (approximately 1.61) has a phosphoric acid concentration of 4
After adjusting to 0mM, N
Purified by aCl 0-0.5M salt gradient. VI
Approximately 400 ml of the column fraction containing PGPEA was
Reversed phase column (1.6 x 10 cm, YMC, AM type, particle size 10 μm, pore size 120 angstroms,
After desalting and concentrating using C18), it was newly subjected to reverse phase high performance liquid chromatography (YMC-AM324) for
Separation and purification was performed using a ~35% acetonitrile concentration gradient. The purified VIPGPEA was 160.2 mg (purity 99
．． 6%) was obtained. The amino acid sequence of VIPGPEA was converted to H-His-S by a peptide sequencer.
er-Asp-Ala-Val-Phe-Thr-As
p-Asn-Tyr-Thr-Arg-Leu-Arg
-Lys-Gln-Leu-Ala-Val-Lys-
Lys-Tyr-Leu-Asn-Ser-Ile-L
eu-Asn-Gly-Pro-Glu-Ala-OH
It was confirmed that

iv) Culture method ■ Fed-batch culture using pGMVG1-trp/C600gaK- Recombinant pGMVG1-trp/C600ga
K-, ampicillin 0.1 g/l, tryptophan 0.
1g/l, leucine 0.5g/l, threonine 3g/1
, 3 in FB-B medium containing 0.1 g/l thiamine hydrochloride.
Cultured overnight at 0°C. Add 500 ml of this culture solution to a 10 liter fermenter and add 0.05 g of ampicillin.
/l, tryptophan 0.3g/l, leucine 1g/l
The cells were added to 5 liters of FB-B medium containing 6 g/l of threonine and 0.1 g/l of thiamine hydrochloride and cultured at 37°C. Stirring speed is 200-900 rpm, ventilation speed is 5-900 rpm.
The dissolved oxygen concentration was maintained within a range of 15 l/m. Further, the pH of the culture solution was controlled to 7.0 with 5M NaOH. From 3 hours after the start of culture, ampicillin 0.0
．． 1g/l, tryptophan 1g/l, leucine 5g/l
l, threonine 30g/l, thiamine hydrochloride 0.1g/l
FB-F medium containing was gradually added, and culture was continued. 0.5 g of IAA was added 9.5 hours after the start of the culture, and the culture was continued, and the culture was terminated 14 hours after the start of the culture. Cultured bacterial cells were centrifuged (×8,000g, 10 minutes, 4℃
) (total wet weight 751 g, dry weight 23 g
/l). SDS-PAGE a part of cultured bacterial cells at each time
After adding buffer solution and heating at 95℃ for 5 minutes, 12.5% SD
S-PAGE and CBB staining confirmed that 40-60% of the fusion protein was expressed after 14 hours.

■ Fed-batch culture using pGMVGH1-trp/W3110 The recombinant pGMVGH1-trp/W3110 was cultured overnight at 30°C in FB-B medium containing 0.1 g/l ampicillin and 0.5 g/l tryptophan. . Add 500 ml of this culture solution to a 10 liter fermenter and add 0.05 g/l of ampicillin and 1 g/l of tryptophan.
The cells were added to 5 liters of FB-B medium containing 5 liters of FB-B medium and cultured at 30°C. The stirring rotational speed was controlled within the range of 200 to 900 rpm, and the aeration rate was controlled within the range of 5 to 15 l/m to maintain the dissolved oxygen concentration. In addition, the pH of the culture solution was adjusted to 7.0 with 10M NaOH.
was controlled. From 6.1 hours after the start of culture, ampicillin 0.1 g/l and tryptophan 5 g/l were added while measuring the turbidity (O.D. 550) of the culture medium, the glucose concentration in the medium, and the acetate concentration produced. FB containing l
-F medium was gradually added and culture was continued. Start of culture 8.8
After ~9.4 hours, the culture temperature was raised from 30°C to 37°C, and the culture was continued. The culture was terminated 13 hours after the start of the culture. The cultured bacterial cells were centrifuged (×8,000
g, 10 min, 4°C) (total wet weight 217 g
dry weight 9.6 g/l). SDS-PAGE buffer was added to a portion of the cultured cells at each time, and after heating at 95°C for 5 minutes, 12.5% SDS-PAGE was performed and CBB staining was performed. After 13 hours, more than 60% It was confirmed that the fusion protein was expressed, and that expression could be effectively induced by increasing the culture temperature from 30°C to 37°C.

[0036]

[0037]

[0038]

[0039]

[0040]

Effects of the Invention The method for producing peptides of the present invention can be used to produce peptides with a molecular weight of 2,000 to 10,00 that do not contain L-methionine in the molecule.
This is an excellent method for inexpensive mass production of 00 peptides.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the construction of a plasmid encoding the galK-VIPGPEA fusion protein. Details of the construction are described in the Examples. Plasmid pVIPGP1D
The NA fragment (containing the VIPGPEA gene and the rrnB terminator region derived from pKK22-3) was transformed into pDR540.
galK protein 2 by introducing it into the MluI site of
VIPGPEA is added to the C-terminal portion of a total of 266 amino acid residues, including 64 amino acid residues and 2 amino acid residues derived from the restriction enzyme site.
A plasmid pGMVG1 is constructed which encodes a fusion protein in which the two are connected via Met. This plasmid pG
The expression of MVG1 is controlled by the tac promoter. In addition, the promoter region of pGMVG1 was transferred to pDR7
pGMVG1-trp by replacing with 20
can be configured. The expression of this plasmid pGMVG1-trp is controlled by the trp promoter.

[Figure 2] (pGM of pGMVG1-trp (pBR system)
It is a diagram showing conversion to VGH1-trp (pUC system). ) The HindIII short fragment of pGMVG1-trp was isolated from the trp promoter to VIPG
It contains DNA up to the PEA gene, but the terminator region is deleted. Therefore, pUC-based plasmid p
pUCSTP1 is constructed by introducing into UC18 the EcoRI-ScaI fragment of pSTP1 having the same terminator as pGMVG1-trp. pGMVG1,t shown earlier in this pUCSTP1
By introducing the HindIII short fragment of rp, pGMVGH1-trp is constructed. pGMVG1-trp and pGMVGH1-trp both have the same trp promoter, galK-VIPGPEA
It has a gene and a terminator region, and the only difference is whether the underlying plasmid is pBR or pUC.

Claims (3)

[Claims] Claim 1: A new method for producing a peptide with a molecular weight of 2,000 to 10,000 that does not have L-methionine in the molecule, using a trp promoter to
C of galK protein equivalent to 00 to 300 amino acid residues
At the end, L-methionine and the above molecular weight 2,000 to 10
,000 peptides linked together is expressed in prokaryotic cells, the fusion protein is purified, and the peptide mixture obtained by decomposition with bromcyanide is further purified to produce the above-mentioned peptides with a molecular weight of 2,000 to 10,000. Method. 2. A peptide having a molecular weight of 2,000 to 10,000 and having no L-methionine in the molecule has the following amino acid sequence H-His-Ser-Asp-Ala-Val.
-Phe-Thr-Asp-Asn-Tyr-Thr-
Arg-Leu-Arg-Lys-Gln-Leu-A
la-Val-Lys-Lys-Tyr-Leu-As
n-Ser-Ile-Leu-Asn-Gly-Pro
-R (wherein R represents OH, Glu-OH, or Glu-Ala-OH), the method of (1) above. [Claim 3] (1) above, wherein the culture method is fed-batch culture.
the method of.