JPH03280893A - New production of peptide - Google Patents

Abstract

NEW MATERIAL:A peptide having an amino acid sequence expressed by the formula (Hse is homoserine). USE:Production of vasoactive intestinal polypeptide(VIP) derivatives, etc. PREPARATION:A gene is inserted into the C-terminal of a structural gene of human TNF and expressed as a fused protein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a novel method for producing peptides.

[Prior Art and Problems] Currently, bioactive peptides are generally obtained by extracting and separating them from nature, by organic chemical synthesis, or by manufacturing methods using genetic recombination. There is.

However, it is not cost-effective to produce bioactive peptides with a molecular weight of 2,000 to 10,000 in large quantities by extraction from animal organs or organic chemical synthesis, and production by genetic recombination is also not possible. When expressed in prokaryotic cells, it is degraded and difficult to obtain, so it cannot be obtained using inefficient production methods such as producing a fusion protein with β-galactosidase etc. in advance and then degrading and purifying it. I had no choice.

[Means for Solving the Problems] As a result of intensive studies on production methods using genetic recombination with high economic efficiency, the present inventors found that a method of production using genetic recombination with high economic efficiency has been developed. In the production of 10,000 Pe.
Instead of the usual fusion protein production method in which L-methionine and the above-mentioned peptide with a molecular weight of 2,000 to 1000 are arranged, an amino acid sequence corresponding to the collagenase recognition site is arranged at the C-terminus, and this L-methionine leads to the collagenase recognition site. Expressing a fusion protein in which the sequences are repeatedly linked about 1 to 50 times in prokaryotic cells, purifying the fusion protein,
From the peptide mixture obtained by decomposition and digestion with bromcyan and collagenase, the above molecular weight 20
They discovered that mass production is possible by purifying 00 to 10,000 peptides, and completed the present invention.

That is, the present invention is directed to a compound having a molecular weight of 2,000 to 1,000 that does not have L-methionine or a collagenase recognition site in its molecule.
This is a novel method for producing a peptide of 0, in which L-methionine, the above-mentioned peptide with a molecular weight of 2,000 to 10,000, and an amino acid sequence corresponding to a collagenase recognition site are added to the C-terminus of a polypeptide that can be expressed in prokaryotic cells. This is a method in which a fusion protein linked several times is expressed in prokaryotic cells, the fusion protein is purified, and the peptide mixture obtained by decomposition and digestion with bromcyan and collagenase further purifies the above-mentioned peptide with a molecular weight of 2,000 to 10,000. .

In the method of the invention described above and in the following description,
His is L-histidine, Ser is L-serine, A
sp is L-aspartic acid, Aha is L-alanine, Val is L-valine, Phe is L-phenylalanine, Thr is L-threonine, Asn is L-asparagine, Tyr is L-tyrosine, Arg represents L-arginine, Leu represents L-leucine, Lys represents L-lysine, Gln represents L-glutamine, 11e represents L-inleucine, and Pro represents proline, respectively.

Below, a novel method for producing the peptide of the present invention will be explained.

l) Structural gene i) Gene design The method of the present invention is a new method for producing peptides, in which a fusion protein that does not exist in nature is produced in advance, and the desired peptide is obtained by decomposing it. A gene and a gene having a collagenase recognition site must be synthesized. Among the DNA sequences encoding the amino acids constituting the desired peptide, a sequence that is highly utilized by E. coli is selected, and A-T base pairs are selected. The enriched regions are not continuous, and the synthetic fragments that are the parts for synthesizing this structural gene do not have intramolecular self-complementary base sequences, do not have repetitive sequences, and are approximately 30 bases in size. Design with this in mind.

Therefore, preferred specific examples of the structural gene of the desired novel peptide used in the present invention and the gene having a collagenase recognition site connected thereto are those having the nucleotide sequence shown in the Examples below (in the figure, this structural gene is , His
from the CAC corresponding to Asn to the AAC portion corresponding to Asn, and the collagenase recognition site extends to GGTCCG leading to it).

As a method for expressing this structural gene and a gene having a collagenase recognition site, for example, this gene can be inserted into the C-terminus of the human TNF structural gene and expressed as a fusion protein.

That is, first, a Met (L-methionine) codon for cyanogen bromide decomposition is provided at the 5' end of the structural gene.

ii) Synthesis To synthesize the gene designed as described above, for each of the ten-rearrangements, divide it into several seven fragments, chemically synthesize these, and combine each of the seven fragments. According to

Each strand consists of 18 to 30 bases, and is preferably divided into 7 fragments of 8 degrees of freedom, each of which overlaps by 10 to 12 bases.

Methods for synthesizing each of the seven fragments include a solid phase method and a liquid phase method, but solid phase synthesis using a phosphoramidite method is most preferred.

1ii) Purification When synthesizing a 7-ragment, the longer the chain length, the more impurities there will be, making purification generally difficult. Oligonucleotides with hydroxyl-protecting groups were purified by reversed-phase high-performance liquid chromatography (reverse-phase HPLC).
This can be achieved by fractionating by LC), removing the protecting group, further fractionating by using anion exchange column chromatography, and then desalting.

iv) Each of the 7 fragments except the phosphorylated and linked 5'-terminal fragments are T4-
After phosphorylating with kinase and inactivating the enzyme in each reaction, the 5′-terminal fragment was added.
Mixed and desalted. The desalted 7-lactone is concentrated and 9
It is possible to obtain a gene fragment having a structural gene and a collagenase recognition site by heating it at 0 to 100'O, slowly cooling it to anneal it, and then reacting it with T I-DNA ligase to bind it. .

V) Preparation of a vector having a human TNF expression system In the present invention, for example, various vectors that have an expression system such as a TNF structural gene derived from human chromosomal DNA and a promoter for its expression and that can be propagated in E. coli are used. Plasmids can be used, and these plasmids can be prepared according to known conventional methods.

That is, an appropriate location in the structural gene of human TNF (in particular, C
A plasmid that expresses a fusion protein with human TNF is created by inserting the structural gene of the peptide of interest and a gene having a collagenase recognition site into a desired terminal region.

3) Plasmid recombination molecule For example, the above-mentioned human TNF structural gene is separately linked to the downstream of the expression system including the promoter to the structural gene of the target peptide and a gene having a collagenase recognition site. By integrating it into a plasmid prepared in
Construct a plasmid recombinant molecule for the entire fusion protein.

4) Transformation i) Host cell The host cell to be transformed using a plasmid recombinant molecule incorporating the gene of the fusion protein thus obtained, such as pTGV8, is E. coli obtained RRIAM15 (AT
A lac iq host that is a derivative of Escherichia coli K12 strain such as CC35102), JMIol, or JM105 and has high lactose operon repressor activity is desirable.

ii) Transformation Transform by conventional methods.

5) Production of peptide j) Cultivation of transformants For culturing transformants, L-medium, M9 medium,
M9-casamino acid medium, high phosphate medium, high phosphate-casamino acid medium, etc. can be used, and by adding isopropylthiogalactoside (IPTG) to this and further culturing, the fusion protein can be mass-produced. can.

ii) Purification of fusion protein The fusion protein expressed in E. coli is purified by ultrasonic disruption of E. coli.
It can be obtained as a water-insoluble fraction by centrifugation, and by this operation it is possible to separate it from many water-soluble proteins in E. coli.

ii) Degradation of fusion protein In order to degrade the fusion protein and excise the desired peptide, the Met bond of the fusion protein purified as described above is degraded using brom cyanide, and the peptide is further digested with collagenase.

11]) Purification of the peptide of the present invention In order to purify the compound of the present invention from the peptide mixture obtained by decomposing the fusion protein as described above, the compound of the present invention can be purified by reverse phase HPLC and cation exchange high performance liquid chromatography (cation exchange high performance liquid chromatography). HPLC), but furthermore,
Efficient purification can be achieved by subjecting it to immunoaffinity column chromatography using an anti-VIP antibody.

iv) Confirmation of the peptide of the present invention The peptide of the present invention thus obtained 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.

[Example] Production of a structural gene of a desired peptide and a gene containing a BrCN reaction site and a collagenase recognition site but without a stop codon i) Synthesis of a fragment VIP derivative H-Hi 5 selected as a desired peptide
-3e r-As p-A] a-Va l-Phe
-Thr-Asp-Asn-Tyr-ThrArg-L
eu-Arg-Lys-Gln-Leu-A la-
Va 1-Ly 5-Ly 5-Tyr-Leu-
A s n-5e r-1] e-L e u-A s
A gene that contains the n-OH structural gene, BrCN reaction site, and collagenase recognition site and does not have a stop codon (
See FIG. 1 below, hereinafter referred to as gene 1. ) consists of the 8 fragments shown below, and the synthesis of these fragments was performed using DNA synthesis a! (Applied Biosystems, 380A) was used for solid phase synthesis by the phosphoramidite method.

1) 5'-AATTCA GCACTCTGA
CG2) 5'-CAGTGAATACAGCGT
CAGAGTGCATG3)5'-CTGTATTC
ACTGACAACTACACTCGTC4)5'-T
GTTTACGCA (1, ACGAGTCTAGTTC
,T5)5'-TGCGTAAAACAGCTGGCAG
TTAAG6)5'-TCAGGTATTTCTTA
ACTGCCAGC7)5'-AAATACCTGAA
CTCTATCCTGAACGGTCCGGA8)5
'-AGCTTCCGGACCGTTCAGGATAG
For the purification of 7 fragments of A (1, T or more), after completing the solid phase synthesis using a DNA synthesizer, the ammonia solution containing the obtained base and oligonucleotide with a 5' hydroxyl protecting group was heated at 55°C at 15°C. Heat for a while,
After the base protecting group was removed and the ammonia was distilled off under reduced pressure after the reaction, the oligonucleotide with only the 5' hydroxyl protecting group was fractionated by reverse phase HPLC. 80% of the 7 lactyone obtained.

The protective group for the 5' hydroxyl group was removed by treatment with acetic acid at room temperature for 15 to 30 minutes, and after distillation under reduced pressure, the anion exchange column (DEAE
-2iw) to obtain an oligonucleotide fraction. This was desalted using a gel filtration column (Sephadex G25) to obtain each fragment.

11) Phosphorylated 800 pmof2 each of the fragments 2) to 7) above in 60 WM Tris-HCl buffer (pH 7,5),
In addition to 10 iM M g C (22,15 cod, 2-mercaptoethanol, 0.08 i MATP (buffer solution 1), 25 units of T,-kinase (BR
(manufactured by Company L) was added thereto, and the mixture was reacted for 37° 01:15 hours.

After the reaction, heat at 65° for 15 minutes to inactivate the enzyme, then mix 1/2 volume and l of each reaction solution and 800 pmof2 each of the oligonucleotide fragments in 8), and transfer to a gel filtration column (Sephadex G50). ) and desalted.

111) After concentrating the fraction after binding and desalting of fragments using a centrifugal concentrator, further
01M Tris-HCl buffer (pH 7,6), 10 i
Add 25Δ of buffer consisting of M M g Cl22 and
After heating for 0.013 minutes, the mixture was slowly cooled and annealed. After cooling, dithiothreitol was added to the reaction solution to give a concentration of 201M, and then AT was added to the reaction solution to give a concentration of 1M.
P was added thereto, and the mixture was reacted at 16°C for 122 hours using 875 units of T4 DNA ligase (manufactured by Takara). After the reaction, the enzyme was inactivated by heating at 65°C for 15 minutes.

iv) The purified reaction solution was subjected to 8% polyacrylamide electrophoresis (PAGE) with a thickness of 2 mm, and a band of the desired length (100 bp) was cut out. NA-4 is electrically extracted from the cut out gel.
After adsorbing the DNA on 5 paper (manufactured by S&S), the paper was soaked in 1M NaCI2.20 Tris-HCl buffer (
Elution was carried out by heating at 65° C. for 50 minutes in a high salt concentration buffer consisting of 0.1 i MEDTA (pH 8.0).

This solution was mixed with 7 enol and phenol-chloroform,
After removing impurities with chloroform, 2.5 times the volume of ethanol was added to perform ethanol precipitation.

■) Phosphorylated thus obtained! = Genes are not phosphorylated at their 5'' ends, so they were mixed with 603!M Tris-HCl buffer (pH 7.5) for 10J!MgCQ2.15
In addition to a mixed solution consisting of 2-mercaptoethanol and 0.081 MATP, 62.5 units of T4 kinase (manufactured by BRL) were added, and the mixture was reacted at 37°C for 2.5 hours. After the reaction, heat at 65°C for 115 minutes to inactivate the enzyme, and then apply gel filtration column (Sephadex G50).
and further, plasmid pTZ18R30# was added to 1
(ljIM Tris-HCl buffer (pH 7,5), l
Oi + M M g CQ2.1
Add 400 units of indlII, 37°C, 2
After reacting for 30 minutes, the reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, a band of approximately 2.9 kb was cut out, the gel was dissolved by heating at 65°C for 5 minutes, phenol was added, and centrifugation was performed. After separation, the upper layer was further stirred using phenol chloroform and chloroform, and after centrifugation, 1/10 amount of 3M sodium acetate, 2.5
Add twice the amount of ethanol, perform ethanol precipitation, and
A fragment (pT218R(EH)) was obtained.

pTZ18R(E-H) 300xg and gene 111ng
in ligation buffer with T.

Using 350 units of DNA ligase, the reaction was carried out at 16'0° for 19 hours and 30 minutes, and the plasmid (pTZ18R-
A reaction solution containing gene 1) was obtained. Using this reaction solution,
According to a conventional method, E. coli strain RRIAM was extracted by the CaCu5 method.
Transformants were obtained as white colonies on a single plate containing 50 AII/mM ampicillin.

Furthermore, single-stranded DNA was extracted from the obtained transformant by a conventional method.
was extracted, the base sequence was confirmed using the dideoxy method, and the following gene fragment (VIPGP) AATTCATGCA was obtained.
CTCTGACGCTGTATTCACT(, ACAA
CTACACGTACGTGAGACTGCGACAT
AAGTGACTGTTGATGTGTCGTCTGC
GTAAACAGCTGGCAGTTAAGAAAATA
CCTGAACAGCAGACGCATTTGTCGA
CCGTCAATTCTTTATGGACTTGTCT
ATCCTGAACTGATAGAGATAGGACT
TGACTATCCTAG (VIPGF)-containing plasmid (pTZl8R-VIPGP
) was confirmed to be a plasmid containing the desired gene 1, and the recombinant RRIΔM15 (pTZ18R-VI
PGP) was obtained.

vi) Vector adjustment plasmid pKK223-3 (7 Almasia) 3
Skin and fin Tris-HCl buffer (pH 7,5) 101M
MgCI22, lxH dithiothreitol, 100x
In addition to the mixture consisting of 10Oxρ, restriction enzyme E
coRI 42 units and restriction enzyme H4ndm 6
After adding 0 units and reacting at 37°C for 2 hours and 40 minutes,
The reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, a band of about 4.5 kb was cut out, the gel was dissolved by heating at 65°C for 5 minutes, phenol was added, centrifuged, and the upper layer was further purified with phenol. - After stirring and centrifuging using chloroform and chloroform, add 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol to the upper layer,
Perform ethanol precipitation to obtain DNA fragment (pKK223-3
(E-H)) was obtained.

pKK223-3(E-H) 1100n is a DNA 7 fragment (contains 3 stop codons that are out of reading frame) prepared in advance using a DNA synthesizer, 9) 5'-AATTCAGATCTCCAAGCT
TAAGTGACTAGl 0 ) 5'-AGCTC
TAGTCACTTAAGCTTGAGATCTG6.
T, -D in ligation buffer with 7 ng
16°C using 350 units of NA ligase
The reaction was carried out for 117 hours. Using this reaction solution, Escherichia coli strain RRIΔM15 was transformed by the CaCO2 method according to a conventional method, and a transformant was obtained on a plate containing 50 Aa/mQ of ampicillin.

Furthermore, a plasmid (pSTPl) was extracted from the obtained transformant by the alkaline lysis method, and 10x MMg (12,1
After adding it to a mixture consisting of 1M dithiothreitol and 100xM10Ox, it was treated with restriction enzymes EcoR1, Hindnl and BgQl.
Confirm that each cut is made in one place, and cut the recombinant RRIΔ
It was confirmed as Ml 5 (psTP 1).

This plasmid (psTPl) contains about 2Ali, 11
01t Tris-HCl buffer (pH 7,5), LOINMg
Cl,,I J! M dithiothreitol, 1Ocl+
In addition to the mixture consisting of MNaCQ, restriction enzyme E
coRI 56 units and restriction enzyme HindIII
After adding 50 units and reacting for 2 hours at 37°O%,
The reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, the gel was dissolved by heating at 65°C for 5 minutes, phenol was added, centrifuged, and the upper layer was further mixed with phenol-chloroform and chloroform and stirred. After centrifugation, 1/10 volume of 3M sodium acetate and 2.5 equivalents of ethanol were added to the upper layer, and ethanol precipitation was performed to obtain a DNA fragment (pSTPI (E-H)).

In addition, plasmid (pTZ I 8R-VI PGP)
Approx.
lO*MMgcL, 1 toge dithiothreitol, 100z
In addition to the mixture consisting of M100z, restriction enzyme E
coRI 56 units and restriction enzyme HindIIl
After adding 50 units and reacting at 37°C for 2 hours, the reaction solution was separated by 3% low melting point agarose gel electrophoresis.
Melt the gel by heating for 5°05 minutes, add phenol, centrifuge, add phenol-chloroform and chloroform to the upper layer, stir, centrifuge, and add 1/10 amount of 3M sodium acetate to the upper layer. Add 2.54 volumes of ethanol and perform ethanol precipitation to obtain DNA fragments (
VIPGP) was obtained.

p STP 1 (E-H) about 50ng and VIP
Approximately 10 ng of CP was added to 50 volumes of M Tris-HCl buffer (pH
7,5) lO cod MgCQ2.101M dithioithreitol, 25Jl[MNaCI2, lzMATP, and in addition, T, -DNA ligase 17
Add 5 units and get 16°C! , and reacted for 18 hours. According to a conventional method, this reaction solution was used to transform E. coli strain RRIΔM15 by the CaCQ2 method to obtain a transformant.

Furthermore, the plasmid was extracted from the obtained transformant by the alkaline lysis method, added to a mixture consisting of 10z14 Tris-HCl buffer (pH 7.5), 1OzMMgcffz, 1 cod dithiothreitol, and loOzMNacff. Enzyme EcoRI and restriction enzyme H4nd
100bp of gene 1 can be obtained when IllI is produced, and it does not cut when treated with BgQI.
When treated with , Pvul[, it was confirmed that the recombinant RR1ΔMI5 (pVIPGPl) cut at two places.
(containing one VIPGP gene) was obtained.

vii) Construction of tandem vector Plasmid (pVIPGPl) Approximately 2 to
M Tris-HCl buffer (pH 7,5), 10 fin MgCQ,
, IIIM Dithiothreitol, 100zM100z
In addition to this, restriction enzyme BamHI
56 units and restriction enzyme HindI [150 units were added and allowed to react at 37° for 0.2 hours; after that, the reaction solution was diluted to 2%
Separated by low melting point agarose gel electrophoresis, approximately 0.5kb
I took the band. The gel was dissolved by heating at 65°C for 5 minutes, phenol was added, centrifuged, and the upper layer was further stirred using phenol-chloroform and chloroform.
After centrifugation, 171O amount of 3M sodium acetate and 2.5 times the amount of ethanol were added to the upper layer to perform ethanol precipitation, and the DNA fragment (pV I PGP 1 (B-H)
) was obtained.

In addition, approximately 24 liters of plasmid (pVIPGPl)
01M Tris-HCl buffer (pH 7,5), 10ff
M10ff, 11M dithiothreitol, 100
In addition to the mixture consisting of xMN10Ox, 56 units of the restriction enzyme EcoRI and the restriction enzyme BamHI3
After adding 0 units and reacting at 37° C. for 2 hours, the reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, and a band of about 4.4 kb was obtained. The gel was dissolved by heating at 65°C for 5 minutes, phenol was added, centrifuged, the upper layer was further stirred with phenol-chloroform and chloroform, and after centrifugation, 1/10 volume of 3M sodium acetate, 2 Add .5 times the amount of ethanol and perform ethanol precipitation to obtain a DNA fragment (pV I PGP I (
B-E)) was obtained.

pv I PGP 1 (B-H) about 5 ng and p
VIPGPI (B-E) Approximately 10 ng in advance,
DNA7 fragment ([-H linker-), 11) 5'-AGCTGTACTGCl2) prepared using a DNA synthesizer
T,-DNA ligase in ligation buffer with 7.4 ng of 5'-AATTGCAGTAC.
The reaction was carried out using 175 units at 16° C. for 16 hours and 30 minutes. Using this reaction solution, CaCQ,
E. coli strain RR1ΔM15 was transformed by
Transformants were obtained on L-plates containing ampicillin at Ili/m4.

Furthermore, a plasmid was extracted from the obtained transformant by the alkaline lysis method, and 10. wM Tris-HCl buffer (pH 7,5), 10 x HM g CQ2, ld
After adding dithiothreitol to a mixture of 100xMN10Ox, restriction enzyme EcoRI and restriction enzyme H
It was confirmed that a 221 bp gene fragment was obtained by cutting with indI[+ and that a 111 bp (7) gene fragment was obtained by cutting with pvu I[+, and the recombinant RRIΔM1
5 (pVIPGP2) (containing two VIPGP genes) was obtained.

This tandem vector production method was repeated to transform a plasmid (pV I PGF2)' in which eight structural genes of the desired peptide were linked in tandem into a recombinant RRIΔMI5 (pVIPGP 8).
Get it.

viii) Production of expression vector Here, in order to obtain the human TNF gene necessary for expressing the above tandem genes, a human lymphocyte DNA library (EMBL3 phage) was injected into Escherichia coli NM53g.
Using 25 LB plates, 3 x 104 PFU were seeded on each plate, and after culturing at 37°C overnight, the hephage was transferred to a nitrocellulose filter. On this transferred filter (7
) 7 arge to 1.5M NaC (1,0.5M NaO
The denatured filter was denatured with 1.5M NaC.
(2, Neutralized with 0.5M Tris-HCl solution (pH 8.0). After neutralization, phage DNA was fixed on the filter by vacuum drying at 80°C for 2 hours. (Ma
niatis T. , Molecular cloni
ng p331 (1982)) Furthermore, the base sequence of the human TNF gene that has already been reported (Diane Pennica, Nature, 312
, 724 (1984)) 1 3 ) 3'-GTTTGGGAGTT
CGACTCCCC: CGAGGTCA14) 3
″-GGGTAGATAGACCCTCCCCA15)
3'-CCCGTCCAGATGAAACCCTAG
Oligodeoxynucleotides of TAA were synthesized by the phosphoramidite method using a DNA synthesizer and used as a probe for the human genome TNF gene.

These oligodeoxynucleotides contain 100 pmo
100xM Tris-HCl (pH
8.0), 1. OJIM MgCO2, 5-state dithiothreitol, 50μCi (γ-32P)ATP
.

The reaction was carried out in a solution 30y1 consisting of 5 units of T4 polynucleotide kinase at 37°C for 45 minutes, and then phosphorylated by treatment at 65°C for 10 minutes to inactivate the enzyme.
Shipuko.

Labeled probes are separately associated with DNA-immobilized filters. The association reaction contained 7.5 μCi labeled probe and 0.9 M NaCL O. 09M Quen fiNa, 5x1 Denharz, 0.1% SDS, 20
0 skin/-48°C in 200 solutions of denatured salmon sperm DNA! , 16 hours, filter after reaction to 0.9
MNaC12, 0.09M Na citrate, 0.1% SD
40°C in a solution containing S, 15 minutes each for 3 days, and then 0.0
3M NaCQ, 0.003M citric FaNa% O.
Wash at 50°C for 3 days with a solution containing J% SDS.

Radioautograms were taken from the washed filters, and plaques that reacted to the probe were searched for by superimposing the radioautograms of two replica filters. By this method, one type of EMBL 37age reacting with the probe was obtained from 7.5xlO' plaques.

Phage DNA was extracted from phages using the glycerol glycerol step gradient method (Maniatis T.

Molecular cloning p83 (198
2)) Extraction and purification were performed. DNA with restriction enzyme Eco
After cutting with RI (Toyobo) and separating by 0.5% agarose gel electrophoresis, the DNA fragments were transferred from the agarose gel onto a nitrocellulose filter BA85 (S&S) using the Southern blot method. . The 2.9 kb DNA fragment reacted by associating the glove used for phage isolation with this filter was human T.
It completely contained the NF signal peptide to the stop codon.

The DNA fragment recovered as above was digested with restriction enzyme XhoI (
Toyobo) and Hindn [(Toyobo) cut to 626
A bp DNA fragment was recovered. Furthermore, this gene was cut with the restriction enzyme HpaIl (Toyobo), and the following exon 4 was extracted.
A DNA fragment consisting of 511 bases containing a part of (TNF
l) was separated.

110 120 130 140C
GGGCCAATGCCCTCCTGGCCAATG
CGTGGAGCTGAGAGCCGGTTACGGG
AGGACCGGTTACCGCACCTCGACTC
TC+50 160 170
180ATAACCAGCTGGTGGTGCCAT
CAGAGGGCCTGTACCTCATTATTGG
TCGACCACCACCGGTAGTCTCCC,G
ACATGGAGTA190 200
210 220CTACTCCCAGGTC
CTCTTCAAGGGCCAAGGCTGCCCCT
CCGATGAGGGTCCAGGAGAAGTTCC
CGGTTCCGACGGGGAGG230
240 250 260ACCCAT
GTGCTCCTCACCCACATCAGCC
GCATCGCCGTGGGTACACGAGGAGT
GGGTGTGGTAGTCGGCGTAGCGGC2
70280290300 TCTCCTACCAGACCAAAGGTCAACCT
CCTCTCTGCCATCAAAGAGGATGGT
CTGGTTCCAGTTGGAGGAGAGACGG
TAGTT310 320 330
340GAGCCCCTGCCAGAGGGA
GACCCCAGAGGGGGGCTGAGGCCCTC
C:CGGACGTCTCCCTCTGGGGTCT
CCCCCGACTCCGG350 360
370 380AAGCCCTGGT
ATGAGCCATCTATCTGGGAGGGGT
CTTCCTTCGGGACCATACTCGGGTA
GATAGACCCTCCCCAGAAC, G390
400 410 420AG
CTGGAGAAGGGTGACCGACTCAGCG
CTGAGATCAATCGTCGACCTCTTCC
CACTGGCTGAGTCGCGACTCTAGTT
AGC430440450460 GCCCGACTATCTCGACTTTGCCGAG
TCTGGGCAGGTCTACCGGGCTGATA
GAGCTGAAACGGCTCAGACCCGTCC
AGATG470 480 490
500TTTGGGATCATTGCCCTG
TGAGGAGGACGAACATCCAACCAAA
CCCTAGTAACGGGACACTCCTCC,T
GCTTGTAGGTTGG510 520
530 540TTCCCAACG
CCTCCCCTGCCCCCAATCCCTTATT
ACCCCAAGGGTTTGCGGAGGGGACG
GGGTTAGGGAAAATAATGGGG550
560 570 580CTC
CTTCAGACACCCTCAACCTCTTCTG
GCTCAAAAAAGAGGA to GAA TCTGTG
GGAGTTGGAGAAGACCGAGTTTTTC
TC590600610 AATTGGGGGCTTAGGGTCGGAACCC
ATTAACCCCCGAATCCCAGCCTTGG
GTTCGA (TNFI) In addition, DNA corresponding to the remainder of exon 3 and exon 4
The following eight oligodeoxynucleotides 16 to 23) were organically synthesized using a synthesizer using a synthesizer.
TTCATGGTCAGATCATl 7)5'-
GGTTCAAGAAGATGATCTGACCATG
l 8) 5″-CTTCTCGAACCCCGAGTG
ACAAGCCTGTAGl9')5'-AAC
ATGG to CTACA(1;GCTTGTCACTCG
G20) 5'-CCCATGTTGTAGCAAAC
CCTCAAGC21) 5″-TCAGCTTGAGG
GTTTGCTAG22)5'-TGAGGGGC
AGCTCCAGTGGCTGAACCGC23)5'
-CGGCGGTTCAGCCACTGGA to CTGC
CCC each 800 pmo (+ dissolved in sterile water 15-
T, polynucleotide kinase, 400xM ATP,
A 10-fold amount of Kaifunyon buffer was added to make 50 d, and the mixture was reacted at 37°C for 2 hours. Thereafter, the mixture was treated at 65°C for 10 minutes, desalted and recovered, further concentrated, dissolved in annealing buffer 50°C, reacted at 90°C for 3 minutes, and cooled slowly. In addition, 2000 units of T4 phage ligase
(Toyobo) and an equal volume of ligation buffer,
°C] Reacted at night. After completion of the reaction, the mixture was subjected to 55I acrylamide gel electrophoresis and stained with ethidium bromide. The following 102 bp DNA fragment (TNF2) was excised from the gel and separated and purified using an elution buffer.

10 20 30 40AATTC
ATGGTCAGATCATCTTCTCGAACCC
CGAGTGACAATTAAGTACCAGTCTA
GTAGAAGAGCTTGGGGCTCACTGTT
50 60 70 80GCCTG
TAGCCCATGTTGTAGCAAACCCTCA
AGCTGAGGGGCGGACATCGGGTACA
ACA DingCGTTTGGGAGTTCGACTCCCC
90100 CAGCTCCAGTGGCTGAACCGCGTCG
AGGTCACCGACTTGGCGGC (TNF2) Next, p was cut with restriction enzymes EcoRI and Hindll.
KK'223-3 (Pharmacia) and TNFl and T
NF2 was treated with T4 DNA ligase to create the following 60
A plasmid (pKK2233-hTNF) containing a 3 bp structural gene for human TNF (hTNF) was obtained.

10 20 30 40AATTC
ATGGTCAGATCATCTTCTCGAACCC
Add 50 units of CGAGTGACAAmHl to 3
After reacting at 7°C for 2 hours, the reaction solution was separated by 0.7% low melting point agarose gel electrophoresis, the gel was dissolved by heating at 65°C for 5 minutes, phenol was added, centrifuged, and the upper layer was removed. = Phenol-chloroform, add chloroform, stir, centrifuge, then add 1/10 amount of 3M sodium acetate and 2.5 equivalents of ethanol to the upper layer,
The DNA fragment (pVIPGP8
(B-E)'') was obtained.

pKK223-3-hTNF (B-A) approximately 60n
g and pVIPGP8 (B-E) Approximately 60 ng of DNA7 fragment (TNF-VIP linker) prepared in advance using a DNA synthesizer, 24) 5'
-CTACTTTGGGATCATTGCCCTTGG
25) 5'-AATTCCAGGGCAATGA
The mixture was reacted with 40 ng of TCCAAAGT in a ligation buffer at 16° C. for 24 hours using 350 units of T4 DNA ligase. Using this reaction solution, CaC (E. coli strain RR by method 22) was prepared according to a conventional method.
IΔM15 was transformed and 50/I! Transformants were obtained on single plates containing i/mI2 of ampicillin.

Furthermore, the plasmid was extracted from the obtained transformant by the alkaline lysis method, and diluted with 10zM) Lys salt M buffer (10zM).
pH 7.5), 10 Touge MgCQ2.1 xM dithiothreitol, and looxMNacn, and then added restriction enzyme EcoRI and restriction enzyme Hindll.
When cut with B a m HI, hTNF1VIPGP8,
This was confirmed by the detection of 7 fragments of the tac-promoter and the recombinant RRIΔMl 5 (pTVG
8) was obtained.

Recombinant RRIΔM15 (pTVG8) was cultured in L-medium at 37°C overnight, and 50A of this culture was cultured in L-medium.
In culture medium at 37 °C, O, D, 550 = 0.

When the concentration reaches 5 to 0.7, add 2 cod I as the final concentration.
PTG was added and cultured for another 5 hours. From the culture solution 500 years old, the bacterial cells were collected by centrifugation.
A 5DS-PAGE buffer was added, heated at 90°C for 3 minutes, and 12.5% 5DS-PAGE was performed.

When this gel was electroblotted onto nitrocellulose paper and subjected to Western blotting using rabbit anti-VIP antiserum (manufactured by CCRB) and anti-rabbit IgG combined with alkaline phosphatase, only a band with the desired molecular weight developed, and the present invention Using the method described above, it was confirmed that the desired peptide polymer was expressed as a fusion protein with hTNF.

vii) Purified recombinant RRIΔM15 (pTVG8) was purified at 2.4 (
3 in 1L-medium (50tIg/mI2 ampicillin)
Cultured at 7°C, O, D, .・When the final concentration reached 0.6, IIM's T PTG was added, cultured for another 6 hours, and the bacterial cells were collected from the 2Q culture solution by centrifugation. hydrochloric acid(
pH 8,0), lO MMg(, Qi, l IN phenylmethylsulfonyl fluoride l' (PMSF), 5
50ml buffer consisting of JIM2-mercaptoethanol
ff, crushed by ultrasonication, and crushed at 270
Centrifuged at 00g for 30 minutes.

This precipitate (1.5 Q of culture solution) was mixed with 70% formic acid solution at 42.
After dissolving in 6 mρ, add BrCN Ig and incubate at room temperature for 4
It reacted for 8 hours.

After concentrating the reaction solution under reduced pressure, it was diluted with water and freeze-dried, and 0.2M Tris-HCl (pH 7.5) was added to the resulting powder.
After extracting the water-soluble components several times and concentrating the extract, reverse-phase HPL
C (column YMC-pack.

AM312) to obtain H-Hi 5-5 as a peptide having a collagenase recognition site at the C-terminus of the desired peptide.
e r-As p-A ] a-Va l-Phe-T
hr-Asp-Asn-Tyr-ThrArg-Leu
-Arg-Lys-Gln-LeuAla-Va 1-
Lys-Lys-Tyr-Leu-Asn-5e r-
] 1 e-Leu-Asn-GlyPro-G
] u-A ] a-Va ]-Le u-G l
nPhe-Hse (peptide l) and H-Hi5-5e r-As p-A]
a-Va]]Phe-Thr-Asp-AsnT
yr-ThrArg-Leu-Arg-Lys-Gln
-LeuAla-Val-Lys-Lys-Tyr
-LeuAsn-5er-11e-Leu-Asn-G
lyPro-Glu-Ala
-Va 1-Le u-G I n-Phe-
Equilibrium mixture of Hselactone (peptide 2) and H-Hi s-5er-A sp-A I a-V
a IPhe-Thr-Asp-Asn-Tyr-T
hr-Arg-Leu-Arg-Lys-Gln-Le
u-A I a-Va 1-Ly 5-Ly 5-Ty
r-Le uAsn-5er-11e-Leu-As
Both fractions containing the peptide n-Gly-Pro-Glu-Ala (peptide 3) were separated.

After concentrating these fractions, they were further subjected to cation exchange HPLC (A
purified by sahipak ES-502C),
The product was further purified by reverse phase HPLC for desalting to obtain 6.4 mg of an equilibrium mixture of peptides 1 and 2 and 31.8 mg of peptide.

10 μg of each of these peptides was added to 101M 1-Lis-HCl (pH 7.5) containing 100 μg (19 units)/mQ of collagenase (Nguma type ■), 50 zM
The desired VIP derivative H-His-5er-Asp-Ala-ValPhe- is dissolved in a buffer consisting of CaCa2 and reacted at 4°C.
Thr-Asp-Asn-Tyr-ThrArg-Le
u-Arg-Lys-Gln-LeuAl a-Va
1-Ly 5-Ly 5-Tyr-Le u=A
s n-5e r-1] e-L e u-A s n
-OH was obtained quantitatively.

viii) Confirmation The structure of the peptide produced as described above was obtained by subjecting the amino acid sequence to Edman degradation using a peptide sequencer (manufactured by Applied Biosystems), and then using the phenylthiohydantoin (PTH) amino acid obtained by reverse-phase HPLC.
It was confirmed that it had the desired amino acid sequence.

[Effects of the Invention] The method of the present invention is an excellent peptide production method that enables the mass production of peptides with molecules i2000 to 4000 that do not have L-methionine or collagenase recognition sites in the molecule, which was difficult to expect in mass production in the past. It's a method.

[Brief explanation of drawings]

FIG. 11 The details of the construction of the plasmid pVIPGPl incorporating the synthetic V I PGP gene are described in the Examples. The chemically synthesized VIPGP gene does not have a stop codon in its gene sequence, but the V I PGP gene of the finally constructed Saleta plasmid pv I PGP I contains one of the three stop codons in the plasmid psTPl. This results in having a stop codon. This plasmid pv l PGP l is the plasmid p
tac promoter rrnB in polished rice in KK2233
) Contains a transcription terminator ambicillin resistance gene. Figure 2. The details of the construction of plasmids pVIPGP2 and pVIPGP8 in which VIPGP genes are linked in tandem are described in Examples. By synthesizing two types of fragments obtained from pv I PGP I and connecting them using an NA linker,
A plasmid pVIPGP2 was obtained in which two V I PGP genes were linked in tandem. Synthetic NA linker L
HE-1 and HE-2 have the same sequence as the EcoRI or HindI recognition site, but are not cleaved after binding because they are different from the recognition site. The above operation should be done with pVIPGP2 instead of pVIPGPl.
By using pVIPGP4, pVIPGP4 is constructed, and by repeating this, various numbers of VI
A vector having a gene (VIPGPn) to which the PGP gene is linked can be constructed. Figure 3. Plasmid pT expressing TNF-VIPGP8
The details of the configuration of VG8 are described in the embodiment. The final plasmid pGTV8 has an EcoR linkage site downstream of the tac promoter between the full-length TNF gene and the 8 VI PGP genes.
It has a gene that is an I recognition site. There is no stop codon at this connection site, so plasmid p
TVG3 expresses a fusion protein in which 8 VIPGPs are linked to TNF. Fig, 1

Claims (7)

[Claims] (1) A new method for producing a peptide with a molecular weight of 2,000 to 10,000 that does not have L-methionine or a collagenase recognition site in the molecule, wherein L-methionine and the above are added to the C-terminus of a polypeptide that can be expressed in prokaryotic cells. Molecular weight 2
A fusion protein in which a peptide of 000 to 10,000 and an amino acid sequence corresponding to a collagenase recognition site are linked repeatedly about 1 to 50 times is expressed in prokaryotic cells, the fusion protein is purified, and degraded with bromcyanide and collagenase.
A method for further purifying the above-mentioned peptides having a molecular weight of 2,000 to 10,000 from a peptide mixture obtained by digestion. (2) A peptide with a molecular weight of 2,000 to 10,000 that does not have L-methionine or a collagenase recognition site in its molecule has the following amino acid sequence [gene sequence]. ] The method according to (1) above. (3) The method of (1) above, wherein the peptide expressible in prokaryotic cells is TNF. (4) Novel peptide having the amino acid sequence of the following formula [There is a gene sequence. ] (In the formula, Hse represents homoserine.) (5) A new peptide with the amino acid sequence of the following formula [there is a gene sequence]. ] (In the formula, Hselactone represents homoserine lactone.) (6) Novel peptide having the amino acid sequence of the following formula [There is a gene sequence. ] (7) There is a [gene sequence] obtained by decomposing the peptides of (4), (5) and (6) with collagenase. ] Manufacturing method.