JPS5863395A - Preparation of alpha-neoendorphin - Google Patents

Abstract

PURPOSE:To prepare alpha-neoendorphin, by cultivating a strain of the genus Escherichia transformed by a specific plasmid in an aqueous nutrient medium, producing the alpha-neoendorphin as a proteinic component in the culture, and fractionating the resultant alpha-neoendorphin. CONSTITUTION:This is a method of preparing alpha-neoendorphin by the genetic engineering method. A strain of the genus Escherichia transformed by a plasmid having polydeoxyribonucleotide having the nucleotide sequence expressed by the formula (A is deoxyadenylic acid residue; G is deoxyguanylic acid residue; C is deoxycitydilic acid residue; T is thymidylic acid residue; J is A or G; K is T or C; L is A, C,T or G; CTL is substitutive by TTJ; CGL is substitutive by AGJ) integrated therein is cultivated in an aqueous nutrient medium to produce alpha-neoendorphin as a proteinic component in the resultant culture.A hybrid protein containing the alpha-neoendorphin is collected from the culture, and decomposed to fractionate the aimed alpha-neoendorphin from the decomposition product.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing alpha-neo-endorphin by genetic engineering methods.

Evitate hormone (alpha neo-endorphin, hereinafter abbreviated as α-N) is a new brain hormone discovered in the pig hypothalamus by Samukawa, Matsuyo et al. in 1979, and its structure is H2N-Tyr- Gly-Gly
-Phe-Leu-Arg-Lys-Tyr-Pro-
It has been reported that Lys-COOH.

The present inventors completed the present invention with the intention of mass producing α-N, FX, without using difficult-to-obtain natural products such as porcine hypothalamus as raw materials.

The present invention involves synthesizing the α-N and L genes, constructing a plasmid vector, ligating the genes and the vector, and using it to transform a recipient bacterium, and then culturing the transformed bacterium. This invention relates to a series of steps, partial steps, and intermediates for producing protein containing α-N, J in a product, collecting and decomposing it, and separating α-N, J.

The present invention will be explained in the following steps.

Synthesis of α-N,J Gene The gene directly encoding α-N,B can be represented by the following nucleotide sequence.

Yu ΔKl Construction work 2 Dan GI, 3 work ■ 4 Third work 5 Yo upper 6 stop 7 man 8 Work Dan tile 9 difference JIO (A is deoxyadenylic acid, G is deoxyguanylic acid,
C represents a residue of deoxycytidylic acid, T represents a residue of thymidylic acid, J represents A or G, T or C, L represents A, C,
Represents T or G, CTL is TTJ, CGL is AG
J may be substituted respectively) If a desirable triblend (codon) corresponding to each amino acid is selected in the above sequence, for example, TAT
GGCG-GT TTCCTTCGT AAG
An example of this is the person who is a person.

Furthermore, when a bacterium transformed with a vector incorporating the α-N, E, and genes is cultured, α-NE is thought to be expressed as a hybrid protein in the culture, and from this protein, α-N, It is preferable to add methionine to the N-terminus of α-N, E, so that E can be separated. 4 For this purpose, a methionine codon (ATG) is added to the 5-terminus of the gene. Also, gene 3
' At the end, there is a transcription stop codon (T) to terminate transcription.
AA, TAG).

Furthermore, AATT was added to form a restriction enzyme BOOR-engineered site at the 5' end (outer end of the methionine codon) so that α-N, Fi, and genes could be incorporated into the vector.
Add C. Furthermore, CCTAG is added to the 8' end so as to form a BamHI recognized weave site.

The nucleotide sequence designed in this way is shown as a double strand as follows.

AAKZ GAL' ()CL/ TT:II
ATKI () GI/ (in the sequence, T, L, and L have the same meanings as above; J', K', and TI/ represent nucleotides that form base pairs with the opposing J, I, respectively) Desirable embodiments of the double-stranded nucleotide are as follows.

GTACATA CGG CCA AAG Polynucleotides designed as described above are prepared using methods known in the literature, such as "Nucleic Acid Organic Chemistry J (1979)" published by Kagaku Dojin, "Biochemistry Experiment Course, Chemistry of Nucleic Acids 1" published by Tokyo Kagaku Dojin.
11 J P, 338 (1979)], the upper strand and lower strand polynucleotides can be synthesized and then subjected to a ligase reaction to form a duplex.

Through research by the present inventors, the L strand is fl) to (4), and the lower strand is (5) to (8).
The 5'-
Terminal phosphorylated F-2-4 right and F-(3-8 and 5'-terminal unphosphorylated F-1 and ?-
It was found that the above double-stranded polynucleotide could be obtained all at once by reacting 5 with 5 in the presence of ligase.

(Upper strand) Fi “AAT'I'CATGT”Fil
pAKGGLGGLTTKCF 'mp T L
CG L A A J T A K CF Ii
p CL A A J T A A T A
G (Lower Strand) FV "GATOCTATT" FW pAJノT T L'G
G K'T A JITFVI pT
AI/GL'AGK'AALCFv1 pCL'
CCK'TACATG (in the above sequence, T, K,
(L, Jl, K/, II have the same meanings as above) And the eight oligonucleotides corresponding to the above-mentioned desirable double-stranded nucleotide embodiments are the following F1 to F8.

(One strand) Fl "AATTCATGT"F'2 pATGGC()GT, TTCCF3
pTTCGTAAGTATCF4
pCGAAGTAATAG (lower strand) y 5”a A T Ci, T A T T
“F(J ACTTCGGATACTF
7 TA℃GAAGGAAAC78C-G
CCA 'I' A CA T G The present invention includes each of the above-mentioned oligodeoxyribonucleotides and a method of producing a desired double-stranded polydeoxyribonucleotide at once by a ligase reaction using them.

These oligodeoxyribonucleotides are Br0k&
and the method of Crea et al. [Nucl, Ac1dsRes
, Dan5461 (195Q), Pr0c, Natl,
Acad.

SC1,:1i5765 (1978)]. The reaction formula is shown in FIG.

That is, a fully protected bifunctional dimer (Co., Ltd.) is partially hydrolyzed with a hydrolyzing agent such as pyridine-triethylamine-water to change the phosphodiester group at the 3' end into a monoester group (2). ', then 5'-
A completely protected bifunctional trimer is obtained by reacting a monomer (I) having OH and having a silicic acid ester at the 8' end in the presence of a condensing agent such as methylene sulfonic tetrazolide. (2) is obtained.

Next, using these compound groups as raw materials, they are condensed with a certain base sequence by the Pronk condensation method to obtain the oligodeoxyribonucleotides F4 to F■, such as Fl, F.
8 is obtained in a completely protected form, and then completely deprotected by the action of a deprotecting agent such as concentrated aqueous ammonia or 80% acetic acid to obtain the desired oligodeoxynucleotide.

The eight oligonucleotide fragments thus obtained are ligated according to the base sequences of the α-N, E, and genes. The bond is, for example, the 5'-O
After phosphorylation of the H-terminus, equal amounts of each of the 7-ragment mixtures can be subjected to a Ti reaction in the presence of ligase. These reactions themselves are known. In the process of this ligation reaction, it is important to ensure that two or more target genes do not bind to each other at their 5' end adhesive sites (EcoR and BaJI). When using a fragment, Fl
It is preferable to use the 5'-OH end of F5 without phosphorylation.

Phosphorylation of the 5/-OH terminus is caused by, for example, ATP and T4.
It is performed using polynucleotide kinase°.

The ligation reaction can be performed using, for example, T4 DNA ligase.

In this way, a gene (44-mer) can be synthesized into α-N.

Construction of recombinant plasmid and transformation In order to construct a plasmid containing the promoter and β-galactosidase region of the photons λp/ac 5 DNA lactose operon, pBR322 DNA and λp/ac 5 DNA were digested with restriction endonucleases and reacted. subject to a ligation reaction.

Fc as a restriction endonuclease.

It is preferable to use R engineering, and it is preferable to use T4 DNA ligase as the ligation enzyme.

The DNA thus obtained is used to transform a strain of the genus Nijiserisia. Preferred strains are, for example, E, Co/i,
It is 12W3110. A plasmid is isolated from the transformed strain, digested with Hlndll, and then the orientation of the promoter β-galactosidase is examined by agarose gel electrophoresis to select a plasmid having the desired orientation.

This plasmid has two sites that are cleaved by EcoR. Therefore, the site close to the β-galactosidase promoter is removed. For this purpose, the plasmid is partially digested with IecoR, and the desired DNA fragment is separated from the reaction product by agarose gel electrophoresis. This fragment has a single-stranded portion generated by gcoR-mediated cleavage at the end;
The single stranded portion is made into a double strand by the action of NA polymerase, and then the ring is closed by the action of T4 DNA ligase,
A plasmid with one cleavage site by ECoR is obtained. Using this plasmid, CO/1K
12W8110 was transformed, a tetracycline-resistant strain was selected, plasmid DNA was isolated from the colony of the strain, digested with gcoR and Hlndll, the size of the generated DNA fragment was examined by agarose gel electrophoresis, and plasmid DNA was isolated from two locations. It is determined which of the existing EcOR cleavage sites has been deleted, and a plasmid in which the desired one site has been deleted is selected.

Next, the above plasmid is completely digested with EcoR and BamHI, and large DNA fragments are separated by agarose gel electrophoresis. -10,000, α-N obtained by the above method, D
The 5'-○H end of NA is phosphorylated using, for example, ATP and T4 polynucleotide kinase, and this is subjected to a ligation reaction with the above DNA fragment. Preferably, the reaction is performed using T4 DNA ligase and ATP.

The DNA obtained by this reaction is, for example, E.

A strain of Elysia such as Co/1K12WA802 is transformed as a recipient strain, and the transformed strain is screened using an ampicillin-containing medium and then a tetracycline-containing medium to select ampicillin-insensitive, tetracycline-sensitive colonies. Some of these colonies were arbitrarily selected and plasmid D obtained from them.
NA to Wc.

When the size and base sequence of the small NA fragment produced after digestion with R-E and BamHI was examined, it was found that the tested gold side corresponded to α-N, DNA. The strain obtained by transformation of WA802 strain was named J-Co/ix12sEIM102.

When the strain transformed as described above is cultured, α-N is produced within the bacterial cells. When the plasmid is constructed in the manner described above, α-N, and the gene are inserted into the β-galactosidase structural gene, so α-N, and β-
It is thought to be expressed as a hybrid protein with galactosidase, but as mentioned in the α-N, E gene design, the amino terminal of α-N, E, which is expressed by the addition of ATG to the 5' end. Methionine is inserted into the site.

Therefore, it is expected that α-N and E can be separated by cleaving this hybrid protein with, for example, cyanogen bromide. It was found that α-N-B could be separated as expected.

The above transformed strain is cultured in, for example, L-pros (glucose is removed and ampicillin is added), and cyanogen bromide is applied to the cells obtained by centrifugation from the culture.
α-N and B can be separated by dissolving the lyophilized product of the reaction solution in, for example, dilute acetic acid and performing adsorption chromatography.

The thus obtained α-N exhibited data consistent with natural products in elution pattern in reversed-phase HP LCI, radioimmunoassay, amino acid analysis, amino acid sequence analysis, and biological activity. Moreover, the yield is much superior to those previously reported in the field of genetic engineering methods.

Example 1 Chemical Synthesis of α-N, Fi, and Gene Fragments Regarding the methods and raw materials for the chemical synthesis of eight oligodeoxyribonucleotides named Fl and Fg, respectively, as shown in the text, the following modifications are noted. Except for
Basically, the method reported by Bron force and flare etc. (Brolca, C.).

et al (195Q) Nucl, Ac1ds, Re
s, Q5461 and Crea, R1, et al.
(1978) Proc, Nat/, Acad, Sci.
USA, E5 5765] was used.

The fully protected bifunctional trimer (see Figure 2) is
It was synthesized on an 8-5 mmol scale using the improved method described below. That is, the fully protected bifunctional dimer 1
1 (1 molar equivalent) in pyridine triethyl atone-water (
The corresponding 8'-phosphoric acid diester component was converted using a mixed solution (8: 1: IV/V), the 5'-hydroxyl component (1.5 molar equivalent) was added thereto, and the condensing agent, mesitylenesulfonyltetra The condensation reaction was carried out using zolide (MsTe, 9-8 molar equivalents). The products were isolated and purified by chromatography on silica gel and reverse phase chromatography on C18.
Table 1 shows the synthesis yields of two 3'-terminated gimers and four 3'-terminated tomers whose -hydroxyl groups were protected with benzoyl groups.

Table l Spicy Completely preserved trinucleotide **3' terminal dimer, trimer block n・-1 or 2 Starting from this group of compounds, 8 with a certain base sequence
oligodeoxyribonucleotides, namely F1 to F8
was synthesized using the Glock condensation method.

An example of the mode of the condensation method for FB is as follows.

(-) Fully protected AGTQ obtained by the above method, 95m
m0! After treatment with triethylamine □, 0.375
mmojF(7)f! Add OA T C40B Z
, after adding pyridine and azeotroping, M 8 T e Q, 5
m! No/ was added and reacted at room temperature for 1 hour, then water was added to stop the reaction. The solvent was distilled off under reduced pressure, toluene was added and azeotropy was repeated three times, and the residue was dissolved in chloroform:methanol (7:8 V) containing dibenzenesulfonic acid.
/V)gO1nI! After reacting at 0°C for 10 minutes, it was neutralized by adding 5% NaHCOB, the chloroform layer was concentrated under reduced pressure, and AG was analyzed by silica gel chromatography.
T·ATC-OBZ was purified and separated (yield: 93 cm).

(b) After treating fully protected ()TAOJ15mmO/ synthesized from fully protected TA and fully protected G with triethylamine, the above AGT@ATC-OBZ 0.21m
After adding m0/ and azeotroping by adding pyridine, MBTeQ,
42 mmol was added, reacted at room temperature for 1.2 hours, and treated in the same manner as in (a) to obtain HOGTA@AGT*ATC.
-OBZ was obtained (yield 80%).

(C) Next, after treating the fully protected TTCQ, 3 mmo/ with triethylamine, the above HOGTA@AGTI
After adding IATC-OBZQ, l 5mmo/, adding pyridine and repeating azeotropy, MsTe Q, Qmmo
/ was added and reacted at room temperature for 1 hour. The reaction was stopped by adding water, the solvent was distilled off under reduced pressure, the residue was extracted with chloroform, and the chloroform layer was extracted with 591. NaHCOg
The residue was separated and purified by silica gel column chromatography to obtain fully protected "TT3' CGTAAGTATC (yield: 60 cm)".

Each of the seven fragments of Fl*'2m F4 to F8 were also produced in the same manner as above except that the combination of nucleotides was changed. From each of the eight fully protected 7-ragments thus obtained, all protecting groups were removed by treatment with concentrated NH4OH for 55' CIO hours, followed by treatment with 80% acetic acid for 10 minutes at room temperature. Purification of the final product was performed using high performance liquid chromatography (1'1PLC). Kono H
PT and +C were performed using an ALTEX nod13rIIOA liquid chromatograph. Solvent A (0,00
5M, KH2PO4, pH40) and solvent B (Q, Q
5M, KH2PO4-1, QM KC/; pHHO
Using the linear concentration gradient method according to 2,
AAX (Ji x Hong) Crab 7 A (0,21X50(
! II+) to separate and purify the compound. A gradient was created by starting with solvent A and adding 8 units of solvent B every minute. Elution was performed at 58° C. and a flow rate of 2 me per minute. The peaks of interest were collected, desalted by dialysis, and lyophilized. The homogeneity of the 8 completely deprotected oligodeoxyribonucleotides obtained in this way was (h
The omogenity was determined using two types of HPLC, namely ion exchange [Permanphase AAX (Shubon) Go, and reverse phase [μ-Bondapak C+a (
(see Table 2)] which was confirmed by the fact that a single peak was obtained in each column chromatography.

Table 2 Retention time of synthetic oligonucleotide Retention time 0 minutes) FI AATTCATGT 6.2
11.8F2 ATGGCGGTTTCC8,01
1,2F3 TTC()TAAGTATCg,9
11゜2F4 CGAAGTAATAG
8.6 11.175 ()ATCCTAT
T 6,8 11.4F13 ACT
TCGGATACT 9.0 11.6F7
TACGAAGGAAAC8,910,4 filling
Permaphase AAX MBOndapak
CIB color A O, 21X50CIIl
O, 4X25c +++ flow rate 2C min
Nisaki temperature 58℃ Room temperature Furthermore, the base sequence of eight oligodeoxyribonucleotides was determined using T4 polynucleotide kinase [γ-P
After labeling the 5'-end with l-ATP and partially thawing using nuclease, electrophoresis of paa:'5 using cellulose acetate and homochromatogram a/(1974) NuC! using DEAF-cellulose was performed. , AC1
d8 ReB, 1 83139, a-N,! ! ,
Δ11at10n of DNA Eight oligodeoxyribonucleotides consisting of Fl and F8, whose base sequences were confirmed as described above, were ligated using T4I) NA ligase by the method described below.

F2#F'! 3#F4tF6. F7. 5/-0 for each of the 72 segments of F8 (the ends were each phosphorylated separately with T4 polynucleotide kinase).

[7-PIATP (7400C1/mfno/)200
p” was evaporated to dryness in an empendorftube and 10 μ2 of the fragment was purified with T4 polynucleotide kinase l.
O units and a total volume of 60 μl of 50 mM) Lis-HCl buffer (
pH8,0), 10mM My C/2,10mM
The reaction was carried out in dithiothretol/l/(DTT) for 20 minutes at 87°C. Next, in order to phosphorylate all 57-OK ends, 1 Q n no/ATP and 1 O units of T4 polynucleotide kinase were added to the reaction solution, and 3
The reaction was further allowed to react at 7°C for 1 hour and then stopped by heating at 90°C for 5 minutes. The performance of phosphorylation was confirmed by one-dimensional homochromatography followed by radioautography.

The Fl and F5 fragments are attached to the 5' end sticky site (EcoR) from the gene of interest during the next lidarion reaction.
5 to avoid combining two or more pieces with
/-og terminus was not phosphorylated. The thus obtained phosphorylated fragment) F2°F81 F4 , F
Unreacted ATP was removed by putting 2.5μ2 of each of 6, F71Q, Fl, and F5 into a dialysis tube and dialyzing against %l/water at 4°C overnight.

One after another, the dialysate solution was added to IJS hydrochloric acid buffer (final concentration 20mM)
1) H7,6), prepared to 10mM MPC12, heated at 95°C for 2 minutes, then heated at 80°C for 2 minutes, 3
After standing at 7°C for 10 minutes and at 25°C for 2 minutes, the following ligation reaction was performed. Pour the reaction solution into a 20m tube with a total volume of 80fit.
M) Lis-HCl buffer (1)) (7,6), 70mM
MgCl2, lQmM DTT, Q, 4mM AT
P was prepared and carried out at 11°C using 25 units of T4 DNA ligase. After the start of the reaction, aliquots of the reaction solution were taken every 1 hour, 3 hours, 7 hours, and 20 hours, and using a 15% polyacrylamide gel containing 7M urea, 90mM trisulfocate buffer (1) H8) 4mM EDTA, The progress of the reaction was examined first by electrophoresis and then by autoradiography. As a size marker, FX17
41F DNA was digested with HtnfI and dephosphorylated with bacterial alkaline phosphatase, which was then converted into [γ
- After 24 hours from the start of the ligation reaction using the DNA fragment labeled with PIATP and T4 polynucleotide kinase, the reaction solution was heated at 65° C. for 5 minutes to stop the reaction. The reaction solution was subjected to the above-mentioned 15% polyacrylic^ gel electrophoresis to obtain the desired α-N, 1! :,
The DNA gene (44-mer nucleotide) and unreacted fragments were separated. The part corresponding to the 44-mer nucleotide was cut out from the gel, placed in a polypropylene econocolumn (Bio-Rad Laboratory) whose lower end was connected to a dialysis tube, and electrophoresed in the above-mentioned electrophoresis buffer at a concentration of 1/10. This DNA was eluted. After elution, the dialysis tube was removed from the tube and dialyzed against water to desalt it. The target DNA was precipitated by adding 3 times the volume of Elafur, centrifuged, and dissolved in 20 μl of distilled water. The yield was 1.4 μ2.

3. Construction of recombinant plasmid Figure 3 shows a method for constructing a recombinant plasmid containing the α-N, E, and genes, which will be described in detail below.

A. Construction of plasmid pKO3 A plasmid in which the λp/ac5 DNA tract, promoter of the operon, and β-lactosidase region was constructed as follows. Plasmid pBR822I
) NA 0.5μP and λplac 5 DJ! JA L
OIII' each 801110100mM ) Liss hydrochloric acid! Solution 17 (pH 7°3), 5mM M9C! Digestion was performed for 60 minutes at 87° C. using 4 units of restriction endonuclease library in 2 and 5 QmM NaC/solution. The reactions were heated at 65° C. for 30 minutes to inactivate the enzyme, and 2.5 volumes of ethanol was added to each reaction to precipitate plNA. Each precipitate is ']'4DNA
Ligase buffer [2.0mM] Liss-HCl buffer (1)
H7,6), 10mM Myc12], and then mixed each solution and added ATP and DTT to final concentrations of 0.4mM and 10mM, respectively.
The reaction was carried out at 14° C. for 20 hours using 2 units of DNA1 nogase. Next, add 2.5 times the volume of ethanol and
Precipitate the NA and add the resulting DNA precipitate to 100mM ToI.
J stflfl [Solution (I) H7,8) I 50
mM IJaCl.

5mM M? After dissolving 102 μl of a solution consisting of C12, 100 μl of the solution was added to strain E, Co/1W.
Transformation into 8110 (N.
Lactose medium (Polypeptone 82.Yeast extract 1.O
y, IJaCl 5y+K2H”042.5y+Eosin 360y, Methinonpur-60t, Lactose 1
09. Six colonies with a dark metallic color were selected and named pKOl-pKo(l). Plasmid DNA was isolated from these colonies and the orientation of the promoter β-galactosidase was investigated. Approximately 0.5μ2 of plasmid DNA was added to lOmM Tris-HCl buffer (pH'7.0).

5 QmM Na(:/, l QmM MgCl2
Restriction enzyme H
Five units of i n d ■ were added to digest the DNA. By analyzing the obtained DNA fragment by 0.7% agarose gel electrophoresis, the Ec of the pBR822 plasmid was determined.
The orientation of the promoter β-galactosidase of λp/ac 5 inserted into the oRI site was investigated. As a result, it was found that the desired direction, ie, transcription of β-galactosidase proceeds toward the tetracycline resistance gene, is pKol, pKo8+pKO5. Of these, pKo3 was used in subsequent experiments.

(Note) Transformation method A strain of N. coli was inoculated with 30vte of L-gloss (polypeptone 1Oy, yeast extract 5y*Ne, C15y + glucose 5y, water 1,000me, pH 7,2) and 87℃ OD (3 (Culture until 3Q-0,7,
Bacteria were collected by centrifugation at 0° C. for 6.000 rpm tlO minutes. Suspend the bacterial cells in 0.1 M MyC/280me and incubate at 6°000r.
pm, and further centrifuged at 0°C for 10 minutes, and the collected cells were centrifuged at 0°C.
It was suspended in IMCaC/215me and left in water for 1 hour. Then 6,000 rpm.

Centrifuge at θ°C for 10 minutes and collect the collected cells to 0. I M
It was suspended in CaC12L5me. FicoR engineering reaction buffer (100mM Tris-HCl, 5QmM NaC
/, 5mM M7C/2. 100 ttl (pH 7,3) was added and left at 0°C for 60 minutes. 0.8 me of this mixture containing CaC/2-treated bacterial cells and DNA was added to the L-
In addition to the 2.5 me gloss, shaking culture was performed at 37°C for 90 minutes. The resulting culture was diluted, pre-coated on a selective medium, and cultured at 37°C for 1 day to obtain transformed colonies.

The selection medium was BBL Nutrient Agar 1.5% with final concentrations of ampicillin and tetracycline of 4 and lOμy/-, respectively, unless otherwise specified. / m
Ampicillin-resistant strains and tetracycline-resistant strains were selected using the mixture added so that e.

The following experiment was conducted to remove one of the two Rco R sites in the Uni1Ω and Ku pKo3 plasmid, that is, the Eco R site near the β-galactosidase promoter. pKO3 plasmid DNA I O μ restriction enzyme E
After partial digestion using 1.6 units of coR engineering, the partially digested DNA fragments of the reaction solution were separated using 0.7% agarose gel electrophoresis. DNA fragments were confirmed by staining with ethidium bromide (0.5 μ?/d) and mulching. Purpose D from Guru
- The NA fragment was eluted by electrophoresis, and ethidicumpromide was extracted and removed using phenol. The desired DNA fragment was obtained as a precipitate from the aqueous phase using an ethanol precipitation method. Add T4 DNA polymerase buffer [100mM], IJS hydrochloric acid buffer (pH 8,8), lOmMMyc/2,25mM (N
H4) 2 804, lQmM BDTA 25.0
fi9/me bovine serum algumi>)4. After adding and dissolving Opl, aATP, dTP, dcTP
After adding 5 μl of each 4 mM solution of dGTP and 5 μj of 100 mM β-mercaptoethanol, the mixture was incubated at 37°C and 80°C using 1 unit of T4 DNA polymerase.
Allowed to react for minutes. Next, the reaction solution was heated at 65° C. for 10 minutes, and 2.5 times the volume of ethanol was added to precipitate the DNA. The obtained DNA precipitate was mixed with T4 DNA ligase buffer (2QmM), lithium-hydrochloric acid (1)H7,6), 1
0 m M M? C/2) Dissolve in 40μl, A
TP and DTT were each added to a final concentration of 0.4mM IlOmMe, and T4DNA! Add 2 units of J gauze to 1
The reaction was carried out at 4°C for 20 hours. E after precipitating the DNA with ethanol.

It was used to transform Co/i strain W3110. Tetracycline-resistant colonies were selected by culturing on a nutrient agar medium supplemented with 10 μf/me of tetracycline. Plasmid DNA was isolated from the colonies that appeared, digested with restriction enzymes B'QOR and Hlndill, and the size of the resulting DNA fragments was examined by agarose electrophoresis. We were able to clarify which part of the body was missing. As a result plasmid 1) KO
18 expects Eco t't, I site i.e. /
It was confirmed that the FicoR engineering site close to the ac promoter had disappeared. This plasmid was used for the next alpha neo endorphin gene cloning.

Plasmid 1) KO13 has one restriction enzyme cleavage site each for ECOR and BamH, and two genes with cohesive sites (cohθ5ive end) for EcoR and BamH obtained chemically or from plasmids were combined. It can be easily introduced into plasmid 1) KO18 using a Schion reaction. Furthermore, by transforming the strain Jco/1K12 with this plasmid, the desired clone can be easily selected as an ampicillin-resistant and tetracycline-sensitive strain.

“Construction of C plasmid PαNFX2 α-N, E!, DNA Q, 7μ2 obtained by the above method was 2Q
mM) Lis-HCl buffer 1) H7,6,lQmM M9
CI2° solution 28μ! After cooling in ice water at 95°C for 2 minutes, ATPl, j3nmo/, 5 units of T4 polynucleotide kinase, and DTT I QmM were added.
The reaction was carried out at 7°C for 20 minutes to phosphorylate the 5'-0)i end of the DNA. To phosphorylate all 57-OH termini, the reaction was reheated at 95°C for 2 min, cooled in water, and then added with 1/10 volume of the reaction volume of 10QmM DTT and 5 units of T4 polynucleotide kinase. In addition, 8
The reaction mixture was further reacted at 95°C for 20 minutes, then heated at 95°C for 2 minutes, and then slowly returned to room temperature. Next, plasmid pKO13DNA5μ2 was digested with restriction enzyme FicoR and Ba
0.7 when completely digested using 10 units of each mH.
The DNA fragments were separated by thiagarose gel electrophoresis, and large DNA fragments were recovered by electrophoresis. pK obtained in this way
The KgOR engineering of O18, the BamH engineering fragment DNkip9 and the previously obtained α-N, L gene DtJA25ny phosphorylated at the 5'-OH end were mixed in 20mM Tris-HCl buffer, 6 + 10mM MrC/2tlomM DTT.
, dissolved in 30 μl of a solution consisting of 0.4mM ATP,
4 1 unit of DNA ligase was used to react at 5° C. for 16 hours.

Twice the volume of ethanol was added to the reaction solution to obtain DNA as a precipitate. This DNA precipitate was dissolved in 100mM Tris-HCl buffer (pH 7, (1), QmM M7C/2.66mM Na
Dissolved in a solution 100/Z/ consisting of C1, K, co/1
The cells were transformed into strain WA802. The transformed strain received ampicillin 40μfi! Colonies generated after overnight culture at 87°C on nutrient agar containing 0.5 μf/me and glucose were replicated onto nutrient agar containing 15 μf/me of tetracycline to select tetracycline-sensitive colonies. Of the 118 ampicillin-insensitive colonies, 100 were tetracyclyi.
It was a susceptible strain. For verification purposes, 8 colonies independently isolated from the 100 ampicillin-insensitive and tetracycline-sensitive strains obtained here were selected as WA802/PαNEl・・・・・・WA802
/PαNE13. Plasmids obtained from these colonies I) NA were digested with restriction enzymes EcORT and Ba
In all 8 cases, the size of the small EcORT and BamH fragments of these recombinant plasmids was smaller than that of the α-N, E fragments produced in vitro.
, the size was similar to that of DNA. Of these, strain W
I) NAW base sequence of the small EcORT/BamH fragment obtained from the A802/pαNK2 plasmid, \A (Maxam) and Gi/I/Bar
) (Gilbert) [Proc, Nat, Acad
, Sci, USA, (1977) 74560], the pαNE2 plasmid contained the desired α-N,
It was confirmed that it had the TL DNA base sequence (7th
(see figure). This WA802/1) αNK2 strain is Ei
, Co/1K12 SBM102 and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (deposit number FBRMP-a).
osi).

The E. coli strain obtained by the above method was treated with α-N in 12SBM102.
, the R gene is inserted within the β-galactosigusase structural gene. Therefore, α-N,E, should first be expressed as a hybrid protein consisting of β-galactosidase and α-N,E. However, as mentioned in the initial α-, N, and E-gene design, α-
By inserting methionine into the amino-terminal sites of N,E, this expressed β-galactosidase and α-N
It is expected that α-N, F- can be obtained by cleaving the 71-brid protein of 71 with cyanogen bromide. Therefore, 12B was added to the E. coli strain by the method detailed below.
The expression of α-N, Fi, in BM102 and its identification were performed.

A, Identification of α-N, B. strain using HPLC@ E,
co/iK12sBMIQ9 and a-NJ as a control.
, E, co/i WA802/pKo that does not have the gene
13 and 40μ9 of ampicillin after removing glucose.
L-pross (Luria-broth) with 7me added
) were grown to 4 × 10 cells/me at 37 °C in ZME. Next, isopropyl β-D-thiocalictoviranoside (PTG) was added to 1 mM, and growth was continued for an additional 2 hours. 1 me of the culture was centrifuged in an Enbendorf centrifuge and the resulting pellet was mixed with 500 μj of 70 thiformic acid containing 5”t/me of cyanogen bromide.
The mixture was suspended in a dark place at room temperature for 24 hours. After freeze-drying the reaction solution, the residue was dissolved in 200 μj of 1M acetic acid. Of these, 150fit was applied to a SEP-pakClg (Quarters) column, and after washing the column with 1M acetic acid 2me and water 2-, the substance adsorbed on the column was washed with 1QmMHcOOON.
Elution was performed with a mixture of H4 and acetonitrile (50:50 vA) for 2.5 me. After concentrating this eluate to dryness, the residue was dissolved in 200 μl of 1M acetic acid. 100 of these
HPLC using p-Bondapak'C-1f3 (quarters) column (0.4 x 25 cm)
It was then analyzed. Elution was 5 Q m M KH2PO4
CHBCN was applied in (TIH2,0) by applying a linear concentration gradient from 10 to 90 chi. The elution pattern was monitored by UV absorption at 210 nm.

As is clear from Figure q a and b, the HPIIC elution pattern obtained from 128BM102 for strain E and Co71 contains strain E and Co/1WA8 at the position indicated by the arrow in the figure.
It was found that a new peak not seen in 02/pKO18 existed. The elution position of this peak completely coincided with the elution position of α-N and F- obtained by chemical synthesis under the same conditions. Furthermore, the results of radioimmunoassay described below also indicate that this peak is α-N.

It is easy to understand that E.

B, Identification of α-N, F- using radioimmunoassay α-N
, N for standard radioimmunoassay (RIA);
Minamino, et a/[Biochem.

Biophys, Res, Commun, in pr
ess] method was used.

The HPLC fractions in Figure q a and b are 1rId! A 20 μl aliquot was taken, 4 μl of bacitracin was added thereto, and the mixture was dried under reduced pressure. Residual winter pHs.

1 M) IJS hydrochloric acid 20 μl and R engineering A standard buffer (pH 7, 4 phosphate buffer solution 50 mM, 0.25% bovine serum alzomine, NaC/8.QmM, EDTAg 5m
14,380 μl was added and stirred. Next, part 10
90 μl of R Engineering A standard buffer was added to μl to make 100 μl, and RIA was performed using α-L Fli and antibody.

As is clear from Figure φC, strain I Co/iK12S
HPLC elution obtained from BM102), α-N, E,
radioimmunoactivity appeared. In addition, no radioimmune activity against α-N and E was observed in the strains Fi and ColI WA802/pKO13 No. 0 used as controls.

Ampicillin 40μy/rut! strain in L-Gloss 51 supplemented with 0 co/i K128BM102.
D66〇-〇, cultivated to 7, and then cultivated PTG to an east longitude concentration of 1m.
M was added and cultured for an additional 2 hours. Bacteria were collected from this culture solution, and 10.9 y of wet microbial cells were obtained. This is 1°52
The suspension was suspended in 80 mel of a 70% formic acid solution containing cyanogen bromide, and reacted at room temperature for 24 hours. Distilled water was added to this reaction solution to make 5600me, and BP-Sephadic C-25
column. Column at 0° 1M acetic acid 11. water 50
After washing the ivy with 2j of 2M pyridine water, remove the adsorbed substance by 1
MNH4OH800mete was eluted. This 1MN
After freeze-drying the H4OH fraction, the residue was dissolved in 1M acetic acid 15WLe.
3X151 filled with Sephatex G-25
cm column.

The eluate was collected in 15me portions using 1M acetic acid.

Monitor each fraction with 280 nm UV, roughly dilute a 20 μl portion of each fraction, and analyze the amount of α-N and F- contained in each fraction using the R7IA method. did(
(See Figure 7). In FIG. 7, a total of 75me from fraction numbers 47 to 51 was collected and freeze-dried. 410mM remaining
It was dissolved in HCOONH48me and applied to a carboxymethyl cellulose column 0.9X48C+l+. Elution from the column is l Q m M HCOONH4 QQQm
A linear concentration gradient was performed using e and 500mM HCOONH4 200me. The eluate was fractionated by bme,
Monitored with UV at 280 nm. Roughly take a 10 μj aliquot from each fraction, dilute it appropriately, and calculate the amount of α contained in each fraction.
-N.

E. The amount of was measured using the RIA method. The results are shown in the 7th section.
Figures & d are shown. Q, 35M HCOONH4 eluted fractions had Immuturi activity, and α-N as a single peak. It was thought that W was eluted.

Therefore, this active fraction, fractions 66 to 69,
By collecting and freeze-drying, we were able to obtain four chemically pure α-N and L molecules. Table 3 shows the bacterial strain, K
, α-N obtained from 51 cultures of coji K12SBM102. The yield at each purification step of E is shown. Calculating from these results, strain E, coli K12B
BM102 at least 5X10 molecules of α-N per cell. (See Table 3).

Table 8 α-N from E, Co/i wAso2/pαNEi2
, Fi, purification 1, CNBr treatment a 1271 6.6
100 (Note) E, coji strain wA802/
Wet bacterial cells 10.1 from culture solution 51 of pαN]1Th9
11 and total protein 1271 extracted from the bacterial cells.
1t was treated with cyanogen bromide and used for purification of α-N, E.

Calculated from rice amino acid analysis data + Calculated from Radioimz Ansei Mix the α-N, F- fractions and fractions 66 to 69 separated and purified in Figure 7a, and add 180 μl of 1M acetic acid to 20 μl of the mixture. Mix and add a portion of 4°ttl to p
-Bondapak CHB (Quarters) 0.4X
Analyzed by HPLC using a 25 cm column, heftide was monitored at a UV wavelength of 210 nm.

As is clear from FIG. 8, the analyzed α-N, K showed a single peak. Moreover, this elution position is chemical. It's better than that. -N, F, (7) Complete elution position 8. Agreed. Furthermore, as a result of measuring each fraction obtained by HPLC with R Engineering A using α-N-L antibody, α-N and E were detected at a position that completely coincided with the peak obtained by monitoring at a UV wavelength of 210 nm. , B, Determination of amino acid composition and amino acid sequence α-N constructed by the above method, 11! , 4μ2 in 6N containing 0.1'% phenol, 0.02% 2-mercaptoethanol
After dissolving in 100 μl of hydrochloric acid and hydrolyzing at 110°C for 20 hours, each amino acid was analyzed using an amino acid analyzer (Hitachi≠835
), Pro l, Q, Gly
2. ■,Lev 1. Q, Tyr 2. Q.Ph.
el, 0. L78 2. A result of 1.0 was obtained, which was in good agreement with the natural product. The amino acid sequence was then determined using the Dansil-Edman method (4aney/-1aman) [Gl
ay, W, R.

(1972) Method in Enzymol
When analyzed using oyy, 25°383], )(
-Tyr-Gly-017-Phe-Leu-krf-
The sequence Lys -Tyr-Pro-Lys-〇H was obtained, which completely matched the naturally occurring α-N, O.

α-N purified by the above method. Using the inhibitory effect of electrically stimulated contraction of the guinea pig ileal slave muscle to suppress the morphine activity of E.
W, D, H, z < 7 The method of Paton et al. [Paton.

W, D, Het a/ (19Q13) J, Phys
iol.

194, G3]. When the concentration at 50% inhibition of contraction (CoC30) was determined, 'W, Co11
α-N purified from K12SBM102, Fl! , the standard α-N, which had an engineering C50 value of 5.2 nM,
E, value engineering Co.

= 5. (In good agreement with M.

[Brief explanation of drawings]

Figure 1 shows one embodiment of the double-stranded polydeoxyribonucleotide containing the α-N, L gene used in the present invention and the oligodeoxynucleotide CF used for its synthesis.
'1 to F'8), Figure 2 shows the reaction formula for the synthesis of fully protected bifunctional trimeric deoxyliponuthaleotide, and Figure 8 shows the method for constructing a recombinant plasmid containing α-N, E, and genes. , Figure 4 a and b are B and Co/i, respectively.
K12WA802/1) K013 and B, C
O/i K12BBM102 bacterial cells were digested with cyanogen bromide and purified by adsorption method - elution pattern by HPLC; Figure C shows the elution pattern by standard radioimmunoassay (R Engineering A) for the fraction indicated by the arrow in b. The pattern is shown in Figure 5. After decomposing cozI K128BM102 cells with cyanogen bromide, they were adsorbed onto a column of sp-Sephadic C-25, and the fraction eluted with 1M NH40H was further applied to a column of Sephadex G-25. Figure 7 shows a photograph of eluting with l MHAc, m, co/i K12SBM.
This photo shows the nucleotide sequence of a small fragment obtained by digesting a plasmid isolated from IQ2 with a restriction enzyme using the Maxam-Gilbert method. Applicant: Suntory Limited; (aqn1/6u) uHl, IdJopua-oiu
-cya → 1 ・゛yama CD, ,,, , , ,, 'l', j:) Umbrella 1". 11 Continued from page 1 0 Inventor Takehiro Oshima 3-4 Bessho Nakanomachi, Takatsuki City 0 Author: Masaharu Tanaka 1-8-46, Minamikanedamachi, Suita City 0 Author: Osuewa Hiro 1-7, Inaba-cho, Ibaraki City 0 Author: Toshiyuki Matsuo, 766 Oaza Ki, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture 1: 3 1. Indication of the case 1982 Patent Application No. 463303
No. 2, Name of the invention: Process for producing Alpha Neo Endorphin 3, Relationship with the case of the person making the amendment Patent applicant address: 2-1-40-4 Dojimahama, Kita-ku, Osaka-shi, Osaka Prefecture;
Agent address: 4-46-8 Kitahama, Higashi-ku, Osaka, 541 Contents of amendment (1) "(See Figure 7) on page 37 of the specification, line 7 from the bottom.
”, and in the 9th line of page 42, ``Ta (see Figure 7).Lean to the side of Figure 7'', and in the 2nd line from the bottom of the same page, ``The result -'' was changed to 1. As a result, ” and the last line of the same page “Figure 7a
Shown below. ", and in the 10th line of page 44, ``In Figure 7 a'' was corrected to ``as above,'' and in the 5th line from the bottom of the same page, ``As is already clear in Figure 8,'' Delete page 47, lines 10-13 “
Figure 7 shows the ``analyzed photo''. (2) Engraving of Figure 6-1 to 8 of the drawings (changes in content)
As shown in the attached sheet A! T's ('J ■ O - Fable - 916 Figure-6 ■ gsm-a F8 G

Claims (1)

[Claims] 1. The following nucleotide sequence
-2ε and 93 yxo (In the sequence, A represents deoxyadenylic acid, G deoxyguanylic acid, C deoxycytidylic acid, T represents thymidylic acid residue, J represents A-i or G, (T or C1L represents A, C, T or G, 1 and 1 can be replaced with 22, and 1 and 2 can be replaced with AGJ, respectively) transformed with a plasmid incorporating a polydeoxyribonucleotide. A method for producing alpha-neo-endorphin 2, which is characterized by culturing a strain of the genus Elicia in an aqueous nutrient medium and producing alpha-neo-endorphin as a protein component in the culture.・Method 3 according to claim 1, in which a hybrid protein containing neo-endorphin is collected, it is decomposed, and alpha-neo-endorphin is separated from the decomposed product. Method 5 according to claim 1, wherein the recipient bacterium for transformation is N. coli K1.2, wherein the transformed bacterium is N. coli K1.2. The method 6 according to claim 1, wherein the polydeoxy oligonucleotide has the following nucleotide sequence: Method 7 according to claim 1, wherein the plasmid for incorporating polydeoxynucleotide is p
BR822 and λplac5 (7) A plasmid created by treating each DNA with the restriction enzyme KcoR and ligation, with the two WcOR cleavage sites having been deleted, as described in claim 1. Method 8, Nijieri E/7 e:l! J K12SBM1
0291 The following nucleotide sequence TATI GeO2GGTB TTC4CCT5
CGT6AAG7 TATB CCGg AA
Polydeoxyribonucleotide 10 with G16, oligodeoxyribonucleotide 5' with each of the following sequences (11AATTCATGT"5' (2) pAKGGLGGLTTKcs: 5' + 3) pT IJ CG L A A , r T
A K C"5' 141 pCLAA, TTAATAG"6' (51G A T CCT A T T "5' +61 1) AJ/TTI/()GK/TAJ/T"
5/, 51 (7) The following nucleotide sequence A A J T, A A T A G "TT, characterized by reacting pTvcGvAGK/AAvc and 5'(81pCI/CCK'TACATG" in the presence of ligase) T/
ATTATCCTAG" (In the above sequence, J represents A or G, J' represents T or C so as to form a base pair with the opposing J in the above double-stranded sequence, and in the same manner, K represents D or G. 11, the following nucleotide sequence: 12 oligodeoxyribonucleotides with the nucleotide sequence "AATTCATGT", 14 oligodeoxyribonucleotides with the nucleotide sequence "ATaatoarrTcc", 14 oligodeoxyribonucleotides with the following nucleotide sequence 5'CGAAGTAATAG", 15 oligodeoxyribonucleotides with the following nucleotide sequence "G A Oligodeoxyribonucleotide 16 has the following nucleotide sequence "ACTTCGGATACT"; Oligodeoxyribonucleotide 18 has the following nucleotide sequence 5'TAoGAAGGAAc3'; oligodeoxyribonucleotide with