JP2621903B2 - Escherichia coli ribonuclease H expression vector - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to the production of Escherichia coli ribonuclease H by a DNA recombination method, and more specifically, an expression vector capable of efficiently expressing an Escherichia coli ribonuclease H gene, and the expression vector The present invention relates to a transformant containing a vector, and a method for producing Escherichia coli ribonuclease H using the transformant.

[Prior art and its problems] Ribonuclease H was first isolated from calf thymus by Stein (H.) and Hausen (P.) (Science, 166 , 393-395 [1969]). Later, it was found that it was widely distributed in all kinds of organisms from Escherichia coli to humans.

Among them, E. coli ribonuclease H was first isolated by Miller, HI et al. (Journal of Biological Chemistry, 248 , 2621-2).
645 [1973]), and the gene was transformed by Horiuchi et al.
H.) (Proceedings
Of the National Academy of Sciences, 78 , 3770-3774 [1981], and its entire nucleotide sequence was determined by Kanaya, S. and Crouch, RJ (Journal of the Biological
Chemistry, 258 , 1276-1281 [1983]). Escherichia coli ribonuclease H is a hydrolase consisting of 155 amino acids and having a molecular weight of 17 kd, and has a substrate specificity of specifically cleaving only the RNA strand of a DNA / RNA hybrid by an endo action. Based on such substrate specificity, it has extremely high utility value in the field of biochemistry. In particular, in genetic engineering, which has been remarkably developed in recent years, it is widely used as an enzyme having various actions as described below.

1) Removal of template mRLA during cDNA cloning.

2) Removal of the poly A region of mRLA.

3) Fragmentation of RNA.

Although the importance of this enzyme has been increasing with the development of genetic engineering, its production in Escherichia coli is extremely low at a maximum of about 10 μg per culture in its natural state, and the shortage of supply to demand is evident. is there. Therefore, an attempt has been made to produce a cloned enzyme using Escherichia coli using the DNA recombination method, and the products thus obtained have already been commercialized by various manufacturers [eg, Bethesda Research.・ Laboratory (Bethesda Research Labora)
tory), Pharmacia, and Takara Shuzo]. However, the yield in the production of the cloned enzyme is only about 20 times that in the natural case, at most 200 μg /, and does not supply a large amount of E. coli ribonuclease H. Therefore, it has been desired to develop an expression system that has a high productivity and enables mass production of ribonuclease H.

Further, in the conventional method, in addition to low productivity in the E. coli host, there has been a problem that the yield of the produced enzyme during the isolation and purification step is reduced. That is, conventional methods for isolating and purifying ribonuclease H are described in Miller et al. (Miller, HI, Journal of Biological Chemistry, 248 , 262).
1-2645 [1973]), Ito and Tomizawa (Itoh, T.an
d Tomizawa, J., NAR, 10 , 5949-5965 [1982]) and Arendes, J., Journal of Biological Chemistry, 257 , 4719-4722 [198].
As indicated by 2)), these usually consist of disrupting the cells and performing a combination of cation exchange, anion exchange and gel filtration column chromatography. However, in the conventional method, since the amount of enzyme production is extremely low, it is necessary to perform DNAse treatment, PEG precipitation, or ammonium sulfate fractionation prior to column chromatography, which considerably complicates the purification process, and as a result, The yield is to be significantly reduced. In general, it is known that as the production of microorganisms increases, the purification method is dramatically simplified, and thus the recovery of the finally obtained product is also increased. Therefore, the production of ribonuclease H in Escherichia coli is improved, and the increase in ribonuclease H content in the cells reduces the time and economic burden required for extraction and purification of the enzyme.
Enzymes can be supplied in large quantities at affordable prices.

[Means for Solving the Problems] In order to increase the production of ribonuclease H in Escherichia coli, the present inventors aimed to construct an expression vector capable of efficiently expressing the ribonuclease H gene (rnh). studied was added result, efficiency P _L promoter tac promoter or bacteriophage lambda, and E. coli host cells transformed with a plasmid containing the structural gene of E. coli RNase H in the domination of the promoter, the present enzyme They have found that they produce well, and have completed the present invention.

That is, the present invention contains the structural gene of E. coli RNase H in the domination of the P _L promoter, and the tac promoter tac promoter or bacteriophage lambda, provides replication and expression vector capable of expressing in E. coli Is what you do.

The present invention also relates to a method for producing Escherichia coli ribonuclease H, which comprises transforming an Escherichia coli host with the above expression vector, and culturing each of the obtained transformants under conditions suitable for expression of the Escherichia coli ribonuclease H gene. And isolating and purifying ribonuclease H in each culture solution.

The present invention also provides a transformant containing the expression vector of the present invention.

Furthermore, the present invention relates to a method for isolating and purifying Escherichia coli ribonuclease H from a culture solution, wherein after the culturing of the transformant, the cells are collected from the culture solution, and the lysozyme-ED
The present invention provides a method comprising lysing by TA and sonication, and subjecting the obtained cell lysate to column chromatography in which a DE-52 column and a P-11 column are connected in this order.

Expression vectors of the invention, plasmids pDR600 and pPL8
01 corresponds to the present inventors (Kanaya and Crouch, Journal of Biological Chemistry, 258 , 1276-1).
281 [1983]) and the plasmid pSK750 containing the structural gene (rnh) of Escherichia coli ribonuclease H (hereinafter simply referred to as ribonuclease H) and its promoter region, cloned from the E. coli chromosome by rnh. It was constructed using it as a source (see FIG. 1).

That is, the 820 bp EcoR I-Pst I fragment of plasmid pSK750 is cut out and inserted into the EcoR I-Pst I site of commercially available plasmid pUC19 to obtain plasmid pSK820. This plasmid pS
K820 cut with restriction enzyme HgiA I, 801 bp containing rnh gene
An HgiA I fragment was obtained and treated with T4 DNA polymerase to 3 ′
The protruding portion is smoothed off. Next, using the blunt-ended HgiA I fragment, an expression vector of the present invention is constructed as follows.

Construction of plasmid pDR600 The above 801b pHgiA I fragment was inserted into the Sma I site of plasmid pUC18, and plasmid pUC1
A plasmid having the lach promoter and the rnh gene in the opposite direction was named plasmid pKS801 and used for the subsequent experiments.

After deleting the 230 bp EcoR I fragment from plasmid pKS801, the EcoR I site was converted to a sal I site with a sal I linker,
Obtain plasmid pKS600. Then, a 600 bp Xba I-Sal I fragment containing the rnh gene from the plasmid pKS600, a 4.3 kb plasmid containing the ptr gene of the plasmid vector pDR42S (Patent Application No. 62-218973) for expressing E. coli protease III.
By substituting the XbaI-SalI fragment, one expression vector of the present invention, plasmid pDR600, is obtained. FIG. 2 shows a restriction map of plasmid pDR600. In plasmid pDR600, the lac promoter and the tac promoter are located in tandem, and dominate the rnh gene 3 'downstream. Incidentally, since the promoter of the rnh gene has an HgiA I site in its −35 region, a part of the HgiA I fragment containing the rnh gene was cut out from the plasmid pSK820 when the HgiA I fragment was cut out. Conceivable.

Construction of plasmid pPL801 The above-mentioned 801bpHgiA I fragment was obtained from commercially available plasmid pPL-lamb.
was inserted into the unique (sole) Hpa I site of da (Pharmacia), among the resulting plasmid, and the P _L promoter
Select those with the same orientation of the rnh gene, and
Name it 801. The restriction map of plasmid pPL801 is shown in FIG. In plasmid pPL801, P _L promoters of bacteriophage lambda dominates the rnh gene of the 3 'downstream. In the case of this plasmid as well, it is considered that the promoter of the rnh gene does not function for the same reason as in the case of the plasmid pDR600.

Expression of ribonuclease H in Escherichia coli Using the above plasmid pDR600, Escherichia coli K-12 strain JM10 was used.
9 (recA1, Δlacpro, endA1, gyrA96, thi-1, hsdR1
7, SupE44, relA1, F'traD36, proAb, lacIgz △ 15)
Is transformed. Desired transformant, E. coli JM109 / pDR
600 are selected based on ampicillin resistance, and ribonuclease H is expressed by culturing the transformant with isopropyl β-D-thiogalactoside (IPTG) during its logarithmic growth phase.

On the other hand, E. coli that produces a temperature-sensitive cI repressor using the plasmid pPL801, for example, E. coli K-12 strain N483
0 (f ^- su ^{°, his -, ilu -, galK} -, △ 8 (△ chiD-pg)
[Λ △ Bam, N, cI857HI] is transformed. The P _L promoter is located in the temperature region where the c I repressor is stable (32
(Less than 40 ° C)
Transformants selected based on ampicillin resistance, E. coli N4830 / p
When PL801 is cultured in the logarithmic growth phase by increasing the culture temperature from 30 ° C. to 42 ° C., the promoter functions, the expression of ribonuclease H is induced, and the desired enzyme ribonuclease H is mass-produced.

Isolation and purification of ribonuclease H Isolation and purification of ribonuclease H in the method of the present invention are performed in the following order.

1) Lysozyme-EDTA treatment of E. coli cells 2) Ultrasonic treatment 3) DE-52-P-11 column chromatography 4) Sephacryl S-300 (Super Fine) column chromatography The present invention obtained as described above. After collecting the high ribonuclease H-producing bacteria, the cells are subjected to lysozyme-EDTA treatment and ultrasonic treatment to solubilize the ribonuclease H in the cells. Centrifuge the cell lysate and centrifuge supernatant (crude extract)
After dialysis, DEAE cellulose column chromatography,
A purified sample is obtained by applying the same to a P-11 cellulose column chromatography and a Sephacryl S-300 column chromatography in this order. Here, since the ribonuclease H does not adsorb to the DEAE cellulose, the sample is applied by connecting the elution port of the DEAE cellulose column and the injection port of the P-11 cellulose column (DE-52-P-11 column chromatography), After washing the column, it is possible to remove and treat the DEAE cellulose column. In this case, the substantial column chromatography operation can be regarded as two steps instead of three steps, and the entire ribonuclease H purification step is simplified to three steps together with the extraction operation.

[Effects of the Invention] A transformant containing the vector of the present invention is cultured as described above, and the yield of ribonuclease H isolated and purified from the resulting culture broth according to the method of the present invention is determined by using these vectors. Yield in E. coli not containing (0.005mg /
) About 4000 times 20.0 mg / (for Escherichia coli N4830 / pPL801) and about 1000 times 5.0 mg / (Escherichia coli JM109 / pDR600)
Case) and was significantly improved. Thus, according to the present invention,
Enzymes for genetic engineering useful in the field of biochemistry can be obtained efficiently and in high yield.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Construction of plasmid pDR600 pSK750 was used as the rnh gene source. This plasmid p
SK750 (10 μg) was digested with 50 units each of Pst I and EcoRI in a 100 μ reaction solution at 37 ° C. for 1 hour. The digest was then subjected to 1.5% agarose gel electrophoresis. r
The 820 bp EcoR I-Pst I fragment containing the nh gene was extracted from agarose gel by electroelution (electroelution), and then purified by DE-52 column chromatography. 820bp EcoR I recovered by ethanol precipitation
The amount of the -Pst I fragment was about 1 μg. On the other hand, the plasmid vector pUC19 (manufactured by TOYOBO) (2 μg) was placed in a 50 μ reaction solution at 37 ° C. with 10 units of Pst I and EcoRI.
The digest was digested completely for 1 hour and subjected to 0.7% agarose gel electrophoresis, and the 2.7 kb EcoRI-PstI fragment was eluted and purified in the same manner as the 820 bp fragment. The recovery was almost 100%.

The 820 bp and 2.7 kb EcoR I-P obtained as described above
0.1 μg of each stI fragment was mixed, and mixed with 5 units of T4 DNA ligase (manufactured by TOYOBO) at 16 ° C. in a 20 μ reaction solution.
For 30 minutes to effect plasmid cyclization. Next, E. coli JM109 was transformed with the circularized plasmid,
Plasmid pSK820 was obtained from the transformant.

Next, this plasmid pSK820 (10 μg) was
Was digested with 100 units of HgiA I at 37 ° C. for 1 hour. After phenol-chloroform extraction, DNA was recovered by ethanol precipitation, and 2.5 units of T4 DNA polymerase was added in a 20 μl reaction solution, and the mixture was added at 37 ° C. for 1 hour.
The reaction was performed for 5 minutes. After terminating the reaction by treating at 65 ° C. for 5 minutes, the reaction solution was subjected to 1.5% agarose gel electrophoresis, and the 801 bp HgiA I fragment containing the rnh gene was replaced with 820 bp.
It was eluted and purified as in the case of the bp EcoR I-Pst I fragment. The recovered amount of DNA was 2 μg. On the other hand, the plasmid vector pUC18 (manufactured by TOYOBO) (2 µg) was completely digested with 10 units of Sma I in a 50 µ reaction solution at 37 ° C for 1 hour, and then 1 unit of Escherichia coli alkaline phosphatase was added. The reaction was performed at 37 ° C. After terminating the reaction by the phenol-chloroform extraction method, the DNA was recovered by ethanol precipitation. The recovery was almost 100%.

The 801 bp HgiA I fragment obtained as described above and Sma
After digestion with I, 0.1 μg of each of pUC18 treated with Escherichia coli alkaline phosphatase was mixed, and mixed with 5 units of T4 DNA ligase at 16 ° C. in a 20 μl reaction solution.
The reaction was allowed to proceed for 0 minutes to effect cyclization of the plasmid. Next, E. coli JM109 was transformed with the circularized plasmid,
PKS801 was obtained from the transformant.

Next, this pKS801 (2 µg) was completely digested with 10 units of EcoRI in a 20 µ reaction solution at 37 ° C for 1 hour. After collecting the DNA by ethanol precipitation,
After adding 2 units of DNA Polymerase (Klenow) in the reaction solution of the above, the mixture was reacted at 37 ° C. for 30 minutes, and then treated at 65 ° C. for 10 minutes. 20μ of DNA recovered by ethanol precipitation
After adding 1 unit of Escherichia coli alkaline phosphatase in the reaction solution and treating at 37 ° C. for 30 minutes, the reaction solution is
It was subjected to 0.7% agarose gel electrophoresis. The 3.3 kb DNA fragment was eluted and purified in the same manner as the 820 bp EcoR I-Pst I fragment. D
The recovered amount of NA was about 1 μg. 0.1 μg of the obtained DNA
Mix with Sal I linker (Takara Shuzo) 0.01μg (molar ratio 1: 5)
0) Then, 5 units of T4 DNA ligase were added to the reaction solution of 20 µ and treated at 16 ° C for 30 minutes. Escherichia coli JM109 was transformed with the plasmid thus cyclized, and plasmid pKS600 was obtained from the transformant.

Finally, the ptr gene in the E. coli protease III expression vector pDR42S was replaced with the rnh gene as follows. First, plasmid pKS600 (10 μg) was completely digested with 50 units of XbaI and 50 units of SalI in a 100 μl reaction solution at 37 ° C. for 1 hour, and then subjected to 1.5% agarose gel electrophoresis. A 600 bp Xba I-Sal I fragment was cut out and eluted and purified in the same manner as for the 820 bp EcoR I-Pst I fragment. The amount of DNA recovered by ethanol precipitation was about 1 μg.

On the other hand, plasmid pDR42S (Patent Application No. 62-218973)
5 μg was mixed with 50 units of Xba I and 100 μl of the reaction solution.
After digestion for 1 hour at 37 ° C with unit Sal I, 0.7
% Agarose gel electrophoresis. 3.0kb Xba I-Sal
The I fragment was cut out and eluted and purified in the same manner as in the case of the 820 bp EcoR I-Pst I fragment. The amount of DNA recovered by ethanol precipitation was about 1 μg.

The 600 bp Xba I-Sal I fragment obtained as above.
1 μg and 0.1 μg of the 3.0 Kb Xba I-Sal I fragment were mixed, and
5 units of T4 DNA ligase in the reaction solution
C. for 30 minutes to circularize the plasmid. Escherichia coli JM109 was transformed with the cyclized plasmid, and plasmid pDR600 was obtained from the transformant (see FIG. 1). A detailed restriction map of the desired plasmid pDR600 obtained as described above is shown in FIG. It was shown to. The transformant Escherichia coli ( E. coli ) JM109 / pDR600 has been deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology (Deposit No. 9707, deposited on November 1, 1987). .

Example 2 Construction of plasmid pPL801 DNA containing the rnh gene in the same manner as described in Example 1.
A 801 bp HgiA I fragment was prepared. This 801 bp HgiA I fragment was inserted into the vector plasmid pPL-Iambda (Pharmacia) by the following method.

10 μg of the vector plasmid pPL-I ambda was completely digested with 100 units of Hpa I in a 100 μ reaction solution at 37 ° C. for 1 hour, then 1 unit of Escherichia coli alkaline phosphatase was added, and the mixture was further reacted at 37 ° C. for 30 minutes. .
After terminating the reaction by the phenol-chloroform extraction method, the DNA was recovered by ethanol precipitation. DNA recovery was almost 100%. After digestion with HpaI, 0.1 μg each of pPL-I ambda treated with Escherichia coli alkaline phosphatase and the above-mentioned blunt-ended 801 bp HgiA I fragment were mixed, and mixed with 5 units of T4 DNA ligase at 20 ° C. at 16 ° C. For 30 minutes to effect plasmid cyclization. Next, Escherichia coli N4830 was transformed with the cyclized plasmid, and plasmid pPL801 was obtained from the transformant.

The restriction map of the desired plasmid pPL801 obtained as described above is shown in FIG. The transformant Escherichia coli N4830 / pPL801 has been deposited with the Institute of Microbial Industry and Technology, National Institute of Advanced Industrial Science and Technology (Microorganism Bacteria No. 9709, deposited on November 14, 1987).

Example 3 Culture of transformant and preparation of ribonuclease H-containing cells 1. Transformed Escherichia coli JM109 / pDR600 Escherichia coli K-12 strain JM109 (phenotype described above) transformed with plasmid pDR600 described in Example 1 was 80 μg / μ
And shaking culture at 37 ° C. in an LB medium containing ampicillin.
When the culture broth grew until the turbidity reached a Kret value of around 100, 0.8 mM IPTG was added, and the mixture was further shaken for 4 to 6 hours and then collected. The obtained cells were stored frozen at -20 ° C.

2. Transformed Escherichia coli N4830 / pPL801 Escherichia coli K-12 strain N4830 (phenotype described above) transformed with the plasmid pPL801 described in Example 2 was shake-cultured at 30 ° C. in an LB medium containing 80 μg / ampicillin. When the turbidity of the culture broth grew to a Kret value of around 100, the culture temperature was raised to 42 ° C., and the mixture was further shaken for 4 to 6 hours.
The bacteria were collected. The obtained cells were stored frozen at -20 ° C.

Example 4 Extraction and Purification of Ribonuclease H from Transformant 1. Ribonuclease H from E. coli N4830 / pPL801 cells
Extraction and purification of N4830 / pPL80 collected from the 16 cultures obtained in Example 3.2
1 cell about 50g 10m containing 0.1M NaCl and 25% sucrose
After suspending in 300 ml of M Tris-HCl buffer (pH 8), a 0.3 M Tris-HCl buffer containing 300 mg of lysozyme and 0.1 M EDTA (p
H8) 75 ml were added. After standing on ice for 5 minutes, the bacteria were disrupted by sonication. Then, at 15000 rpm for 30 minutes, 4
The mixture was centrifuged at 0 ° C., and the supernatant was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM β-mercaptoethanol, 1 mM EDTA and 10% glycerol (buffer A). By the above operation, the ribonuclease H in the bacterial cells is almost
100% solubilized. Conventional polyethylene glycol (P
In the method using EG) (without sonication), the recovery of the enzyme in the solubilized fraction was extremely low at 3% or less, so the above method has been significantly improved in this respect.

Next, the dialyzed sample solution was applied to DE-52 and P-11 columns equilibrated with buffer A. The column sizes of DE-52 and P-11 are φ2.6 × 20 cm (〜110 ml) for the former and φ1.8 × 16 cm (〜40 ml) for the latter. Frozen in 11 column inlets and tubes. Under these conditions, ribonuclease H is adsorbed on the P-11 column through the DE-52 column, so that there is no problem in connecting the two columns in this way, and as a result, it is necessary for the column chromatography operation. The time can be significantly reduced.

After application of the sample, the column was washed with about 200 ml of buffer A (about twice the DE-52 column volume), the DE-52 column was removed, and about 40 ml (P-11 column volume) of buffer solution was further removed. A washed the P-11 column. Then, the NaCl concentration was 0.5 M
The enzyme was eluted by linearly increasing to.
The recovery of ribonuclease H in this DE-52 and P-11 column chromatography was about 80%, and the purification purity was increased from 20% to 90%.

Finally, the eluted fraction from the P-11 column was added to 1 mM DTT, 1 mM
Sephacryl S equilibrated with 10 mM Tris-HCl buffer (pH 7.5) containing M EDTA, 0.08 M Nacl and 10% glycerol
-300 Sperfine column chromatography (φ
1.6 × 190cm) to give ribonuclease H
Was prepared until SDS-PIGE showed a single band.

The recovery of the present enzyme in this Sephacryl S-300 column chromatography was about 90%. Therefore, the purification yield of ribonuclease H throughout the entire purification process is 72
%. The yield of the purified preparation is about 20 mg /
The yield from E. coli N4830 transformed with the plasmid pPL-Iambda without the
It corresponds to 0 times.

2. Ribonuclease H from E. coli JM109 / pDR600 cells
Extraction and Purification of Ribonuclease H Using the bacterial cell solution prepared in Example 3.1, ribonuclease H was extracted and purified in the same manner as in 1 above.

The yield of the purified preparation from JM109 / pDR600 is about 5 mg /, which is lower than that from N4830 / pPL801, but still pD
This is equivalent to 1000 times that of JM109 without R600.

 The above results are summarized in Table 1.

· N4830 / pPL-Iambda, N4830 / pPL801 is activated the P _L promoter by increasing the 42 ° C. from 30 ° C. The culture temperature logarithmic growth phase.

-JM109 / pUC18 and JM109 / pDR600 activated the tac promoter by adding IPTG during the logarithmic growth phase.

[Effect of the Invention] As described above, ribonuclease H is an enzyme having a high value as a reagent. Until now, cloned Escherichia coli ribonuclease H has been commercially available, but its expression level in Escherichia coli is not so high as about 0.2 mg /. Therefore, it is difficult to purify it in large quantities. It is one. The present invention provides an expression level of this cloned ribonuclease H in E. coli of 20 mg.
This level enables the enzyme to be supplied at a lower cost to promote the development of research utilizing the enzyme.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the construction of plasmids pPL801 and pDR600. FIG. 2 is a restriction map of plasmid pDR600. FIG. 3 is a restriction map of plasmid pPL801.

Continued on the front page (51) Int.Cl. ⁶ Identification number Reference number in the agency FI Technical indication (C12N 9/22 C12R 1:19)

Claims (13)

(57) [Claims] 1. An expression vector containing the replication origin derived from the plasmid pUC18, a tac promoter and a structural gene of Escherichia coli ribonuclease H under the control of the tac promoter, and capable of replication and expression in Escherichia coli. 2. The expression vector according to claim 1, which is a plasmid pDR600. Wherein it is contained the structural gene of E. coli RNase H in the domination of the P _L promoter and the P _L promoters of bacteriophage lambda, replication and expression vector capable of expressing in E. coli. 4. The expression vector according to claim 3, which is a plasmid pPL801. 5. The expression vector according to any one of claims 1 to 4, which contains an ampicillin resistance gene as a selection marker. 6. A transformant obtained by transforming an E. coli host with the expression vector according to any one of 1-5. 7. The transformant according to claim 6, wherein the host is E. coli K-12 strain JM109 or E. coli K-12 strain N4830. 8. Escherichia coli JM109 / pDR600 or Escherichia coli N4830 / pP
8. The transformant according to item 6 or 7, which is L801. 9. The method according to claim 6, wherein the transformant is cultured under conditions suitable for expressing the E. coli ribonuclease H gene, and E. coli ribonuclease H is recovered from the resulting culture. A method for producing Escherichia coli ribonuclease H comprising: 10. Escherichia coli JM109 / pDR600 is lysed with isopropyl-
10. The method according to claim 9, comprising culturing in the presence of β-D-thiogalactoside. 11. The method according to claim 9, comprising changing the culture temperature of Escherichia coli N4830 / pPL801 from 30 ° C. to 42 ° C. during the culture. 12. After the completion of the culture, the transformant cells are solubilized by sonication, and the resulting solution is subjected to column chromatography to isolate a desired peptide. The method according to any one of -11. 13. The method according to claim 13, wherein DE-
13. The method according to claim 12, wherein a column in which 52 columns and P-11 columns are connected in this order.