JPH0790000A - Separation of peptide - Google Patents

Abstract

PURPOSE:To separate a peptide in a short time at a low cost without using protein denaturation agent and surfactant by producing granules containing a specific fused protein by E.coli, separating the granules from the E.coli and contacting with a protease at a specific pH to effect the decomposition of the fused protein. CONSTITUTION:A gene coding a fused protein of the formula [L is amino acid sequence enabling the formation of granule by fused protein in E.coli; P is objective peptide; X is amino acid (sequence) containing X-P bond digestible by protease] is expressed in E.co1i, granules containing the fused protein of the formula are produced by the E.coli, the granules are separated from the E.coli, the separated granules are made to contact with a protease at a pH to effect the (partial) dissolution of the granule, promote the partial digestion of the fused protein with the protease and not to cause the deactivation of the protease and the objective peptide separated from the fused protein is further purified.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a granular fraction containing a fusion protein produced by Escherichia coli genetically engineered, without the use of a protein denaturing agent or a surfactant for the target peptide in the fusion protein. The present invention relates to a method for isolation and purification.

[0002]

2. Description of the Related Art In recent years, genetic engineering methods have been widely used for producing physiologically active peptides. However, the peptide having a relatively short chain is decomposed by being digested by the protease in the host cell, resulting in a low yield. Therefore, a method of connecting a gene of a bioactive peptide to a gene of a leader protein (Leader protein) to produce a long-chain fusion protein, and then isolating the bioactive peptide from this fusion protein is used (glucagon : JP-A-3-254692, brain natriuretic peptide (BNP):
15th Annual Meeting of the Molecular Biology Annual Meeting, December 1992, Abstract No. 4243; Japanese Patent Application No. 3-43641, etc.). When the expression efficiency of the leader protein itself used is high, it is expected that the fusion protein will be expressed with the same high expression efficiency.

It is generally known that when a desired physiologically active peptide is highly expressed in Escherichia coli, excess physiologically active peptide accumulated in Escherichia coli forms poorly water-soluble granules. This is also the case when the peptide of interest is expressed as a fusion protein. The poorly water-soluble granules containing this fusion protein have the drawback that they are less susceptible to degradation by the protease required to release the desired peptide from the fusion protein. Therefore, in order to isolate the target peptide from the granule, the following means (1) or (2) is used.

(1) A method of isolating and purifying a fusion protein from granules and then further isolating and purifying a desired peptide from the purified fusion protein. The purified fusion protein obtained from the above granules generally has higher solubility in water than the state of the granules. Therefore, various proteases are allowed to act on the purified fusion protein to cleave the leader protein, and the target peptide is isolated. Let go. This method has the drawback that it requires a complicated purification step of purifying the fusion protein from the granules and further purifying the target peptide from the purified fusion protein.

(2) After the granules are dissolved by adding a protein denaturant, a surfactant or an organic solvent,
A method for isolating a desired peptide by allowing various proteases to act. In order to dissolve the above granules, guanidine hydrochloride, urea, sodium thiocyanate, etc. are used as protein denaturants, sodium dodecyl sulfate, triton, etc. are used as surfactants, and organic solvents are used.
Ethanol, methanol, acetonitrile, dimethylformamide, dimethyl sulfoxide, etc. are used. This fusion protein solubilization method has been reported many times and is most widely used, but it has the following problems.

First, in order to allow the protease to act on the granule containing the fusion protein in the presence of the protein denaturant or the organic solvent, it is essential that the protease used is not inactivated in the presence of the protein denaturant or the organic solvent. Is. However, various known amino acid-specific proteases have different activities to some extent in the presence of protein denaturants or organic solvents. Therefore, this low activity is compensated by adding an enzyme amount more than necessary. As a result, the amount of protease used as a reagent increases and the overall cost increases.

Secondly, when the desired peptide is purified,
Although usually purified by ion exchange chromatography and high performance liquid chromatography (HPLC), the presence of large amounts of protein denaturants, organic solvents or detergents often interferes with these purification steps. That is, ion exchange chromatography becomes impossible due to high ion concentration due to protein denaturant or surfactant, or polarity change due to the presence of organic solvent, or the separation pattern is disturbed during chromatography due to the same cause. Occurs. Therefore, when the protease reaction is carried out in the presence of the protein denaturant or the surfactant, the protein denaturant or the surfactant must be removed by gel filtration or the like after the reaction is completed.

Thirdly, when a protein denaturant is industrially used, for example, guanidine hydrochloric acid is expensive, and further recovery equipment is required for recovery, resulting in high cost. Furthermore, the amount of urea used for ion exchange chromatography for peptide purification is enormous, and environmental pollution must be taken into consideration when it is discarded. Overall, the introduction of steps to remove protein denaturants increases the cost of producing the peptide of interest.

[0009]

DISCLOSURE OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, in which a target peptide in a fusion protein is extracted from a granule fraction containing the fusion protein produced by Escherichia coli genetically engineered. Another object of the present invention is to provide a method for isolation / purification without using a protein denaturant or a surfactant.

[0010]

[Means for Solving the Problems] The present inventors have proposed a method of degrading a fusion protein by allowing various proteases to act directly on granules containing the fusion protein without using a protein denaturant or the like to release the desired peptide. We have conducted intensive studies to develop As a result, the pH of the solution containing the granules
In order to complete the present invention, it was found that the target peptide can be efficiently released by setting the range to a pH range in which at least a part of the granules is dissolved, and is easily digested by protease, and protease is not inactivated. I arrived.

The method for isolating a peptide of the present invention includes the following steps (1) to (4) without using a protein denaturant: (1) A gene encoding a fusion protein represented by the following formula is used in E. coli. Expressing in E. coli to produce granules containing the fusion protein in E. coli: Formula: LXP (where L is an amino acid sequence that allows the fusion protein to form granules in E. coli, P is the peptide of interest, and X is the amino acid or amino acid sequence in which the XP bond is digestible by a protease), (2) separating granules from E. coli, (3) separating granules , Contact the protease,
A step of releasing the peptide P of interest from the fusion protein, wherein the pH range is such that at least a part of the granules can be dissolved, and the fusion protein is susceptible to selective digestion by the protease, and the protease is not inactivated.
Steps performed in the pH range, (4) Steps of collecting the released target peptide.

The method of the present invention is characterized in that not only a protein denaturant necessary for dissolving granules, but also a surfactant, an organic solvent and the like are not used.

In a preferred embodiment, the pH range is on the alkaline side of the isoelectric point of the fusion protein.

In a more preferred embodiment, the above pH range is pH 8.5 to 11.5.

The leader protein L used in the method for isolating peptides of the present invention is a protein having an amino acid sequence that enables the fusion protein to form granules in E. coli, and is, for example, human γ-interferon (H .
Teraoka et al., J. Biochem., 102, 607-612 (1987)), Delta-Cys-short human γ-interferon [4-92] (Teraoka et al., Basic and Clinical, 20, 4029-4034 (1986)), Human interleukin 2 (R. Devos et al., Nucl. Acids Res., 11,
4307-4323 (1983)), mouse interleukin 2 (N. K.
ashima et al., Nature, 313, 402-404 (1985)), human α-
Interferon 80A2 (European Patent Publication 173887), β-galactosidase (DV Goeddel et al., Proc. Natl. Acad.
Sci. USA, 76, 106 (1979)) and aminoglycoside 3'-phosphotransferase II (European Patent Publication 2641).
All or part of 18) (about 70 to about 170 amino acid residues) can be used.

The target peptide P obtained by the peptide isolation method of the present invention is a peptide having a relatively short chain length,
For example, human angiotensin I (Hum
an), H. Akagi et al., Chem. Pharm. Bull., 30, 2498 (198
2)), human atrial natriuretic peptide (Atrial Nat
riuretic Peptide (ANP, Human 1-28), K. Kangawa et al., B
iochem. Biophys. Res. Commun., 118, 131 (1984)),
Bradykinin (Bradykinin, DF Elliott et al., Bioche
m. J., 74, 15 (1960)), bovine β-casmorphine (β
-Casmorphin (Bovine), V. Brantl et al., Hoppe-Seyler's
Z. Physiol. Chem., 360, 1211 (1979)), C-type Natriuretic Peptide (CN
P, Human 1-22), T. Sudoh et al., Biochem. Biophys. Res.
Commun., 168, 863 (1990)), Delta Sleep-Inducing Peptide (DSIP), GA Sch
Oenenberger et al., Proc. Natl. Acad. Sci., USA, 74, 12
82 (1977)), Human dynorphin A (Hu
man), A. Goldstein et al., Proc. Natl. Acad. Sci., USA,
76, 6666 (1979)), β-Endorph
in, S. Horikawa et al., Nature, 306, 611-614 (1983)),
α-Endorphin, N. Ling et al., Proc.
Natl. Acad. Sci. USA, 73, 3942 (1976)), human endothelin-1 (Human), M. Yanagisawa
Et al., Nature, 332, 411 (1988)), Mouse Vasoactive Intesti
nal Contractor (VIC, Mouse), N. Ishida et al., FEBS let
t., 247, 337 (1989)), Met-Enkephalin (Met-Enke
phalin, M. Noda et al., Nature, 297, 431-434 (1982)),
Fibronecti Active Fragment (Fibronecti
n Active Fragment (RGDS), MD Piershbacher et al., Na
ture, 309, 30 (1984)), Galanin (Hum
an), M. Bersani et al., FEBS Lett., 283, 189 (1991)),
Glucagon-like pe
ptide, C. Orskov et al., J. Biol. Chem., 264, 12826 (19
89)), Magainin 1, M. Zasloff, Proc.
Natl. Acad. Sci. USA, 84, 5449 (1987)), α-Mating Factor, D. Stoetzler
Et al., Eur. J. Biochem., 69, 397 (1976)), porcine α-Neo-Endorphin (Porcine), K. K.
angawa et al., Biochem. Biophys. Res. Commun., 99, 871.
(1981)), peptide T (Peptide T, MR Ruff et al., FEB.
S Lett., 211, 17 (1987)), Sarafotoxin S6B
(Sarafotoxin S6B, C. Takasaki et al., Toxicon, 26, 543
(1988)), somatostatin (Somatostatin, P. Brazea
u et al., Science, 179, 77 (1973)), and tuftsin (K. Nishioka et al., Biochim. Biophys. Acta,
310, 230 (1973)).

X in the fusion protein is an amino acid or amino acid sequence whose XP bond can be digested by a protease, and is determined by the type of protease (described later) used. For example, Lys, Glu, Arg, Ile-Glu-Gly-A
An amino acid or amino acid sequence selected from the group consisting of rg, Asn and Phe. X does not exist in P but may exist in L, and when it exists in L, L is decomposed during digestion of the fusion protein, and the fusion protein acts as a protease. It is easy to receive and may be preferable.

L-X-P is expressed in Escherichia coli by the usual gene recombination technique. Generally, when a foreign peptide is highly expressed in Escherichia coli by genetic engineering, the excess peptide accumulated in Escherichia coli forms poorly water-soluble granules, and this LXP is also produced as granules. The method of highly expressing the fusion protein in Escherichia coli by genetic engineering to form granules is a general method for those skilled in the art, for example, JP-A-3-254692, 15th Annual Meeting of the Molecular Biology Annual Meeting, 1992. 12
Month abstract number 4243 (Japanese Patent Application No. 3-43641), European patent publication 26411
8, etc.

In order to separate the above granules, a usual method may be used. For example, after culturing Escherichia coli, cells are collected, washed with a buffer, and then treated with lysozyme. Then, the reaction solution is crushed by a cell crusher and centrifuged (3000 to 7000 × g, preferably about 5000 × g, 15 to 30
To prepare the granules.

Then, the separated granules are contacted with a protease having a specificity for X to release the target peptide P from the fusion protein. The pH condition in this step is a pH range in which at least a part of the granules can be dissolved, and a pH at which the fusion protein is susceptible to selective digestion by protease and protease is not inactivated.
It is a range.

The pH range in which at least a part of the granules can be dissolved greatly depends on the properties of the fusion protein and the granules, and therefore cannot be unconditionally defined. You can decide. Usually on the alkaline or acidic side of the isoelectric point of the fusion protein. For example, granules containing the fusion protein γ-IFN-Glu-Glucagon (computer-predicted isoelectric point is 9.75) are on the alkaline side of the isoelectric point without the use of protein denaturants, detergents or organic solvents. Part of the solution dissolves in the pH range of 10 to 11 so that it becomes cloudy and becomes a suspension, which is completely dissolved at about pH 12.
Similarly, in the case of other fusion proteins as well, generally, when the pH condition is set to the alkaline side of the isoelectric point, at least a part of the granule can be dissolved. In the above example, the pH in the alkaline range
Was selected, but in some cases, the dissolved pH on the acidic side is suitable.

The pH range in which the fusion protein is liable to be selectively digested by the protease is a pH range in which at least a part of the granules is dissolved, and the pH range in which the protease can act. For example, in the case of the above fusion protein,
It can be presumed that the pH range of at least 10 in which at least a part of the granules is dissolved is in the pH range that is easily digested by protease. When this fusion protein is digested with the above BLase, the stable pH range of BLase is 4-11, so pH 10-1
It turns out that the reaction should be performed in 1. When using other fusion proteins or other proteases, pH conditions can be set in the same manner. When the pH is on the alkaline side, pH 8.5 to 11.5 is usually preferable.

During the digestion reaction of the fusion protein with the protease, the pH is controlled within the pH range defined above. However, as the digestion reaction progresses, the granules are gradually decomposed to increase their solubility, and it is not always necessary to control the pH during the digestion reaction. Therefore, the pH may be adjusted only at the beginning of the reaction, or the pH may be controlled during the reaction.

As the protease used in the method for isolating the peptide of the present invention, for example, when granules containing a fusion protein are dissolved on the alkaline side, generally any neutral / alkaline protease can be used. In a preferred embodiment, the following amino acid specific proteases may be used.

Acidic amino acid-specific proteases can be used when the peptide of interest, which does not contain acidic amino acids, is expressed as a fusion protein via acidic amino acids. For example, when expressed as a fusion protein via glutamate, a protease produced by Bacillus licheniformis known as a glutamate-specific protease (hereinafter referred to as BLase, S. Kakudo et al., J. Biol. C
hem., 267, 23782-23788 (1992)), Mpr (A. Sloma et al.,
J. Bacteriol., 172, 1024-1029 (1990)), Bacillus s
Protease produced by ubtilis (hereinafter referred to as BSase, T.
Niidome et al., J. Biochem., 108, 965-970 (1990)), V8.
-Protease (GR Drapeau et al., J. Biol. Chem., 247, 6
720-6726 (1972), Sigma Chemical Co.), Streptomyce
Protease produced by s fradiae (hereinafter referred to as SFase,
K. Kitadokoro et al., Biochim. Biophys. Acta, 1163, 14
9-157 (1993)), a protease produced by Staphylococcus aureus (hereinafter referred to as SPase, K. Yoshikawa et al., Bioc.
him. Biophys. Acta, 1121, 221-228 (1992)) and the like can be used.

When the target peptide containing no aromatic amino acid is expressed as a fusion protein via an aromatic amino acid such as phenylalanine, tyrosine or tryptophan, the above-mentioned aromatic amino acid portion of these fusion proteins is cleaved. Proteases can be used. For example, α-Chymotrypsin (Si
gma Chemical Co.) and proteases with similar properties can be used.

When the target peptide containing no basic amino acid is expressed as a fusion protein via a basic amino acid such as lysine or arginine, a protease that cleaves the above basic amino acid portion of these fusion proteins is used. Can be used. For example, trypsin: Tr
ypsin (Sigma Chemical Co.), arginyl endopeptidase: Arginylendopeptidase (Takara Shuzo), and lysyl endopeptidase: Lysylendopeptidase
(Wako Pure Chemical Industries, Ltd.) can be used. In addition, an enzyme with trypsin-like specificity (Factor Xa: Factor-X
a (Takara Shuzo Co., Ltd.), Plasmin: Plasmin (Sigma Chem
ical Co.), Kallikrein (Sigma Chemic
al Co.), thrombin: Thrombin (Sigma Chemical C
o.), trypsin-like enzyme from actinomycetes: Trypsin-like Pro
teases from Streptomyces (Sigma Chemical Co., N. Yo
shida et al., FEBS Lett., 15, 129 (1971)) and papain group (Sigma Chemical Co.)) can be used.

In the case of granules which dissolve on the acidic side, proteases which are stable on the acidic side can be used. For example, as a protease that cleaves hydrophobic amino acids such as phenylalanine and tyrosine, pepsin (Sigma Chemic
al Co.), asparaginyl endopeptidase: Aspara
Ginylendopeptidase (Takara Shuzo Co., Ltd.) or the like can be used.

Other enzymes used on the alkaline or acidic side include thermolysin known as metalloproteases which cleaves the N-terminal side of leucine, isoleucine, etc .: Thermolysin (DAIWA KASEI K.
K.) can be used.

Table 1 shows the specificity and stability of the above proteases.
Indicates pH range and optimum pH. The fusion protein in the granule is a protease that is stable in a pH range where it is easily digested by protease, and a protease having specificity for the above X can be appropriately selected. If its specificity, stable pH range and optimum pH are known, it can be used depending on the properties of the fusion protein.

[0031]

[Table 1]

In general, the protease has increased stability as the concentration of the fusion protein as a substrate increases, and as a result, the stable pH range may be widened. Therefore, it may be usable outside the stable pH range.

The amount of protease used is 1/500 to 1/10000 weight or more, preferably about 1/1500 weight, of the granular protein. The reaction temperature varies depending on the protease used, but usually room temperature is preferred. The reaction time is
A sufficient time for the peptide of interest to be released from the fusion protein, ie 1 to 16 hours, preferably about 3 hours.

Purification of the desired peptide released from the fusion protein may be carried out by a conventional method, for example, isoelectric point fractionation method and crystallization using the difference in isoelectric point between the desired peptide and other products. Use If necessary, conventional methods such as HPLC and ion exchange chromatography may be used in combination.

Taking the fusion protein γ-IFN-Glu-glucagon (hereinafter abbreviated as γ-IFN / Glucagon) as an example,
The present invention will be described in more detail.

Granules containing γ-IFN / Glucagon (isoelectric point is 9.75 according to computer prediction, primary structure is shown in FIG. 1 and SEQ ID NO: 1 in the sequence listing) are more alkaline than isoelectric point.
It dissolves at (about pH 12), and even in the range of pH 10 to 11, it becomes a suspension in which some of the granules are dissolved, and is in a state in which it can sufficiently undergo an enzymatic reaction. Therefore, in this state, BLase
Digest with to isolate glucagon. The outline of this method will be described below.

BLase is the synthetic substrate benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide.
Its pH stability is 4.0 to 10.0, and the optimum reaction pH is about 8.0. However, since the stability region for proteinaceous substrates is different and that the stability of the enzyme is expected to increase when the substrate is a concentrated solution, we tried to act BLase at around pH 10. Granules (γ-IFN / Glucagon; final concentration 40-50 mg / ml) are completely dissolved at about pH 12, but pH 10
A lot of cloudiness occurs in the vicinity. If the reaction with BLase (1/1000 to 1/1500 weight of granular protein) is performed in this state, the fusion protein (γ
-IFN / Glucagon) disappeared and glucagon was produced (confirmed by analytical HPLC).

As the purification method, mainly an isoelectric point fractionation method utilizing the difference in isoelectric point of the product and crystallization were used. The glucagon isolated and purified by this method had exactly the same amino acid composition and amino acid sequence as natural glucagon. The purity of this glucagon was 99% or more in the HPLC purity test. This yield is about the same as in the case of the conventional method (JP-A-3-254692) in which guanidine hydrochloride, which is a protein denaturant, is added.

As described above, according to the method of the present invention, the desired peptide can be obtained easily and efficiently. Such a simple method has never been invented
It is considered that the granules are insoluble at the optimum pH of action of various proteases and are extremely unlikely to be affected by the action of protease, and the activity of the protease is reduced when the pH is out of the optimum action, so that the efficiency of the enzymatic reaction is also reduced. It was because it had been.

According to the method of the present invention, peptides containing no acidic amino acid such as glutamic acid (eg, glucagon, brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) are used. ) Etc.) is expressed as a fusion protein via glutamic acid, and BLase, BSase, V8 protease, SFase, SPase, etc. are used as glutamate-specific proteases to easily and efficiently obtain the peptide of interest.

[0041]

The present invention will be further described by the following examples.

(Example 1) (γ-IFN / Glucag using BLase
Isolation and purification of glucagon from on) Granules containing glucagon (fusion protein; γ-IFN / G
lucagon, the amino acid sequence of which is shown in FIG. 1 and SEQ ID NO: 1 in the sequence listing) was prepared as follows. After culturing the glucagon-producing bacterium (JP-A-3-254692) for 24 hours, centrifugation (Sharpless centrifuge (Kansai Centrifuge Co., Ltd.), 10,0
The cells were collected at 00-20,000 × g). Buffer the cells (8 g / l sodium chloride, 0.2 g / l potassium chloride, 2.9 g / l
Suspended in disodium hydrogen phosphate dodecahydrate, 0.2 g / l potassium dihydrogen phosphate, 5 mM EDTA, 10 mM β-mercaptoethanol and 10 mM benzamidine, pH 6.5),
After thoroughly stirring, the mixture was centrifuged (5000 xg, 30 minutes) to collect washed bacterial cells. The cells were suspended again in the above buffer solution, lysozyme was added thereto so that the final concentration was 2 mg / g, and the cells were reacted at 35 ° C. for 1 hour. Then, the reaction solution is sonicated or treated with a cell disruptor and centrifuged (5000 xg, 30
The insoluble fraction was precipitated by the following procedure and collected as a washed pellet. 5 g of this washed pellet was suspended in 25 ml of distilled water. The protein concentration of this suspension was 125 mg / ml (Bi
oRad Protein Assay). The protein concentration was further diluted with distilled water to about 40 mg / ml. Calcium chloride was added to this solution to a final 2 mM and then adjusted to pH 10.5 with 1N sodium hydroxide. BLase was immediately added to 1/1500 weight of the total protein amount, and the enzyme reaction was started at room temperature. Since the pH decreased with the progress of the reaction, the pH was controlled at 8.5 to 10.5.

The reaction progress was observed by HPLC. HPLC conditions were using a Vydac protein C4 column (4.6 x 150 mm),
Elution was performed by equilibrating the column with 37% acetonitrile containing 0.1% trifluoroacetic acid (TFA) in advance, adding the sample, and
It was performed as 47% acetonitrile containing 0.1% TFA per minute, and the flow rate was 1 ml / min. Detection was at 220 nm and 280 nm.
Under these conditions, glucagon elutes in 17-18 minutes. Two to three hours after the initiation of the enzymatic reaction, the starting fusion protein completely disappeared, and glucagon was produced at the same time. Immediately pH with 1N hydrochloric acid
The reaction was terminated at 2.5-3.0, and then impurities were removed by centrifugation (5000 xg, 10 minutes). The supernatant was adjusted to pH 11.0 to 11.5 with 1N sodium hydroxide. Immediately 1N
By adjusting the pH to 9.5 with hydrochloric acid and leaving it at room temperature for about 2 hours, impurities having an isoelectric point different from that of glucagon were precipitated. Impurities were removed by centrifugation (5000 xg, 10 minutes).

The obtained supernatant was adjusted to pH 3.0 with 1N hydrochloric acid. Then pH 5.5-7.0 with 0.5N sodium hydroxide.
And glucagon was precipitated as an isoelectric precipitation. 4 ° C
It was left for 8 to 24 hours to complete the precipitation of glucagon.
Glucagon was collected by centrifugation (3500 xg, 10
Minutes). Glucagon suspension of about 1 mg / ml, pH 10.5-1
After adjusting to 1.0 and dissolving completely, immediately pH 8.0 to 8.2
When the mixture was adjusted to, and allowed to stand at room temperature for 16 hours with slow stirring, crystals were precipitated. The crystals were collected by centrifugation or filtration to give 90 mg of crystals. The crystals were identical to natural glucagon in both amino acid composition and amino acid sequence (Table 2). Figure 2 shows the obtained glucagon HP
The chart analyzed by LC is shown.

[0045]

[Table 2]

(Example 2) (γ-IFN-Glu-B using BLase)
Isolation / purification of BNP from NP) A granule containing a brain natriuretic peptide (BNP) (fusion protein; γ-IFN-Glu-BNP, the amino acid sequence of which is shown in FIG. 3 and SEQ ID NO: 2 in the sequence listing), BNP was isolated and purified from the granules as follows according to the 15th Annual Meeting of the Molecular Biology Society (December 1992), Abstract No. 4243. The washing pellets were prepared according to Example 1 above. About 1 g of washed pellets
Suspended in 1 ml of 2 mM calcium chloride and adjusted to pH 10.2 with sodium hydroxide. The protein concentration of this solution is 45 mg / ml
(BioRad Protein Assay). BLase was immediately added at 1/500 weight of the total protein amount, and the enzyme reaction was started at room temperature. Since the pH decreased with the progress of the reaction, the pH was adjusted to 9.5-10.5 with 1.0N sodium hydroxide. Reaction progress is HPL
Observed by C. The HPLC conditions were a Cosmosil 5C18 column (4.6 x 150 mm), and elution was performed with 0.1% TFA in advance.
After equilibration with 10% acetonitrile containing 10% acetonitrile, the sample was added, and 30 minutes was performed as 25% acetonitrile containing 0.1% TFA, and the flow rate was 1 ml / min. Detection was at 220 nm and 280 nm. BNP is eluted in 25 to 26 minutes under the above conditions. The above reaction was analyzed over time and BNP was observed at 1.5-3.0 hours.
This peak that seems to be BNP was collected by HPLC under the same conditions as above. FIG. 4 shows a chart obtained by analyzing BNP by HPLC. As a result of amino acid analysis, it was the same as natural BNP (Table 3). About 100 g of BNP was obtained from about 1 g of the washed pellet.

[0047]

[Table 3]

(Example 3) (Isolation and purification of glucagon by various proteases) Glucagon-containing granules (fusion protein; γ-IFN / Glucag)
on, the amino acid sequence of which is shown in FIG.
Each of the proteases of BSase, BLase, α-chymotrypsin, trypsin, lysyl endopeptidase, and thermolysin was allowed to act on each other. The washed pellet (Example 1) was suspended in distilled water to obtain a protein concentration of 125 mg / ml (Bi
oRad Protein Assay). The protein concentration was further diluted with distilled water to about 40.0 mg / ml. Calcium chloride was added to this solution to a final 2 mM and then adjusted to pH 10.5 with 1N sodium hydroxide. Immediately, various proteases were added at 1/500 weight of the total protein amount, and the enzyme reaction was started at room temperature. Since the pH decreases with the progress of the reaction, adjust the pH to 9.5-1.
Adjusted to 0.5. The reaction progress was observed by HPLC. HPLC
The same conditions as in Example 1 were used. Approximately 3 to 16 hours after the initiation of the enzymatic reaction, the fusion protein as a raw material almost disappeared depending on the enzyme, and simultaneously glucagon or a fragment derived from the fusion protein was produced. 5, 6 and 7 show the results of HPLC analysis of each fragment generated by enzymatic degradation. Raw fusion protein (γ-I
FN / Glucagon) elutes at 27-29 minutes. The numbers shown above the peaks in FIGS. 5, 6 and 7 indicate the amino acid sequence numbers (SEQ ID NO: 1 in the sequence listing) of the starting fusion protein. This indicates that each of the proteases used reacted under alkaline conditions to generate glucagon and peptide fragments. Therefore, it is shown that, if the physiologically active peptide is appropriately arranged in the fusion protein, it can be similarly produced using various proteases.

Example 4 (Isolation / Purification of Various Peptides) Granules containing a fusion protein in which various target peptides were joined to a leader protein via an appropriate amino acid or amino acid sequence were prepared and carried out. According to Example 1,
The protease was allowed to act in the appropriate pH range. Tables 4 and 5 show combinations of these peptides, amino acids or amino acid sequences, leader proteins, proteases and reaction pH. In any case, the target peptide could be efficiently obtained.

[0050]

[Table 4]

[0051]

[Table 5]

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, from a granular fraction containing a fusion protein genetically engineered by Escherichia coli, the target peptide in the fusion protein can be obtained without using a protein denaturant or a surfactant. It can be isolated and purified in a short time and at low cost. By this method, the target peptide can be effectively produced industrially.

[0053]

[Sequence list]

[0054]

[SEQ ID NO: 1] Sequence length: 172 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn 1 5 10 15 Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile 20 25 30 Leu Lys Asn Trp Lys Glu Leu Ser Asp Arg Lys Ile Met Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln 50 55 60 Glu Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys 65 70 75 80 Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 85 90 95 Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu 100 105 110 Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys 115 120 125 Arg Lys Arg Ser Lys Ser Leu Val Asp Pro Phe Arg Ser Ser Glu His 130 135 140 Ser Gln Gly Thr Phe Thr Ser Asp Tyr Ser Lys Tyr Leu Asp Ser Arg 145 150 155 160 Arg Ala Gln Asp Phe Val Gln Trp Leu Met Asn Thr 165 170

[0055]

[SEQ ID NO: 2] Sequence length: 175 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence Gln Asp Pro Tyr Val Lys Glu Ala Glu Asn Leu Lys Lys Tyr Phe Asn 1 5 10 15 Ala Gly His Ser Asp Val Ala Asp Asn Gly Thr Leu Phe Leu Gly Ile 20 25 30 Leu Lys Asn Trp Lys Glu Leu Ser Asp Arg Lys Ile Met Gln Ser Gln 35 40 45 Ile Val Ser Phe Tyr Phe Lys Leu Phe Lys Asn Phe Lys Asp Asp Gln 50 55 60 Glu Ile Gln Lys Ser Val Glu Thr Ile Lys Glu Asp Met Asn Val Lys 65 70 75 80 Phe Phe Asn Ser Asn Lys Lys Lys Arg Asp Asp Phe Glu Lys Leu Thr 85 90 95 Asn Tyr Ser Val Thr Asp Leu Asn Val Gln Arg Lys Ala Ile His Glu 100 105 110 Leu Ile Gln Val Met Ala Glu Leu Ser Pro Ala Ala Lys Thr Gly Lys 115 120 125 Arg Lys Arg Ser Lys Ser Leu Val Asp Pro Phe Arg Ser Ser Glu Ser 130 135 140 Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg 145 150 155 160 Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 165 170 175

[Brief description of drawings]

FIG. 1 shows the amino acid sequence of fusion protein γ-IFN / Glucagon.

[Fig. 2] B for granules containing the fusion protein γ-IFN / Glucagon
2 is a chart showing the results of HPLC analysis of glucagon isolated and purified by allowing Lase to act.

FIG. 3 shows the amino acid sequence of the fusion protein γ-IFN-Glu-brain natriuretic peptide (BNP).

FIG. 4: Isolation of granules containing fusion protein γ-IFN-Glu-brain natriuretic peptide (BNP) by applying BLase.
2 is a chart showing the results of HPLC analysis of purified BNP.

FIG. 5 shows the results of HPLC analysis of fragments generated by the action of BSase on granules containing the fusion protein γ-IFN / Glucagon (a) and the results of HPLC analysis of fragments generated by the action of BLase (b). It is a chart shown.

FIG. 6 shows the results of HPLC analysis of fragments produced by the action of α-chymotrypsin on granules containing the fusion protein γ-IFN / Glucagon (a) and the results of HPLC analysis of the fragments produced by the action of trypsin (b). )
It is a chart showing.

FIG. 7 shows the results of HPLC analysis of fragments produced by the action of lysyl endopeptidase on granules containing the fusion protein γ-IFN / Glucagon (a) and the results of HPLC analysis of the fragments produced by the action of thermolysin (b). ) Is a chart showing

 ─────────────────────────────────────────────────── --- Continuation of the front page (72) Inventor Koichi Matsumoto 1-6-401 Mukogaoka, Toyonaka City, Osaka Prefecture (72) Inventor Hiroshi Teraoka 2-47-10 Takakuradai, Sakai City, Osaka Prefecture

Claims (7)

[Claims] 1. A method for isolating a peptide which does not use a protein denaturant, which comprises the following steps (1) to (4): (1) A gene encoding a fusion protein represented by the following formula is produced in E. coli. Expressing and causing the E. coli to produce granules containing the fusion protein: Formula: LXP (where L is an amino acid sequence that allows the fusion protein to form granules in E. coli. , P is a peptide of interest, and X is an amino acid or amino acid sequence in which the bond of X-P can be digested by a protease), (2) separating the granules from the E. coli, (3) the separating A step of releasing the peptide P of interest from the fusion protein by contacting the granules with the protease, wherein at least a part of the granules can be dissolved
a step in a pH range in which the fusion protein is susceptible to selective digestion by the protease and the protease is not inactivated, and (4) a step of collecting the released target peptide P . 2. The method for isolating a peptide according to claim 1, wherein the pH range is on the alkaline side of the isoelectric point of the fusion protein. 3. The method for isolating a peptide according to claim 1, wherein the pH range is pH 8.5 to 11.5. 4. The amino acid of a protein wherein L is a protein selected from the group consisting of human γ-interferon, human interleukin 2, mouse interleukin 2, human α-interferon, β-galactosidase and aminoglycoside 3′-phosphotransferase II. The method for isolating a peptide according to claim 1, which has all or part of the sequence. 5. The X is Lys, Glu, Arg, Ile-Glu-Gly-A.
The method for isolating a peptide according to claim 1, which is an amino acid or an amino acid sequence selected from the group consisting of rg, Asn and Phe. 6. The P is human angiotensin I, human atrial natriuretic peptide, bradykinin, bovine β.
-Casmorphin, human C-type natriuretic peptide,
Delta sleep-inducing peptide, human dynorphin A, β-
Endorphin, α-endorphin, human endothelin-1, mouse vasoactive intestinal contractor, Met-enkephalin, fibronectin active fragment, human galanin, glucagon, glucagon-like peptide, magainin 1, α-mate factor, porcine α-neoendorphin , Peptide T,
The method for isolating a peptide according to claim 1, which is a peptide selected from the group consisting of Sara Photoxin S6B, somatostatin and tuftsin. 7. The protease is a lysyl endopeptidase, a protease produced by Bacillus licheniformis, or a protease produced by Bacillus subtilis, Strep.
Staphylococ, a protease produced by tomyces fradiae
Protease produced by cus aureus, thermolysin, trypsin, trypsin-like protease, V8 protease, arginyl endopeptidase, factor Xa,
Thrombin, asparaginyl endopeptidase, α-
The method for isolating a peptide according to claim 1, which is selected from the group consisting of chymotrypsin and pepsin.