JPH06339389A - Production of protein or polypeptide using new host of escherichia coli - Google Patents

Abstract

PURPOSE:To obtain the protein having human immune interferon activity effi ciently and in high yield by culturing a transformant using a specific Escherichia coli as a host. CONSTITUTION:A transformant shown by W3110(M25)/PIN5T4 is cultured by using Escherichia coli (FERM-BP-1,097) deficient in protease activity not blocked by diisopropylfluorophosphate, phenylmethylsulfonyl fluoride, N-d-tosyl-L- lysylchloromethyl ketone, N-tosyl-L-phenylalaninechloromethyl ketone, ethylenediaminetetraacetic acid (EDTA), leupeptin, antipain, alpha2-macroglobulin and chymostatin and inhibited by zinc chloride and copper chloride to give the objective adventitious protein or polypeptide.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention uses novel Escherichia coli as a host of a vector capable of highly expressing an exogenous protein or polypeptide (hereinafter, simply referred to as protein), and obtains a protein from the obtained transformant. A method for obtaining a high yield is provided. Specifically, the present invention provides a method for efficiently obtaining a protein having human immune interferon activity from a transformant using this novel Escherichia coli deficient in human immune interferon degrading activity.

[0002]

2. Description of the Related Art In recent years, genetic engineering, especially recombinant DN
Along with the rapid progress of the A technology, it has become possible to mass-produce a large amount of physiologically active proteins, which used to rely on separation / purification from organisms until then. As a result, it has become possible to use a physiologically active protein, whose existence has been known only in the past, in a drug such as a drug or an agricultural chemical.

Regarding the recombinant technology, many improvements have been made so far on how to efficiently produce a target substance, and an improved technology has been proposed. For example, improvement of vectors, development of new host vector system, improvement of culture medium / culture conditions, and more recently, development of proteins having more stable amino acid sequences using protein engineering has been carried out.

However, in the present situation, even a highly expressed physiologically active protein requires many steps in the extraction / purification step from the transformant. Extraction of useful substances easily and efficiently from transformants that produce useful substances
It goes without saying that establishing a refining technology is industrially useful.

[0005] In extracting and purifying a target protein produced by a recombinant microorganism (transformant), usually, the cultured microorganism is first killed by using a bactericidal agent (the extraction step should be performed in a completely sealed manner). If this is possible, this step may be omitted), the cells are disrupted, the disrupted suspension is centrifuged, and either the supernatant or the precipitate is subjected to a purification process. There is. However, in general, the extract from the supernatant or the precipitate has a strong protease activity, so that the target protein is often decomposed.

For example, when human immune interferon (hereinafter abbreviated as hIFN-γ) is produced using Escherichia coli, when extracting hIFN-γ produced by the bacterial cells,
When the cells are crushed, a large amount of a peptide of 131 amino acids obtained by cleaving the C-terminal side of the originally intended peptide of 146 amino acids is obtained. In order to obtain a peptide having 146 amino acids by suppressing the cleavage at the C-terminal side, a protease inhibitor has been added so far to suppress the decomposition (US Pat. No. 4,476,069). Techniques such as microbial cell crushing and aggregation / precipitation at the same time as microbial cell disruption and precipitation, followed by purification (JP-A-59-161321) are disclosed. A method for efficiently extracting and purifying hIFN-γ is being developed in combination with polyethyleneimine.

[0007] On the other hand, in recent years, by purifying the target substance by secreting and producing the target protein extracellularly (belliplasm or medium), impurities in the extract are reduced or degradation by protease is minimized. Efforts have been made to facilitate this, but techniques for efficient secretion and correct processing of precursors that occur during the secretory process (usually, secretory peptides or proteins are secreted through some degradation process) However, it has not yet been put to practical use.

[0008]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The inventors of the present invention have conducted extensive studies in view of the problem that hIFN-γ expressed in Escherichia coli is easily decomposed particularly in the extraction step from cultured cells. It was found that the above problems can be solved by using Escherichia coli deficient in a specific protease activity as a host, and the present invention has been completed.

[0009]

The target mutants that can be used as the host of the present invention are diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, N
-Α-tosyl-L-lysyl chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, ethylenediaminetetraacetic acid (EDTA), leupeptin, antipain, α ₂ -macroglobulin and chymostatin, zinc chloride and Escherichia coli deficient in copper chloride-inhibited protease activity, more preferably Escherichia coli deficient in protease activity for degrading a polypeptide having human immune interferon activity.

The target mutant strain that can be used in the present invention is obtained as follows.

First, Escherichia coli, which is a host of recombinant microorganisms, is subjected to mutation induction treatment. Next, for example, the cells that have been subjected to this mutation induction treatment are cultured, and the cells are lysed,
After addition of the target protein, analysis of the presence or absence of degradation of the protein by SDS-polyacrylamide gel electrophoresis (hereinafter abbreviated as SDS-PAGE) reveals a mutation lacking the activity of degrading the target protein (eg hIFN-γ). You can select the stock. If Escherichia coli is a transformant that highly produces the target protein, the target mutant strain can also be obtained by directly analyzing the disrupted liquid of the transformant or the culture of the bacterial cell itself by SDS-PAGE. You can Using the mutant strain thus obtained as a host, a target protein (hIFN-γ) expression vector is introduced, transformation is performed, and the obtained transformant strain is cultured to produce the target protein (hIFN-γ). Then, the cultured cells are crushed, and the target protein (hIFN-γ) is extracted and purified.
In the present invention, since the host lacks the activity of degrading the target protein (hIFN-γ), the target protein (hIFN-γ) can be degraded without using any protease inhibitor or protein denaturant in the extraction step. Obtained without.

The Escherichia coli deficient in the degrading activity of hIFN-γ protein and the transformant using the hIFN-γ expression vector as a host will be described in detail below. The scope of the present invention has the above-mentioned properties. Needless to say, this does not apply to Escherichia coli deficient in degrading enzymes.

The novel Escherichia coli obtained by the present invention is
It was named SBM282, and was awarded to the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology, Micro Engineering Research Article No. 1097 (FERM BP-10
97) has been deposited.

[0014]

EXAMPLES Example 1 Isolation of a protease activity deficient strain W3110 (M25) / pI which is a hIFN-γ producing bacterium
N5T4 (ampicillin resistance gene (Ap ^r) and transformed with a plasmid pIN5T4 replaced with the tetracycline resistance gene (Tc ^r) E. coli on a plasmid pIN5GIF54 disclosed in JP-60-24187) LB medium (0.5 % Yeast extract, 1% bacto
37 in tryptone, 0.5% NaCl, pH 7.0)
After culturing overnight at 0 ° C., the cells were centrifuged at 10,000 rpm for 5 minutes to collect the cells, and then M9 medium (0.1% NH 3
₄ Cl, 0.6% Na ₂ HPO ₄ , 0.3% KH ₂ PO
₍₄ , 0.05% NaCl, adjusted to pH 7.4, 2 ml / l of 1M MgSO ₄ , 10 ml / l of 20% glucose, 0.1 ml / l of 1M CaCl ₂ were added to wash the cells) , Suspended in the same medium. Nitrosoguanidine (hereinafter abbreviated as NTG) as a mutagen
Was added at a concentration of 50 μg / ml, left for 30 minutes, and then centrifuged at 10,000 rpm for 5 minutes to collect bacterial cells. The cells were washed with LB medium and then inoculated into the same medium. After culturing this at 37 ° C. overnight, the colonies appearing on the plate were treated with LB medium for 500 m.
1 platinum loop was inoculated into the tube containing 1 and cultured overnight at 37 ° C. After centrifugation at 10,000 rpm for 5 minutes, L
ysozyme-EDTA solution (500 μg / ml L
200 μl of ysozyme, 1 mM EDTA) was added, and the cells were disrupted by the freeze-thaw method. 1mg /
100 μl of hIFN-γ solution adjusted to a concentration of ml was added, and the mixture was incubated at 37 ° C. for 1 hour, and SDS sample solution (10% glycerin, 5% 2-meltcaptoethanol, 23% SDS, 62.5 mM Tris-hydrochloric acid buffer, pH 6.8) 150 μl was added, treated at 100 ° C. for 5 minutes, then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was subjected to SDS-PAGE and the residual amount of hIFN-γ (molecular weight about 18,000) consisting of 146 amino acids. It was investigated. In this way, one mutant strain having no hIFN-γ degrading activity was obtained from about 2,000 strains.

In order to confirm that the deletion mutation of this degrading activity is not a plasmid-derived mutation but a host-derived mutation, the plasmid was then cured. That is, the above mutant strain showing tetracycline resistance due to a gene on the plasmid (pIN5T4) was cultured in a medium not containing tetracycline, and a strain which became a tetracycline sensitive (Tcs) strain was selected to confirm the loss of the plasmid. For this strain, as described above, hIFN-γ protein was added to the disrupted cell suspension to obtain SDS-
As a result of examining the presence or absence of degradation of hIFN-γ protein by PAGE, no degradation activity was observed. This result indicates that this mutant strain is due to mutation of the host chromosome. Next, the strain from which this plasmid had been eliminated was again transformed with the plasmid pIN5T4) (described above), which is a plasmid in which a gene for expressing hIFN-γ was incorporated, according to a conventional method. Using tetracycline resistance as a marker,
A transformant strain is selected, and the presence or absence of degradation of hIFN-γ in the cell disruption / extract of the transformant strain is checked by SDS-PAG.
As a result of examination by E, hIFN-γ was not decomposed at all.

The cured mutant W31
10 (M25) is named SBM282 and has been deposited at the Institute of Microbial Technology of the Agency of Industrial Science and Technology (FERM
BP-1097). Further, the production / selection of the mutant strain by the mutagenesis treatment may be carried out by subjecting Escherichia coli into which the vector has not been introduced to the mutagenesis treatment to select the desired mutant strain and introducing the target protein expression vector into it. it can.

2. Properties of Mutant Strain Obtained by Mutagenesis Treatment Next, what kind of protease is deficient in the hIFN-γ degrading activity-deficient mutant strain obtained by the mutation induction treatment shown in the above 1 I checked if there was. As a result, as shown below, a mutant strain lacking a protease activity that was not inhibited even by using a general trypsin-like enzyme inhibitor, while being inhibited by a copper salt or a zinc salt. there were. W that is a transformant that produces hIFN-γ using a parent strain (W3110) that is not mutated as a host
After culturing 3110 / pIN5T4 in a 500 ml Meyer flask containing 100 ml of LB medium overnight at 37 ° C., the cells were collected by centrifugation at 8,000 rpm for 10 minutes. 1 g of the obtained wet cells was suspended in 30 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.5), homogenized at 20,000 psi using a French press homogenizer, and centrifuged at 8,000 rpm for 20 minutes.

Various protease inhibitors shown in Table 1 were dissolved in 700 μl of 0.1 M Tris-hydrochloric acid buffer (pH 7.5) in which each final concentration was dissolved, and 50 μl of the above supernatant was added.
1 was added thereto, and a hIFN-γ solution (1 mg / ml) 2
After adding 50 μl, the mixture was incubated at 37 ° C. for 1 hour. This solution was subjected to SDS-PAGE and hIFN
-The presence or absence of γ decomposition was examined. The results are shown in Table 1.

[0019]

[Table 1] Of the substances listed in Table 1, hIFN-γ degrading activity was inhibited only by CuCl ₂ and ZnCl ₂ , and hI by other substances which are generally considered to be trypsin-like enzyme inhibitors.
FN-γ degradation activity was not inhibited.

3. Time-course of hIFN-γ degradation after cell disruption
Change Next, transformants W3110 / pI in which the wild-type strain as a host
Transformed strain W311 using N5T4 and the above mutant strain as hosts
0 (M25) / pIN5T4, hIFN-γ productivity, and hIFN-γ after disruption of cultured cells of each transformant strain
The change with time of decomposition was examined.

That is, W3110 / pIN5T4 and W3
Incubate 110 (M25) / pIN5T4 in the same manner as above.
The cells were collected and suspended in a buffer. This suspension (W), the precipitate obtained by crushing the bacterial cells by a French press and then centrifuging (P), and each supernatant after 0, 15, 30, 60 minutes after centrifuging are as described above. Using SDS-PAGE,
Undegraded hIFN-γ protein (molecular weight about 18,0
00) amount and decomposed protein (molecular weight of about 16,000)
0) The amount was examined. The results are shown in Fig. 1.

For W3110 / pIN5T4, 1
Within 5 minutes 50% of the supernatant and within 60 minutes almost 100
% HIFN-γ is isolated while W311
With 0 (M25) / pIN5T4, almost no degradation was observed even after 60 minutes, and hIFN- having a molecular weight of about 18,000 was detected.
γ was kept stable. No difference was observed between the producers of hIFN-γ.

[0023] 4. HI from transformant using mutant strain
Extraction and purification of FN-γ W3110 / pIN5T4 and W3110 (M25) /
pIN5T4 was cultured in 20 liter LB medium in a 30 liter jar fermenter at 30 ° C. for 36 hours.
The obtained culture solution was centrifuged at 8,000 rpm for 10 minutes to collect the cells. 1 kg of each wet bacterial cell was added to 20 mM Tris-
Suspended in 7,000 ml of hydrochloric acid buffer (pH 7.5), and used this as a homogenizer M15 manufactured by Manton Gorin Co.
The cells were disrupted at 8,000 psi. Incidentally, W31
For 10 / pIN5T4, the same buffer containing 1 mM ZnCl ₂ was used instead of the 20 mM Tris-HCl buffer. This crushed liquid is 7,000 rpm,
The supernatant obtained after centrifugation for 20 minutes was supplemented with 15% polyethyleneimine (pH 8.0, adjusted with HCl) to a final concentration of 0.
The mixture was added to 75%, stirred for 10 minutes, allowed to stand at 4 ° C. for 2 hours, and the generated precipitate was removed by centrifugation at 7,000 rpm for 20 minutes. With this supernatant, hI
FN-γ was purified.

That is, the above supernatant was equilibrated with 20 mM N-2-hydroxyethylpiperazine-N'-3-propanesulfonic acid buffer (EPPS buffer) pH 8.6, QAE Sephadex A-25 (Pharmacia). The flow-through fraction obtained by applying the column was
% Containing 2-meltcaptoethanol (2-ME) 20
C equilibrated with mM Tris-HCl buffer pH 7.4
The fraction passed through the M Sepharose CL6B (Pharmacia) column and adsorbed on the column was 0 to 0.5.
Elution was performed with a linear gradient of M NaCl, and the sections with high interferon activity were collected. The interferon activity was measured according to the method described in JP-A-58-201995. Subsequently, ammonium sulfate was added to this fraction so as to be 50% saturated, and salting out was performed to obtain 7,000 r.
A precipitate was obtained by centrifugation at pm for 20 minutes. This is 0.
20 mM with 3M NaCl and 0.1% 2-ME
Sephacryl S-20 dissolved in sodium phosphate buffer (PSB) pH 7.4 and equilibrated with the same buffer
0 (Pharmacia) column was applied to obtain a final purified product having a purity of 99% or more. W3110 / pIN5T
The final yields of hIFN-γ produced by 4 and W3110 (M25) / pIN5T4 were 5% and 12%, respectively.

[0025]

As described above, by using the method according to the present invention, the target protein (hIFN-γ) can be obtained in a yield of 2 times or more even though the expression level is the same in the cells. Further, since the target protein (hIFN-γ) is not decomposed even after cell disruption, it is not necessary to use a protease inhibitor or a protein denaturing agent, and the extraction / purification steps can be simplified and made more efficient.

[Brief description of drawings]

FIG. 1 shows SDS-PAGE for investigating the time course of degradation of hIFN-γ protein. In the figure, W is a suspension of cultured bacterial cells before crushing, P is a precipitate obtained by crushing and centrifuging the suspension, and 0 ', 15', 30 ', and 60' are bacteria respectively. Centrifuge supernatant after crushing the body is 0, 15, 30, 60
3 shows patterns of various proteins after incubation at 37 ° C. for minutes. a is a band corresponding to a hIFN-γ protein, and b is a protein band corresponding to a hIFN-γ degradation product.

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[Procedure amendment]

[Submission date] January 12, 1994

[Procedure Amendment 1]

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[Claims]

[Procedure Amendment 2]

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[0001]

TECHNICAL FIELD The present invention uses novel Escherichia coli as a host of a vector capable of highly expressing an exogenous protein or polypeptide (hereinafter, simply referred to as protein), and obtains a protein from the obtained transformant. A method for obtaining a high yield is provided. Specifically, to provide a method for efficiently obtaining a protein or the like using a novel E. coli lacking human immune interferon degradation activity, the E. coli from transformant as a host with a human immune interferon activity.

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0006

[Correction method] Change

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For example, when human immune interferon (hereinafter abbreviated as hIFN-γ) is produced using Escherichia coli, when extracting hIFN-γ produced by the bacterial cells,
When the cells are crushed, a large amount of a peptide of 131 amino acids obtained by cleaving the C-terminal side of the originally intended peptide of 146 amino acids is obtained. Suppressing the cleavage of the C-terminal side, far in order to obtain a peptide consisting of 146 amino acids, inhibit degradation by adding protease inhibitors (U.S. Patent No. 4,476,0 4 No. 9), using a protein denaturant Disclosed is a technique such as microbial cell crushing and coagulation / precipitation at the same time as microbial cell disruption, followed by purification (Japanese Patent Laid-Open No. 59-161321). We are developing a method to efficiently extract and purify hIFN-γ using salt and polyethyleneimine together.

[Procedure amendment 4]

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The novel Escherichia coli obtained by the present invention is
It is named Escherichia coli SBM282, and is in the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology.
It has been deposited as ERM BP-1097).

[Procedure Amendment 5]

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[0014]

EXAMPLES Example 1 Isolation of a protease activity deficient strain W3110 (M25) / pI which is a hIFN-γ producing bacterium
N5T4 (ampicillin resistance gene (Ap ^r) and transformed with a plasmid pIN5T4 replaced with the tetracycline resistance gene (Tc ^r) E. coli on a plasmid pIN5GIF54 disclosed in JP-60-24187) LB medium (0.5 % Yeast extract, 1% bacto
37 in tryptone, 0.5% NaCl, pH 7.0)
After culturing overnight at 0 ° C., the cells were centrifuged at 10,000 rpm for 5 minutes to collect the cells, and then M9 medium (0.1% NH 3
₄ Cl, 0.6% Na ₂ HPO ₄ , 0.3% KH ₂ PO
₍₄ , 0.05% NaCl, adjusted to pH 7.4, 2 ml / l of 1M MgSO ₄ , 10 ml / l of 20% glucose, 0.1 ml / l of 1M CaCl ₂ were added to wash the cells) , Suspended in the same medium. Nitrosoguanidine (hereinafter abbreviated as NTG) as a mutagen
Was added at a concentration of 50 μg / ml, left for 30 minutes, and then centrifuged at 10,000 rpm for 5 minutes to collect bacterial cells. The cells were washed with LB medium and then inoculated into the same medium. After culturing this at 37 ° C. overnight, the colonies appearing on the plate were treated with LB medium 500 μm.
1 platinum loop was inoculated into the tube containing 1 and cultured overnight at 37 ° C. After centrifugation at 10,000 rpm for 5 minutes, L
ysozyme-EDTA solution (500 μg / ml L
200 μl of ysozyme, 1 mM EDTA) was added, and the cells were disrupted by the freeze-thaw method. 1mg /
100 μl of hIFN-γ solution adjusted to a concentration of ml was added, and the mixture was incubated at 37 ° C. for 1 hour, and SDS sample solution (10% glycerin, 5% 2-meltcaptoethanol, 23% SDS, 62.5 mM Tris-hydrochloric acid buffer, pH 6.8) 150 μl was added, treated at 100 ° C. for 5 minutes, then centrifuged at 10,000 rpm for 10 minutes, and the supernatant was subjected to SDS-PAGE and the residual amount of hIFN-γ (molecular weight about 18,000) consisting of 146 amino acids. It was investigated. In this way, one mutant strain having no hIFN-γ degrading activity was obtained from about 2,000 strains.

[Procedure correction 6]

[Document name to be amended] Statement

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[Correction method] Change

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The cured mutant W31
10 (M25) is named Escherichia col i SBM282, has been deposited with the Agency Fermentation Research Institute (FERM BP-1097). Further, the production / selection of the mutant strain by the mutagenesis treatment may be carried out by subjecting Escherichia coli into which the vector has not been introduced to the mutagenesis treatment to select the desired mutant strain and introducing the target protein expression vector into it. it can.

[Procedure Amendment 7]

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For W3110 / pIN5T4, 1
Within 5 minutes 50% of the supernatant and within 60 minutes almost 100
% HIFN-γ is isolated while W311
With 0 (M25) / pIN5T4, almost no degradation was observed even after 60 minutes, and hIFN- having a molecular weight of about 18,000 was detected.
γ was kept stable. No difference was observed in the productivity of hIFN-γ.

[Procedure Amendment 8]

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[Figure 1]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:19) (C12N 1/21 C12R 1:19)

Claims (3)

[Claims] 1. Diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, N-α
- tosyl -L- Li silk Rollo methyl ketone, N- tosyl -L- phenylalanine chloromethyl ketone, ethylenediamine tetraacetic acid (EDTA), leupeptin, antipain, alpha ₂ - not inhibited by macroglobulin and chymostatin, zinc chloride and copper chloride A method for producing an exogenous protein or polypeptide by culturing a transformant using Escherichia coli deficient in protease activity, which is inhibited by Sec. 2. The method according to claim 1, wherein the foreign protein or polypeptide has human immune interferon activity. 3. The transformant is W3110 (M25) / p.
The method of claim 2 represented by IN5T4.