JPH05211887A - Production of nerve growth factor - Google Patents

Abstract

PURPOSE:To produce a nerve growth factor by using genetically recombined Escherichia coli holding a vector DNA having a signal peptide gene on the upstream side of a nerve growth factor gene. CONSTITUTION:The objective method for producing a nerve growth factor is composed of a step for extracting the nerve growth factor with an extracting agent, a step for purifying the nerve growth factor in the presence of the extracting agent, a step for removing the extracting agent and a step for purifying the nerve growth factor in the absence of the extracting agent. Thereby, the high-purity human nerve growth factor (hNGF) holding the biological activity can be obtained by a genetic manipulating technique using Escherichia coli to supply a large amount of the hNGF at a low cost.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a nerve growth factor, Nerve Growth Factor.
The present invention relates to a method for producing a high-purity nerve growth factor having biological activity by using recombinant Escherichia coli having a vector DNA having a signal peptide gene upstream of r (hereinafter abbreviated as NGF) gene.

[0002]

2. Description of the Related Art NGF is an essential factor for promoting differentiation and survival of peripheral sympathetic nerve cells and sensory nerve cells, and function-maintaining action (Physiol. Rev. 60 , 1284-1335).
(1980), Ann. Rev. Biochem. 5
1 , 845-868 (1982)), a large amount of CNS was detected in the hippocampus and cerebral cortex of rat brain, and a small amount was detected in cerebellum and basal ganglia, and NGF messenger RNA.
It has been clarified that similar results can be obtained from the content (Proc. Natl. Acad. Sci. US
A 81 , 7951-7955 (1984) and EM.
BO J. 4 , 1389-1393 (1985)).

As described above, NGF acts as a trophic factor for central cholinergic nerve cells, and attention has been paid to its association with Alzheimer's disease, which causes marked damage to basal forebrain cholinergic nerve cells. In addition, administration of NGF into the brain of aged rats improves spatial cognitive memory (Nature 329 , 65-68 (1987)).
Have also been reported.

Therefore, NGF, especially human NGF (hereinafter abbreviated as hNGF), which is not antigenic to the human body, is expected as a therapeutic drug for neurological diseases such as Alzheimer's disease, and its mass production has become an important issue. .. .

NGF is present in the mouse submandibular gland, snake venom, guinea pig prostate, bovine semen, musk rat submandibular gland,
Purified (cell growth factor 8-20 Asakura Shoten
(1984)). Among them, a relatively large amount of NGF is present in the mouse submandibular gland, and it has been purified by crushing the submandibular gland, subjecting the supernatant liquid to gel filtration, and performing ion exchange chromatography (Proc. Natl.Aca
d. Sci. USA 64 , 787-794 (196).
9)).

On the other hand, it was reported that hNGF was purified from human placenta (Neurochem. Res. 3,
175-183 (1978)), but purification of a large amount of hNGF from human tissue is very difficult to obtain the material.

Therefore, a method for producing by genetic engineering technology is being studied. When animal cells are used as a host, they are secreted and produced in a hNGF culture solution, and the culture supernatant solution is purified by gel filtration, ion exchange chromatography, and reverse phase chromatography, and has a biological activity equivalent to that of mouse NGF. HNGF has been obtained (Bioch
em. Biophys. Res. Commun. 17
1 , 116-122 (1990)).

However, culture of animal cells generally requires a longer culture period than culture of Escherichia coli and the like, and the production costs are high, such as maintaining the sterility of the culture equipment and the cost of the medium.

Therefore, if hNGF retaining biological activity can be produced by genetic engineering using Escherichia coli or yeast widely used as a host, hNGF can be supplied at a lower cost.

Up to now, when Escherichia coli was used as a host,
The biological activity of the produced mouse NGF is
About 1/1000 of F (Gene. 70 , 57-65
(1988)), 1/10 of mouse NGF in hNGF
There is a report that it is about (JP-A-1-257491).

On the other hand, regarding yeast, there is a report that the biological activity of hNGF is about 1/200 of that of mouse NGF (G
ene. 83 , 65-74 (1989)).

As described above, the method of producing various NGF such as hNGF by Escherichia coli has a problem that the biological activity of NGF is low.

[0013]

SUMMARY OF THE INVENTION In order to solve the above-mentioned problems of the prior art, it is an object of the present invention to produce NGF having high biological activity using genetically modified Escherichia coli.

[0014]

Means for Solving the Problems The present inventors have studied the above-mentioned problems with Escherichia coli into which a hNGF gene having a signal peptide gene has been introduced in order to secrete hNGF into the periplasm. In the drawing method, for example, osmotic pressure method, h
Almost no NGF could be recovered.

It was considered that this is because hNGF behaves together with the membrane component of E. coli. Then, as a result of studying the extraction of hNGF from the membrane component recovered by crushing the bacterial cells, it was found that most hNGF can be recovered.

[0016] Further, if the extractant is removed from the extracted component in the above-mentioned state, insoluble aggregates containing hNGF are formed, and there arises a problem that the recovery rate of hNGF is lowered and subsequent purification by chromatography or the like becomes difficult. It was

Therefore, when hNGF was partially purified by gel filtration using a developing solution containing an extractant and the extractant was removed, the extractant was removed without forming insoluble aggregates in the fraction containing hNGF. It has become possible.

When purified by reverse phase chromatography to obtain hNGF with higher purity, hNGF retains high purity and high biological activity, thus completing the present invention.

In the present invention, a recombinant Escherichia coli carrying a vector DNA having a signal peptide gene upstream of the NGF gene is used, and in the production of hNGF, for example, HB101 (pTRSNGF) (JP-A-3-2)
44385, Microtechnology Research Institute, No. 11285).

There is no particular problem with the NGF gene as long as it is a DNA encoding the amino acid sequence of NGF, but it is preferable to use the NGF gene synthesized in consideration of the frequency of use of E. coli gene codons.

As the signal peptide gene, a signal peptide gene derived from Escherichia coli, for example, a β-lactamase signal peptide gene, an alkaline phosphatase signal peptide gene, a signal peptide gene of various outer membrane proteins and the like are used, and other organisms. A signal peptide gene that is derived or artificially designed and can exert its function in Escherichia coli is used.

There are no particular restrictions on the control genes such as promoters that control the expression of these genes.

As a method for recovering the membrane component, a known method may be used, for example, centrifugation can be performed.

The extractant used in the extraction step is guanidine hydrochloride, urea, sodium dodecyl sulfate, sodium lauryl sarcosinate, pt-octyl phenyl ether,
Sorbitan monolaurate is considered, but NGF
Considering the recovery rate and the simple removal method, guanidine hydrochloride is most suitable. For example, 6M guanidine hydrochloride has a hNGF recovery of 98%, and 8M urea has a recovery of 45%.
%, 6% urea was 30%, and 1% p-t-octylphenyl ether was 3% or less. The concentration of guanidine hydrochloride was 90% at 4M and 65% at 2M when the hNGF recovery rate at 6M was 100%.

The means for gel filtration used in the purification step in the presence of an extractant is not particularly limited as long as a commercially available carrier for gel filtration may be used, but the developing agent may be the extractant used for extraction. It is most suitable to contain such substances, and when different extractants are used, formation of insoluble aggregates may cause problems such as clogging of the column and reduction of NGF recovery rate.

Further, various chromatographies other than gel filtration can be used for partial purification, but in the case of ion exchange chromatography, urea, nonionic surfactant and the like must be used as an extracting agent. For example, hNGF extracted with guanidine hydrochloride as an extractant is added to a 0.1% solution containing 8M urea.
Ion exchange chromatography can be performed by dialysis against 13 M NaCl, 10 mM phosphate buffer (pH 7) (hereinafter abbreviated as PBS), but after dialysis, hN
Insoluble aggregates containing GF were formed, and a decrease in hNGF recovery rate was observed.

As a method for removing the extractant, dialysis, gel filtration for desalting, chromatography with a commercially available resin for removing the extractant, and the like can be used.

The means used in the purification step in the absence of an extractant may be optionally subjected to various chromatography such as reverse phase chromatography, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration and electrophoresis, or the like. It can be purified to purity.

[0029]

According to the present invention, in a method for producing nerve growth factor using recombinant Escherichia coli carrying a vector DNA having a signal peptide gene upstream of the nerve growth factor gene, most of the extraction agent is used. hN
The extractant can be removed without recovering GF and forming insoluble aggregates, and subsequent chromatography allows purification of hNGF with high purity and biological activity retained.

[0030]

EXAMPLE An example of the present invention will be described below, but the present invention is not limited to this.

(1) Step of culturing hNGF gene recombinant Escherichia coli Preculture medium: NH ₄ Cl 1 g, Na ₂ HPO ₄ 6 g,
_{KH 2 PO 4 3g, NaCl 0.5g} , MgSO 4 · 7H
₂ O 0.5 g, CaCl ₂ .2H ₂ O 0.015 g, casamino acid 2.5 g, glucose 5 g, thiamine hydrochloride
0.1 g, proline 0.1 g, yeast extract 1.5 g,
Tryptophan 0.04g, Ampicillin 0.05g
Was dissolved in distilled water, pH was adjusted to 7.0 with NaOH, and the total amount was adjusted to 1 liter. Main culture medium: NH ₄ Cl 1 g, Na ₂
HPO ₄ 6 g, KH ₂ PO ₄ 3 g, NaCl 0.5 g,
0.5 g of MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O 0.
015 g, casamino acid 2.5 g, glucose 5 g, thiamine hydrochloride 0.1 g, proline 0.1 g, ampicillin 0.05 g are dissolved in distilled water and pH is adjusted with NaOH.
It was 7.0 and the total amount was 1 liter.

Cells used: Escherichia coli HB10 plasmid pTRSNGF in which the signal peptide gene of β-lactamase and the hNGF gene were ligated downstream of the trp promoter.
HB101 (pTRSNGF) introduced in No. 1
244385, Microtechnical Research Institute No. 11285) was used.

The above-mentioned bacterial cells were inoculated into two 500 ml Sakaguchi flasks containing 50 ml of a preculture medium, and cultured at 37 ° C. overnight with shaking. 100 ml of this preculture solution was inoculated into a 2 liter Sakaguchi flask containing 1 liter of main culture medium,
The cells were cultured with shaking at 24 ° C for 24 hours.

(2) Extraction step of hNGF The above culture solution was centrifuged at 12,000 × g for 10 minutes to collect the cells, and 10 mM Tris-hydrochloric acid buffer solution (pH 8) 250
The cells were suspended in ml and centrifuged under the above conditions to wash the cells twice. 10mM Tr containing 5mM EDTA
After suspending in 140 ml of is-hydrochloric acid buffer solution (pH 8),
Crushing was performed by intermittently irradiating with ultrasonic waves at 100 W for 3 minutes under ice cooling three times. The disrupted solution was centrifuged at 60,000 rpm for 3 hours (RP70T rotor manufactured by Hitachi, Ltd.), and the precipitate was suspended in 40 ml of 10 mM Tris-hydrochloric acid buffer (pH 8) containing 1 mM EDTA and washed, and then again under the above conditions. It was centrifuged and the precipitate was collected.

This precipitate was mixed with 6M guanidine hydrochloride and 1M.
After suspending in 10 ml of 10 mM Tris-hydrochloric acid buffer solution (pH 8) containing mM EDTA and incubating at 37 ° C. for 1 hour, the supernatant liquid centrifuged at 100000 rpm for 30 minutes (Hitachi: RP100AT rotor) was used for h.
This was the NGF extract.

(3) Step of detecting hNGF with anti-hNGF antibody (A) Preparation of anti-hNGF polyclonal antibody Escherichia coli HB101 (pTRL carrying the hNGF gene)
NGF) (Japanese Patent Application Laid-Open No. 3-244385, Microorganisms Research Institute, No. 11)
283) produces hNGF in the form of insoluble granules.
The cells were disrupted, and hNGF recovered by centrifugation was solubilized with a PBS solution containing 6M guanidine hydrochloride to obtain P
The solution was dialyzed against a BS solution to prepare a protein concentration of 1 mg / ml.

An equal amount of this preparation and Freund's incomplete adjuvant were mixed and emulsified to be used as an antigen. 1 ml of this antigen was subcutaneously administered to the back of a 9-week-old female rabbit (Kb1-JW: Japanese white native breed), and every 14 days thereafter, 0.
5 ml was administered three times. 14 days later, whole blood was collected and 3000
Antiserum was obtained by centrifugation at rpm for 30 minutes.

(B) SDS-PAGE and Immunoblotting According to the method of Laemmli et al. Using 15% polyacrylamide gel, the above-mentioned hNGF extract was subjected to SDS-PA.
I went to GE. Then, the protein in the gel was blotted on a nitrocellulose membrane coated with polyvinylidene fluoride by electrophoresis using 0.192M Tris-hydrochloric acid buffer containing 20% methanol and 0.1M glycine. This membrane was blocked with a blocking agent (BlockAce, manufactured by Dainippon Pharmaceutical Co., Ltd.), and absorbed by a lysate of Escherichia coli HB101 strain.
It was immersed in PBS containing a% blocking agent and incubated at 37 ° C. for 1 hour. P containing 0.1% Tween 20
After washing with BS, peroxidase-labeled anti-rabbit IgG antibody (manufactured by BIORAD) 0.05% and 10%
It was immersed in PBS containing a blocking agent, incubated at 37 ° C. for 1 hour, and color development was detected by a conventional method.

FIG. 1 shows a supernatant liquid obtained by centrifuging cells after cell disruption,
The results obtained by performing SDS-PAGE and immunoblotting under the above conditions on the washing solution and the precipitate and the supernatant solution and the precipitate after centrifugation after treatment with 6M guanidine hydrochloride are shown. In FIG. 1, lanes 1 to 3 are the supernatant of the disrupted bacterial cells shown in step (2), a washing solution and a precipitate, and lanes 4 to 6 are immunoblotting of lanes 1 to 3, lanes 7 and 8, respectively. Is the supernatant and precipitate after centrifugation treated with 6M guanidine hydrochloride shown in step (2). Lanes 9 and 10 are immunoblottings of lanes 7 and 8, respectively.

When the color intensity of immunoblotting was measured by a densitometer, the hNGF recovery rate after cell disruption was 97%, and almost all of the hNGF recovery was possible by guanidine hydrochloride extraction.

(4) Purification Step by Chromatography 1 containing 6 M guanidine hydrochloride and 1 mM EDTA
0 mM Tris-hydrochloric acid buffer solution (pH 8) at 1 ml / mi
Column equilibrated by passing it through at a flow rate of n (Pharmacia: Superdex 75 pg, φ16 × 600 m
4 ml of the above extract was added to m), and fractionation was performed every 4 minutes. Fractions containing hNGF were selected by the same detection method as in step (3) above.

(5) Purification step by dialysis The above fraction containing hNGF was dialyzed into a dialysis membrane (Spectra 1 S).
PECTRUM MEDICAL INDUSTRIES
10 mM acetate buffer (pH 4.5) 50
It was dialyzed 4 times against 0 ml to obtain 15 ml.

The results of SDS-PAGE and immunoblotting of this hNGF were shown in lanes 4 and 8 of FIG.
Shown in. In FIG. 2, lane 1 is a molecular weight marker, lanes 2 and 6 are cells of HB101 (pTRSNGF), lanes 3 and 7 are supernatants after centrifugation with 6M guanidine hydrochloride, and lanes 4 and 8 are in step (5). Gel filtration indicated hN
GF fractions, lanes 5 and 9 are the SDS of the respective hNGF fractions of the reverse phase chromatography shown in step (7).
-Results of PAGE and immunoblotting.

As a result of measuring the gel subjected to SDS-PAGE with a densitometer, the content rate of hNGF was 30%. In addition, a protein concentration measurement kit (Protein A
When using bovine serum albumin (hereinafter abbreviated as BSA) as a standard using SSAY, manufactured by BIORAD,
It was 0.08 mg / ml.

(6) Measurement of biological activity of hNGF PC1 cloned from rat adrenal medulla pheochromocytoma
It was measured by neurite outgrowth of PC12h cells, which is a mutant strain derived from 2 cells. The culture media were E-RDF and RD-1 powder culture media (manufactured by Kyokuto Pharmaceutical Co., Ltd.), 17.5 g and 16.5, respectively.
mg, it was used and filter sterilized dissolved NaHCO ₃ 1.13 g of distilled water 1l.

PC12h cells cultured in a medium containing 10% fetal calf serum added to the above medium were collected by centrifugation and adjusted to a concentration of 3 × 10 ⁴ cells / ml in the above medium, which was then used for collagen (Cellmatrix Type).
100 μl was put into a 96-well plate well coated with e1-C, manufactured by Nitta Gelatin).

The roughly purified hNGF solution obtained in step (5) was added to a 0.2 μm sterilized filter (Millex G).
V, manufactured by Millipore) and sterilized by filtration. From the results of densitometry, P containing 0.2% BSA so that the hNGF concentration was 5 μg / ml with the hNGF content of 30%.
Diluted with BS.

2 μl of the solution was added, and the cells were cultured at 37 ° C. in an incubator under a 5% CO ₂ atmosphere. On the 3rd day of culture, the medium was replaced as described above, and PC1 observed on the 5th day of culture
A phase contrast micrograph of 2h cells is shown in FIG.

5 μg / ml mouse NG as a comparative control
FIG. 4 shows a phase contrast micrograph of PC12h cells on the 5th day of culture, which was carried out in the same manner as the hNGF crudely purified solution using F (Toyobo Co., Ltd.). The neurite outgrowth activities of hNGF and mouse NGF were almost the same.

(7) Purification Step by Reversed Phase Chromatography A column (φ4 × 250 m) packed with spherical silica gel (Recrosphere, manufactured by Kanto Chemical Co., Ltd.) having an octyl group bonded thereto.
m) was equilibrated with distilled water containing 0.1% trifluoroacetic acid and 10% acetonitrile. H obtained in step (5)
NGF was added to the above column, and the mixture was eluted with a gradient at a liquid flow rate of 1 ml / min until the acetonitrile concentration reached 1.67% / min to 90%.

The fractions containing hNGF were subjected to SDS-PAGE and immunoblotting by the method described in step (3), and the results are shown in lanes 5 and 9 of FIG. It was 98% when the purity of hNGF in SDS-PAGE was assayed by a densitometer. PBS containing 0.2% BSA so that the hNGF concentration becomes 5 μg / ml
FIG. 5 shows a phase-contrast micrograph of PC12h cells on day 5 of culture in the same experiment as described in measurement (6) after diluting with.

Comparative mouse NGF and h shown in FIG.
The neurite outgrowth activity of NGF was almost the same.

[0053]

INDUSTRIAL APPLICABILITY According to the present invention, NGF having biological activity can be produced by using Escherichia coli, and hNGF can be supplied inexpensively and in a large amount.

[Brief description of drawings]

FIG. 1 is a photograph of SDS-PAGE and immunoblotting performed by the method described in steps (1) to (3).

FIG. 2 is a photograph of SDS-PAGE and immunoblotting performed by the method described in steps (1) to (3).

FIG. 3 is a phase-contrast photomicrograph of PC12h cells on day 5 of culture, to which the hNGF crude purified solution shown in measurement (6) was added.

FIG. 4 is a phase-contrast photomicrograph of PC12h cells on day 5 of culture, to which mouse NGF was added as shown in measurement (6).

FIG. 5 is a phase-contrast photomicrograph of PC12h cells on day 5 of culture, to which the hNGF fraction solution shown in step (7) was added.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Norio Shimizu Inventor Norio Shimizu 2520 Akanuma, Hatoyama-cho, Hiki-gun, Saitama Stock company Hitachi Research Laboratory

Claims (9)

[Claims] 1. A step of synthesizing nerve growth factor using a recombinant Escherichia coli carrying a vector DNA having a signal peptide gene upstream of the nerve growth factor gene, and then extracting the nerve growth factor gene using an extracting agent. Process,
A method for producing a nerve growth factor, which comprises performing a step of purifying a nerve growth factor gene in the presence of an extractant, a step of removing the extractant, and a step of purifying the nerve growth factor gene in the absence of the extractant. 2. The method for producing nerve growth factor according to claim 1, wherein the signal peptide is a β-lactamase signal peptide. 3. The method for producing nerve growth factor according to claim 2, wherein said signal peptide is a β-lactamase signal peptide of Escherichia coli. 4. The method for producing nerve growth factor according to claim 1, 2 or 3, wherein the nerve growth factor is human nerve growth factor. 5. The method for producing a nerve growth factor according to claim 1, 2 or 3, wherein the nerve growth factor extractant is guanidine hydrochloride. 6. The method according to claim 1, wherein the step of purifying nerve growth factor in the presence of the extractant is gel filtration.
Or the method for producing a nerve growth factor according to the item 3. 7. The method for producing nerve growth factor according to claim 1, 2 or 3, wherein the step of removing the extractant is dialysis. 8. The method for producing a nerve growth factor according to claim 1, 2 or 3, wherein an acetic acid buffer is used as the dialysate. 9. The method for producing a nerve growth factor according to claim 1, 2 or 3, wherein the step of purifying the nerve growth factor in the absence of the extractant is reverse phase chromatography.