JP2664802B2 - Method for producing N-terminal extracellular protein at ion channel direct receptor - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a microbiological production method of an N-terminal extracellular extracellular protein of a direct ion channel receptor by recombinant DNA technology.

[Problems to be Solved by the Prior Art and the Invention] Neurotransmitter receptors in the central nervous system are proteins that play a central role in synaptic signal transmission, and elucidation of their structures, functions, and regulatory mechanisms Is essential for understanding the brain and nervous system. Among them, amino acid receptors have recently attracted much interest in basic research and clinical applications.

Representative amino acids as neurotransmitters are glutamic acid, glycine, γ-aminobutyric acid, and the like, and neuropharmacology and electrophysiological studies have been advanced on the receptors.

However, the content of these receptor proteins in the living body is small, and it is often difficult to obtain them in large quantities. Therefore, it is useful to express a large amount of neurotransmitter receptor using Escherichia coli as a host by genetic engineering,
To date, there has been no such example.

γ-Aminobutyric acid (hereinafter abbreviated as “GABA”) is a typical inhibitory transmitter, and GABA receptors have A and B subtypes. Among them, the GABA _A receptor is conjugated to a Cl- channel and has an α subunit, a β subunit, and the like.

Glycine is also a typical inhibitory transmitter along with GABA, and the glycine receptor constitutes a Cl channel with only the α subunit.

From the amino acid sequences predicted from the cDNA base sequences of the previously cloned GABA _A receptor α subunit, β subunit, and glycine receptor α subunit, a region of about 200 amino acid residues from the N-terminus is a ligand. It was considered to be an extracellular region containing the binding site (N-terminal extracellular site).

However, the analysis of the interaction with the ligand has been performed only for the entire receptor, and the analysis performed using only the N-terminal extracellular site includes the analysis including other neurotransmitter receptors. But never before.

[Means for Solving the Problems] Accordingly, the present inventors have developed a bovine GABA _A receptor α subunit, a β subunit, and a rat glycine receptor α.
The N-terminal extracellular site of each of the subunits was successfully produced by E. coli for the first time, and the present invention was completed.

 Hereinafter, the present invention will be described in detail.

First, cDNA fragments of the bovine GABA _A receptor α subunit, β subunit and rat glycine receptor α subunit used in the present invention are prepared, for example, by the following method.

From bovine brain and rat spinal cord, guanidine thiocyanate-lithium chloride method [cathala et al., DNA
(D.N.A.), 2 , 329, (1983)], preparing RNA having poly (A), using the poly (A) RNA, using a cDNA synthesis system kit manufactured by Amersham, and following the instructions. , And the PCR method [S
cience, 230 , 1350 (1985)]
The cDNA fragment of each subunit is amplified. For example, the plasmid vector pUC19 or pBluescript SK
Insert (+).

Using the thus obtained cDNA fragments of the bovine GABA _A receptor α subunit, β subunit, and rat glycine receptor α subunit, a region encoding the N-terminal extracellular site of the mature protein Was added with a stop codon at its 3 'end and further amplified by PCR,
Insert this into an E. coli expression plasmid vector,
Used for production in E. coli.

Expression plasmid vectors in E. coli use the tac promoter as the promoter and introduce a ribosome binding sequence downstream thereof. As such a ribosome binding sequence, for example, a chemically synthesized consensus sequence of a ribosome binding sequence of Escherichia coli can be used. Escherichia coli outer membrane protein as a signal peptide for secretion further downstream
Introduce the sequence of the signal peptide of OmpF.

In the present invention, the gene encoding the target protein represented by the nucleotide sequences of the formulas (I) to (III) is
Ligation is performed so as to obtain a fusion protein with the OmpF signal peptide.

The expression plasmid thus obtained is introduced into, for example, Escherichia coli MC1061, and the transformant is cultured in a bactopeptone broth according to a conventional method to produce a target protein.

[Examples] Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded.

Example 1 Bovine brain GABA _A receptor α, β subunit secretion production in E. coli at N-terminal extracellular site using E. coli outer membrane protein OmpF signal peptide 1) GABA _A receptor α, β subunit from bovine brain Unit cDN
Cloning of A 1-1) Preparation of poly (A) RNA from bovine brain and synthesis of cDNA RNA containing poly (A) is prepared from bovine brain according to the guanidine thiocyanate-lithium chloride method (described above) as follows. did.

20 g of brain was excised from cattle and immediately frozen in liquid nitrogen. This was put in a warding blender together with liquid nitrogen and pulverized at 3,000 rpm for 2 minutes. This was further crushed with a homogenizer (500 rpm) in 100 ml of a solution consisting of 5 M guanidine thiocyanate, 10 mM EDTA, 50 mM Tris-hydrochloric acid (pH 7) and 8% β-mercaptoethanol, and solubilized. Transfer 20 ml of this lysate into a centrifuge tube.
Gently put on 10 ml of 5.7M cesium chloride solution, Hitachi RPS28-
After centrifugation at 27,000 rpm for 20 hours using two rotors, RNA was recovered as a precipitate. The RNA precipitate was dissolved in 10 ml of a solution containing 0.1% sodium lauryl sulfate, 1 mM EDTA, and 10 mM Tris-hydrochloric acid (pH 7.5), extracted with phenol-chloroform, and recovered by ethanol precipitation. About 3.9 of obtained RNA
5 mg was dissolved in 1 ml of a solution consisting of 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM EDTA. The mixture was kept at 65 ° C. for 5 minutes, and 0.1 ml of 5M salt was added. The mixture is chromatographed on an oligo dT cellulose column (PL Biochemical) with a column volume of 0.5 m.
l).

The adsorbed poly (A) RNA was eluted with a solution containing 1 mM EDTA and 10 mM Tris-hydrochloric acid (pH 7.5) to obtain about 100 μg of poly (A) -containing mRNA.

Using poly (A) mRNA separated and purified as described above, cDNA was synthesized according to the instructions using a cDNA synthesis system kit manufactured by Amersham.

1-2) Amplification of cDNA of bovine GABA _A receptor α, β subunit by PCR method Next, the nucleotide sequence of cDNA of GABA _A receptor α, β subunit determined by Schofield et al. [Nature, 328 , 221 (1987)] and four 17-base synthetic primers 1-4 (FIG. 1) were applied to Applied Biosystem.
(Applied Biosystems) DNA Synthesizer 380
Synthesized by Form A.

Using the cDNA prepared in 1-1) as a template, the GABA _A receptor α and β subunits by PCR using primers 1 and 2 and primers 3 and 4 [Science 230 , 1350 (1985)] Were amplified (α subunit; about 1.7 kbp and β subunit; about 1.4 kbp) (FIG. 2). The PCR at that time was 10 ng of cDNA and 30 p
Using mols primers, 94 ° C for 1 minute in 100 μl system
Heat denaturation, 52 ° C: 2 minutes, annealing, 72 ° C: 3 minutes Complementary strand synthesis was defined as one cycle, and a total of 25 cycles were performed.

In the following, DNA fragment cleavage, modification, and ligation are performed according to the instructions provided with the enzyme, transformation of E. coli, preparation of plasmid DNA from E. coli, electrophoresis of DNA, and separation and purification from agarose gel. Is
Maniatis and colleagues [Molecular Cloning, Cold
Spring Harbor laboratory Press (Molecular Cloning, Cold Spring Harbor Laboratory Press) (1989)].

Further, these two fragments were separated and purified by 1% agarose gel electrophoresis according to a conventional method.
One μg of the obtained DNA fragment was dissolved in T4 DNA polymerase buffer (50 mM Tris-HCl (pH 8.5), 7 mM magnesium chloride, 15 mM ammonium sulfate, 10 mM 2-mercaptoethanol, 0.1 μM EDTA, 33 μM dNTP), and
The DNA termini were blunted by incubating 20 μl of the mixture containing the unit of T4 DNA polymerase (manufactured by Toyobo Co., Ltd.) at 37 ° C. for 30 minutes. In addition, 70μ
l of water and 100 µl of phenol-chloroform were added thereto, and the mixture was stirred by vortex. After centrifugation at 12,000 g for 5 minutes, the supernatant was separated (phenol-chloroform extraction) and 1/10 volume of 3M sodium acetate ( pH 5.2) and 2.5 volumes of ethanol were added, incubated at -20 ° C for 20 minutes, centrifuged at 12,000 g for 15 minutes, and the precipitate was further rinsed with 70% ethanol and dried (ethanol precipitate). Dissolved in water. These are referred to as α subunit; fragment 1 solution, β subunit; fragment 2 solution, respectively.

1-3) Insertion of Bovine GABA _A Receptor α Subunit cDNA into Plasmid Vector pBluescript Next, in order to cut the restriction enzyme Sma I site in plasmid vector pUC19 (Toyobo), 1 μg
PUC19 in a buffer [10 mM Tris-HCl (pH 7.5), 7 mM
Magnesium chloride 20 mM potassium chloride 7 mM 2-mercaptoethanol] dissolved in 20 μl, 10 units of Sma
I (manufactured by Toyobo Co., Ltd.) was added, the mixture was incubated at 37 ° C. for 2 hours, extracted with phenol-chloroform, precipitated with ethanol, dried and dissolved in 10 μl of water. 1μ of this DNA solution
l and 10 µl of the fragment 1 solution obtained in the above 1-2) were added to a buffer TL [6.6 mM Tris-HCl (pH 7.6), 6.6 mM magnesium chloride 10 mM dithiothreitol (hereinafter referred to as DTT).
), And the mixture was ligated overnight at 14 ° C with 5 units of T4 DNA ligase (manufactured by Toyobo Co., Ltd.). Further, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was used to transform it according to the instructions.

Next, plasmid DNA was prepared from the transformant according to a conventional method. Its insert fragment 1.7 kbp - in order to obtain [Sma I (in insert) Eco RI (in pUC19 fragment) fragment, 3
dissolved μg of plasmid DNA to Sma I buffer 20 [mu] l, Sm
a I 10 units was added and 37 ° C. 2 hours incubated. This was precipitated with ethanol and buffer H [100m
M sodium chloride, 50mM Tris-HCl (pH7.5), 10m
M Magnesium chloride 1 mM DTT Dissolve in 20 μl system, E
co RI (Toyobo Co., Ltd.) 10 units was added, was completely digested by incubating 37 ° C. 2 hours. The obtained reaction solution is
% Agarose gel and electrophoresed at 1.7 kbp according to the standard method.
Was separated and purified, and dissolved in 20 μl of water to obtain solution A.

Separately, 1 μg of the plasmid vector pBluescript SK
(+) The Sma I site and the Eco RI site of (Stratagene) were cleaved with 10 units of enzyme in the same manner as above, and phenol-
Extraction with chloroform and ethanol precipitation were carried out and dissolved in 10 μl of water to obtain solution B. Add 10 μl of solution A and 1 μl of solution B to buffer T
L, 20 μl of the system, 5 units of T4 DNA ligase (manufactured by Toyobo Co., Ltd.) were added, ligated at 14 ° C. overnight, and Escherichia coli JM109 was transformed with this according to the instructions. Plasmid DNA was prepared from the transformant according to a conventional method to obtain pGABAI (FIG. 3-A).

1-4) Insertion of bovine GABA _A receptor β subunit cDNA into plasmid vector pBluescript 1 μg of pBluescript SK (+) was added to SmaI buffer 20 μl.
dissolved in l, and incubated for 10 units 37 ° C. 2 hours added Sma I of completely digested, further phenol - chloroform extraction and ethanol precipitation, and dissolved in water 10 [mu] l. Of these, 1 μl and 10 μl of the fragment 2 solution obtained in the above 1-2) were ligated under the same conditions as described above, and Escherichia coli JM109 was transformed using the ligation according to the instructions. Plasmid DNA was prepared from the transformant according to a conventional method to obtain plasmid pGABAII (FIG. 3).
B).

5 'of the inserted fragment of the above plasmids pGABAI and pGABAII
Dideoxy at the base sequence of 30 base pairs at the terminal and 3 'end
As a result of the investigation by the chain termination method, Sc
The nucleotide sequence of the cDNA of the GABA _A receptor α and β subunits determined by hofield et al. [Nature, 32
8 , 221 (1987)].

2) Construction of an expression plasmid at the N-terminal extracellular site of bovine GABA _A receptor α and β subunits 2-1) Construction of an expression plasmid at the N-terminal extracellular site of bovine GABA _A receptor α subunit (see FIG. 4) _The region encoding the 225 amino acid residue at the N-terminal extracellular position of the mature protein of the _A receptor α subunit is 3 ′
In order to amplify a DNA fragment by a PCR method so that a termination codon is added to the end, a synthetic primer 5 (see FIG. 1) and a synthetic primer 6 (FIG. 1) are used in a DNA synthesizer manufactured by Applied Biosystem (Applied Biosystem). Synthesized by Model 380A.

Using the plasmid pGABAI obtained in 1) as a template and primers 5 and 6, the c-terminal
DNA amplification was performed. The conditions of the PCR method are shown below. In a 100 μl system containing 10 ng of plasmid DNA and 30 pmols of each primer, heat denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes, and synthesis at 72 ° C. for 3 minutes.
Cycled. This was separated and purified by 1% agarose gel electrophoresis, and furthermore, buffer L [10 mM
Tris-HCl (pH7.5), 10mM magnesium chloride, 1m
M DTT] and 10 units of Kpn I (Toyobo Co., Ltd.)
And incubate at 37 ° C for 2 hours for complete digestion.
Phenol-chloroform extraction, ethanol precipitation,
Dissolved in 10 μl of water. Furthermore, under the same conditions as performed in the above 1-2), and smooth the 3 'overhang with T4 DNA polymerase, phenol extracted, ethanol precipitated, further dissolved in buffer TL20myueru, restriction enzyme Bgl II (Toyo 10 units were added thereto, and the mixture was incubated at 37 ° C. for 2 hours to completely digest, and then subjected to phenol extraction and ethanol precipitation to obtain fragment 3.

Separately, an expression plasmid vector pUSFA [Appl. Microb
Iol.Biotechnol. (Applied Microbiology logical Biotechnology) 31, similarly 253-528 (1989)], subjected cleavage by Kpn I, smoothing of 3 'protruding terminus by T4 DNA polymerase, cleavage by Bgl II, fragment 4 was obtained.

Fragment 3 and Fragment 4 were added to buffer TL [66 mM
Tris-HCl (pH7.6), 6.6mM magnesium chloride 10m
M DTT, 66 μM ATP]
Add 5 units of DNA ligase (Toyobo Co., Ltd.), 14 ℃
Ligation overnight. This was further introduced into Escherichia coli MC1061 according to a conventional method. Plasmid DNA was prepared from this transformant to obtain pGABAIN (FIG. 4).

2-2) Construction of expression plasmid for N-terminal extracellular site of bovine GABA _A receptor β subunit (see FIG. 5).

GABA _A receptor β subunit mature protein N-terminal extracellular region coding for 220 amino acid residues 3 '
Adding a stop codon at the end, further DNA fragments to mutated Kpn I site 'a ((5'-GGTCCC-3 ( 5'-GGTACC-3)' to) from 360 bases Me in the insert P
To amplify by the CR method, four synthetic primers 7 to 10
(FIG. 1) was synthesized using a DNA synthesizer Model 380A manufactured by Applied Biosystem.

PCR was performed using pGABA II as a template, and fragment 5 was synthesized with synthetic primers 7 and 8,
10 gave fragment 6. The conditions of the PCR method are shown below. Plasmid DNA 10ng, primers 30pmols each
Heat denaturation at 94 ° C for 1 minute in a 100 µl system containing
Annealing for 2 minutes, 72 ° C for 3 minutes Synthesis of complementary strand 1
The cycle was performed for 20 cycles. This was subjected to 1% agarose gel electrophoresis, and fragments 5 and 6 were separated.
Purified.

Furthermore, PCR was performed under the same conditions using fragments 5 and 6 and primers 7 and 10 to obtain fragment 7.

This fragment 7 and the fragment 4 prepared in 2-1) were mixed with a buffer TL [66 mM Tris-HCl (pH 7.6), 6.
6mM magnesium chloride 10mM DTT, 66μM ATP] 20μl
, And 5 units of T4 DNA ligase (manufactured by Toyobo Co., Ltd.) was added, followed by ligation at 14 ° C. overnight. Furthermore, Escherichia coli MC1061 was transformed using this product according to a conventional method.

Plasmid DNA was prepared from the transformant according to a conventional method to obtain pGABAIIN (FIG. 5).

3) Expression of GABA _A receptor α and β subunit N-terminal extracellular site in E. coli 3-1) Bovine GABA _A receptor α subunit N-terminal extracellular site secretory production E. coli MC1061 carrying pGABAIN [Journal of Bacter
iology (Journal of Bacteriology) 143 ,
2, 971-980 (1980)] was cultured overnight at 30 ° C. in bactopeptone broth (20 g of bactopeptone, 10 g of sodium chloride, 2 g of glycerol, 1.6 mg of thiamine and 40 mg of ampicillin in water 1). This culture solution was transferred to 100 ml of a new bactopeptone broth at a dilution of 100-fold and cultured at 30 ° C. for 7 hours, and then isopropyl-β-D-thiogalactopyrase, an inducer of the tac promoter on the plasmid. Nosid (hereinafter, abbreviated as IPTG) was added to a concentration of 1 mM to induce secretory production of the GABA _A receptor α-subunit N-terminal extracellular site.
Cultured for hours. Thereafter, the cells were centrifuged (at 7,000 rpm for 10 minutes) to collect the bacteria. In addition, 10 ml of buffer [20%
Sucrose, 30 mM Tris-HCl (pH 8.0), 1 mM EDT
A], and 8 μl of the suspension was subjected to SDS polyacrylamide gel electrophoresis. As a result, it was confirmed that a considerable amount of extracellular N-terminal GABA _A receptor α subunit was produced.

3-2) Secretory Production of N-Terminal Extracellular Site of Bovine GABA _A Receptor β Subunit Escherichia coli MC1061 carrying pGABAIIN was cultured overnight at 30 ° C. in the above bactopeptone broth. This culture
Transfer to a new 100 ml of Bacto-peptone broth at 100-fold dilution and incubate at 30 ° C for 7 hours, then add IPTG to 1 mM
GABA _A receptor β subunit N
Induces secretory production in the extracellular position of the terminal cells.
The culture was carried out at three different temperatures of 15 ° C. and 42 ° C. for 15 hours. afterwards,
The cells were centrifuged (at 7,000 rpn for 10 minutes) and collected. Further, the suspension was suspended in 10 ml of a buffer [20% sucrose, 30 mM Tris-HCl (pH 8.0), 1 mM EDTA], and 8 μl of the suspension was added.
Was subjected to SDS polyacrylamide gel electrophoresis, and it was confirmed that a significant amount of the GABA _A receptor β subunit N-terminal extracellular site was produced.

[Example 2] Rat spinal cord glycine receptor α subunit using Escherichia coli outer membrane protein OmpF signal peptide Secretory production in E. coli of N-terminal extracellular site 1) Glycine receptor α subunit from rat and spinal cord
Cloning of cDNA 1-1) Preparation of poly (A) RNA from rat and spinal cord and cDN
Synthesis of A From rats and spinal cords, R-containing poly (A) according to the guanidine thiocyanate-lithium chloride method [cathala et al., DNA (DNA), 2 , 329, (1983)].
NA was prepared as described below.

10 g of spinal cord was excised from the rat and immediately frozen in liquid nitrogen. This was put in a wording blender together with liquid nitrogen and crushed at 3,000 rpm for 2 minutes. This was prepared using 5M guanidine thiocyanate, 10mM MEDTA, 50mM
The mixture was further crushed with a homogenizer (500 rpm) in 100 ml of a solution composed of Tris-hydrochloric acid (pH 7) and 8% β-mercaptoethanol, and solubilized. 20 ml of the solubilized solution is gently placed on 10 ml of a 5.7M cesium chloride solution in a centrifuge tube.
After centrifugation at 27,000 rpm for 20 hours in an S28-2 rotor, the RNA was recovered as a precipitate. This RNA precipitate was dissolved in 10 ml of a solution containing 0.1% sodium lauryl sulfate, 1 mM MEDTA, and 10 mM Tris-HCl (pH 7.5), extracted with phenol-chloroform, and recovered by ethanol precipitation. About 3.9 of obtained RNA
5 mg was dissolved in 1 ml of a solution consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. After keeping the temperature at 65 ° C. for 5 minutes, 0.1 ml of 5M salt was added. The mixture was applied to an oligo dT-rose column (PL Biochemical) chromatography (column volume 0.5 ml). The adsorbed poly (A) RNA was eluted with a solution containing 1 mM EDTA and 10 mM Tris-HCl (pH 7.5) to obtain about 100 μg of poly (A) -containing mRNA.

Using poly (A) mRNA separated and purified as described above, cDNA was synthesized according to the instructions using a cDNA synthesis system kit manufactured by Amersham.

1-2) Amplification of rat glycine receptor α-subunit cDNA by PCR method Base sequence of glycine receptor α-subunit cDNA determined by Grennininloh et al. [Nature, 32
8 , 215 (1987)] and two 17-base primers
11 and 12 (FIG. 1) were synthesized using a DNA synthesizer model 380A manufactured by Applied baiosystem (Applied Biosystem).

Using the cDNA prepared in 1-1) as a template, primers 11 and 12
PCR [Science 230 , 1350 (1
985)] to amplify a cDNA fragment (about 1.3 kbp) of the glycine receptor α subunit (FIG. 6).

PCR was performed using 10 ng of cDNA and 30 pmols of each primer in a 100 μl system at 94 ° C. for 1 minute, heat denaturation,
Annealing was performed at 2 ° C. for 2 minutes, and synthesis of a complementary strand was performed at 72 ° C. for 3 minutes, and a total of 25 cycles were performed. Hereinafter, cleavage, modification, and ligation of DNA fragments
The transformation of E. coli, the preparation of plasmid DNA from E. coli, the electrophoresis of DNA, and the separation and purification from agarose gel were carried out according to the conditions attached to the enzyme.
niatis et al. [Molecular Cloning, Cold Sp
ring Harbor laboratory Press (Molecular Cloning, Cold Spring Harbor Laboratory)
Press) (1989)].

Further, the fragments were separated and purified by 1% agarose gel electrophoresis according to a conventional method. Got
1 μg of the DNA fragment was treated with T4 DNA polymerase (Toyobo) buffer (50 mM Tris-HCl (pH 8.5), 7 mM
Magnesium chloride, 15 mM ammonium sulfate, 10 mM 2-
Dissolved in mercaptoethanol, 0.1 μM EDTA, 33 μM dNTP) and added 4 units of T4 DNA polymerase
The DNA ends were blunted by incubating 20 μl of the mixture at 37 ° C. for 30 minutes.

Further, 70 μl of water and 100 μl of phenol-chloroform were added, stirred by vortex, and centrifuged at 12,000 g for 5 minutes. The supernatant was separated (phenol-chloroform extraction), and 1/10 volume of 3M acetic acid was added. Sodium (pH 5.
2) and 2.5 volumes of ethanol were added and incubated at -20 ° C for 20 minutes, followed by centrifugation at 12,000 g for 15 minutes. The precipitate was further rinsed with 70% ethanol and dried (ethanol precipitate), and 20 µl of water was added. (Fragment 8).

1-3) Insertion of Rat Glycine Receptor α Subunit cDNA into Plasmid Vector pUC19 Next, in order to cut the restriction enzyme Sma I site in plasmid vector pUC19 (manufactured by Toyobo Co., Ltd.), 1 μg
PUC19 in Sma I buffer [10 mM Tris-HCl (pH 7.5),
7mM magnesium chloride, 20mM potassium chloride, 7mM β-
Mecaptoethanol] dissolved in 20μl, 10 units of Sm
aI (manufactured by Toyobo Co., Ltd.) was added, the mixture was incubated at 37 ° C. for 2 hours, extracted with phenol-chloroform, precipitated with ethanol, dried and dissolved in 10 μl of water. This DNA solution 1
μl and 10 μl of the fragment 8 solution obtained in 1-2) were added to T4
Ligation was performed overnight at 14 ° C. using 5 units of DNA ligase (manufactured by Toyobo Co., Ltd.) according to the instructions. Further, Escherichia coli JM109 (manufactured by Takara Shuzo Co., Ltd.) was used to transform it according to the instructions. From this transformant, plasmid DN
A was prepared according to a conventional method to obtain pGLYRI (FIG. 7).

2) Construction of expression plasmid for rat glycine receptor α subunit N-terminal extracellular site (see FIG. 8).

In order to amplify a DNA fragment by a PCR method so as to add a stop codon at the 3 'end to the region encoding the 219 amino acid residues at the N-terminal extracellular site of the mature protein of the glycine receptor α subunit, the synthetic primer 13 Ap (14, Fig. 1)
The DNA was synthesized using a DNA synthesizer Model 380A manufactured by plied Biosystem (Applied Biosystem). Using the plasmid pGLYRI obtained in 1) as a template, primers 13 and 14 were used to amplify cDNA at the N-terminal extracellular site by PCR. The conditions of the PCR method are shown below. In a 100 μl system containing 10 ng of plasmid DNA and 30 pmols of each primer, 20 cycles were performed with 1 cycle of heat denaturation at 94 ° C. for 1 minute, annealing at 55 ° C. for 2 minutes, and synthesis of a complementary strand at 72 ° C. for 3 minutes.
This was separated and purified by 1% agarose gel electrophoresis, and further dissolved in buffer H [100 mM sodium chloride, 50 mM Tris-HCl (pH 7.5), 10 mM magnesium chloride, 1 mM DTT] to obtain 10 units. Restriction enzyme Pst I
(Manufactured by Toyobo Co., Ltd.) and incubated at 37 ° C. for 2 hours to completely digest, extract with phenol-chloroform, precipitate with ethanol, and dissolve in 10 μl of water. Under the same conditions as in the above (1-2), the 3 'protruding end was blunted with T4 DNA polymerase, extracted with phenol and precipitated with ethanol. Furthermore, dissolved in buffer H20myueru, restriction enzyme Bgl II (manufactured by TOYOBO) 10 units added, 37 ° C., it was completely digested by incubation for 2 hours,
Phenol extraction and ethanol precipitation gave fragment 9
I got

Fragment 4 and Fragment 9 obtained in 2-1) of Example 1 were dissolved in a buffer TL of 20 μl, 5 units of T4 DNA ligase (manufactured by Toyobo Co., Ltd.) was added, and ligation was performed at 14 ° C. overnight. In addition, E. coli MC10
61 was transformed according to a conventional method. Further, a plasmid DNA was prepared from this transformant according to a conventional method to obtain pGLYRIN.

3) Expression in E. coli of N-terminal extracellular site of glycine receptor α subunit E. coli MC1061 carrying pGLYRIN [Journal of Bacter
iology (Journal of Bacteriology) 143 ,
2, 971-980 (1980)] was cultured overnight at 30 ° C. in bactopeptone broth.

Transfer this culture solution to 100 ml of a new bactopeptone broth at a dilution of 100 times and incubate at 30 ° C for 7 hours. Then, adjust the concentration of IPTG, an inducer of the tac promoter on the plasmid, to 1 mM. In addition, secretory production of the glycine receptor α-subunit N-terminal extracellular site was induced, and the cells were further cultured at 30 ° C. for 15 hours. Thereafter, the cells were centrifuged (at 7,000 rpm for 10 minutes) to collect the bacteria. In addition, 10 ml of buffer [20% sucrose, 30 mM Tris-
HCl (pH 8.0), 1 mM EDTA], and 8 μl of the suspension
When subjected to SDS polyacrylamide gel electrophoresis,
It was confirmed that a significant amount of glycine receptor α subunit N-terminal extracellular site was produced.

[Effects of the Invention] According to the present invention, a protein at the N-terminal extracellular site of an ion channel-directed receptor can be easily produced in Escherichia coli.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequences of the synthetic primers used in Examples 1 and 2. FIG. 2 is a schematic diagram showing cDNA fragments amplified by the PCR method in Example 1 and their relative positions with respect to the α subunit and the β subunit. FIG. 3 is a schematic diagram of plasmid pGABAI and plasmid pGABAII used in Example 1. FIG. 4 is a schematic diagram of a method for preparing the expression vector pGABAIN used in Example 1. FIG. 5 is a schematic diagram of a method for preparing the expression vector pGABAIIN used in Example 1. FIG. 6 is a schematic diagram showing the cDNA fragments amplified by the PCR method in Example 2 and their relative positions to the α subunit. FIG. 7 is a schematic diagram of the plasmid pGLYRI used in Example 2. FIG. 8 is a schematic diagram of a method for preparing the expression vector pGLYRIN used in Example 2.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Tatsuro Shibui 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Inside the Mitsubishi Chemical Research Laboratory (72) Inventor Kenji Nagahari 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical (56) References Neurochemistry International, Vol. 15, No. 1, (1989) p. 33-38 EMBO Journal, Vol. 6, (1989), p. 1665− 1670

Claims (1)

(57) [Claims] 1. A sequence comprising a tac promoter, a ribosome binding sequence, an Escherichia coli outer membrane protein OmpF signal peptide coding sequence, and the following formula (I): _A gene encoding the N-terminal extracellular site of the GABA _A receptor α subunit represented by the following formula (II): _A gene encoding the N-terminal extracellular site of the GABA _A receptor β subunit represented by the following formula (III): In culturing Escherichia coli into which an expression plasmid comprising the gene encoding the N-terminal extracellular site of the glycine receptor α subunit represented by the following formula was introduced, about 7 hours after the start of the culture, isopropyl-β-D-thio A method for producing an ion channel-directed receptor N-terminal extracellular protein, which is induced by using galactopyranoside.