JPH07213289A - Method for genetic engineering mass-production of neutrophic factor - Google Patents

Abstract

PURPOSE:To massively obtain the subject factor by expressing a vector containing a DNA produced by binding the specific polypeptide DNA of Escherichia coli, a factor Xa DNA and a Escherichia coli regulator gene to the 5'-end side of a neurotrophic factor DNA and subsequently treating the produced protein with an enzyme. CONSTITUTION:A DNA coding eight amino acid molecules on the N-end side of the leader polypeptide of the tryptophane operon of Escherichia coli and a DNA coding a factor Xa-recognizing sequence are successmvely bound to the 5'-end upstream side of a DNA coding the polypeptide chain of a neurotrophic factor [e.g. human nerve growth factor(hNGF)]. Further, a vector bound to the regulator gene of Escherichia coli is introduced to the upstream of the DNA coding the leader polypeptide. The produced transformant is cultured, and the fused type neurotrophic factor produced thus is reacted with a protease. Thus, the neurotrophic factor is massively produced by the genetic engineering method.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a fusion neurotrophic factor, in particular, a gene DNA corresponding to a human nerve growth factor (hereinafter abbreviated as hNGF) or a brain-derived neurotrophic factor (hereinafter abbreviated as BDNF), and the gene. And a transformant transformed with the plasmid vector, and a fusion-type neurotrophic factor produced by using the transformant, and a proteolytic enzyme acting on the fusion-type neurotrophic factor to produce a mature nerve A method of producing a nutritional factor.

[0002]

2. Description of the Related Art Neurotrophic factor is known as a polypeptide involved in the survival of nerve cells, the extension of neurites and the differentiation of nerve cells. Among them, nerve growth factor (NG) is known.
F) and brain-derived neurotrophic factor (BDNF) are known. NGF extracted from the adult submandibular gland of a male mouse is a polypeptide having a molecular weight of 140,000 and is called 7SNGF based on its sedimentation constant. The β subunit among them shows NGF activity by itself, is a polypeptide consisting of 118 amino acids, and is called βNGF. On the other hand, human NGF has not been found to have a large amount of tissues such as mouse submandibular gland.

Therefore, a human gene library was screened by using the mouse βNGF cDNA as a probe to isolate the hNGF gene, and the amino acid sequence was determined therefrom (JP-A-60-84299).

The brain-derived neurotrophic factor was isolated from nature by Barde, Y.-A. et al., And then the BDNF gene was cloned by Leibrock, J. et al. (Nature, 341,
149 (1989)).

Recently, it has been possible to mass-produce a heterologous protein in Escherichia coli by using a genetic engineering method, and attempts have been made to produce hNGF and BDNF by the genetic engineering method. For example, JP-A-6
In 0-84299, gene expression was attempted using the natural hNGF gene. For BDNF, Knusel, B. et al. Produced BDNF using animal cells (Proc. Natl. Acad. Sci. USA 88, 961 (1991)),
Negro, A. et al. Produced BDNF with methionine added to the N-terminus by Escherichia coli (Biochem. Biophys. Res. Commu.
n. 186, 1553 (1992)). This method uses hNGF
Gene or BDNF gene is incorporated into an expression vector, and cells containing the vector are cultured to obtain hNG.
It is a mass production of F and BDNF.

[0006]

However, in the above-mentioned known technique, the hNGF or BDNF gene is expressed in a host cell with high expression efficiency, and the vector has high expression efficiency, and transformation with an expression vector is also performed. The cell culture conditions of the treated cells to hNGF or BD
No sufficient consideration has been given to making it suitable for the production of NF, and easy and large-scale production of hNGF and BD
There is a problem that it is difficult to produce NF in host cells, especially E. coli.

The object of the present invention is to provide a fusion hNGF gene DNA or a fusion BDNF gene DNA having a base sequence with high expression efficiency in E. coli host cells, a recombinant plasmid incorporating the DNA together with a promoter with high expression efficiency, and Another object of the present invention is to provide a transformed microorganism transformed with this recombinant plasmid, which has an excellent ability to produce fused hNGF or fused BDNF. Further, the transformed microorganism is cultivated, and at this time, suitable medium conditions are used. Is applied to efficiently produce a fused hNGF, and to act a proteolytic enzyme on the produced fused hNGF to provide a novel method for efficiently obtaining a mature hNGF or a mature BDNF. .

[0008]

Means for Solving the Problems The inventors of the present invention have completed the present invention as a result of intensive research to solve the above-mentioned problems, and have revealed that the neurotrophic factor genes such as hNGF and BDNF Previously, DNA encoding 8 amino acids from the N-terminal side of the tryptophan operon leader peptide (trp L) of Escherichia coli was ligated and expressed as a fusion type protein to enhance the expression of the neurotrophic factor gene. It is something to do. It is possible to obtain a large amount of the target protein by binding the above-mentioned 8 amino acids to the target protein to form insoluble granules in the expressed protein and making it resistant to degradation by proteolytic enzymes. It takes advantage of what you can do.

Furthermore, a recognition sequence for the protease Xa (ILE-
GLU-GLY-ARG) is designed to cause the fused 12-amino acid to act on the produced fusion-type target protein to cleave excess 12 amino acids to obtain the mature-type target protein. Was done. The features of the present invention are as follows (1)
~ (11).

(1) It has a DNA encoding a polypeptide chain of a neurotrophic factor, and the 5'end upstream of the DNA has
A DNA encoding the N-terminal 8 amino acids of the E. coli tryptophan operon leader polypeptide is bound with a DNA encoding the recognition sequence of Factor Xa, and further the DNA encoding the leader polypeptide.
A vector having an Escherichia coli regulatory gene upstream of the vector.

(2) A vector in which the N-terminal 8 amino acid sequence of the leader polypeptide of the tryptophan operon of Escherichia coli and the recognition sequence of Factor Xa linked to the sequence are the following amino acid sequences.

MET LYS ALA ILE PHE
VAL GLU PHE ILEGLU GLY A
RG (3) A vector whose base sequence encoding the amino acid sequence of the N-terminal 8 amino acids of the leader polypeptide of the tryptophan operon of Escherichia coli and the recognition sequence of Factor Xa linked to the sequence is the following DNA.

ATG AAA GCA ATT TTC
GTG GAA TTC ATCGAA GGC A
GG (4) The vector of (1), (2) or (3), wherein the Escherichia coli regulatory gene is a tryptophan regulatory gene.

(5) The neurotrophic factor is nerve growth factor or brain-derived neurotrophic factor (1),
The vector of (2), (3) or (4).

(6) A transformant obtained by transforming Escherichia coli with the vector according to (1), (2), (3), (4) or (5).

(7) The transformant described in (6) is cultured,
A method for producing a neurotrophic factor, which comprises producing the neurotrophic factor in a culture.

(8) A method for producing human nerve growth factor, which comprises reacting the fused neurotrophic factor according to (7) with a proteolytic enzyme to produce a mature neurotrophic factor.

(9) A method for producing a mature neurotrophic factor, which comprises using Factor Xa as the proteolytic enzyme according to (8).

(10) The method for producing a neurotrophic factor according to (7), wherein IA (3-β-indoleacrylic acid) is added as an inducer and the culture is performed.

(11) The method for producing a neurotrophic factor according to (7), which comprises culturing using a medium that does not contain tryptophan.

The present invention will be described in detail below.

The amino acid sequence of hNGF is shown in Ullrich et al. (Natu
re, 303, 821 (1983)) and consists of 118 amino acids. In addition, BDNF has a 119 amino acid sequence, and its gene is the pig BDNF gene by Leibrock, J. et al., Followed by the isolation of human and mouse genes, and their amino acid sequences are identical.

HNGF and BD produced in microbial cells
Since NF is a heterologous protein, it is easily decomposed by proteolytic enzymes in the cells. Therefore, we decided to generate insoluble granules in order to prevent degradation in the cells, and to ligate the DNA encoding the N-terminal 8 amino acids of the trpL polypeptide of the tryptophan operon in front of the hNGF gene or BDNF gene. . In order to convert the produced fused hNGF or fused BDNF into mature hNGF or mature BDNF, the trpL polypeptide N
Terminal 8 amino acids (MET-LYS-ALA-ILE-PHE-VAL-GLU-P
HE) and hNGF or BDNF protein between the recognition sequences of factor Xa which is a protease (ILE-GLU-GLY-ARG)
Inserted. By doing so, the fusion type h produced
When factor Xa is allowed to act on NGF or fused BDNF, the extra 12 amino acids are cleaved, resulting in mature hN.
GF or mature BDNF is obtained.

FIG. 2 shows the expression vector pTRLNG used to prepare the expression vector for the fusion-type hNGF of the present invention.
The structure of F is shown (Japanese Patent Application No. 2-38358, Microtechnology Research Institute, No. 11283). The hNGF gene in which DNA encoding the N-terminal 8 amino acids of the trpL polypeptide of the tryptophan operon was ligated to the tryptophan promoter was ligated, and EcoRI and BamHI of pBR322 were ligated.
It was inserted between. This EcoRI site was later deleted. In addition, the hNGF gene used here was synthesized by organic chemistry.

Therefore, the expression vector pTRLXNGF of the fusion-type hNGF of the present invention is the expression vector pTRLNG.
DNA encoding the 8 amino acids at the N-terminal side of the trpL polypeptide of the tryptophan operon in the structure of F and h
A DNA encoding a recognition sequence for factor Xa (ILE-GLU-GLY-ARG), which is a proteolytic enzyme, between NGF genes
It was prepared by inserting (ATCGAAGGCAGG). FIG. 1 shows the expression vector pTRLXN of the present invention.
The structure of GF is shown. In addition, the expression vector of the fused BDNF is hNG in the structure of the expression vector pTRLXNGF.
It was created by replacing the F gene portion with the BDNF gene. Similarly, FIG. 1 shows the expression vector p of the present invention.
1 shows the structure of TRLXBDNF.

Expression vector pTRLXN obtained here
By using GF or the expression vector pTRLXBDNF to transform host microorganisms such as Escherichia coli HB101 strain, JM105 strain, JM109 strain and the like by a conventional method, a transformed microorganism having an excellent hNGF or BDNF production ability can be obtained.

Further, this transformed microorganism is cultured to produce hN.
When producing GF or BDNF, IA (3-β-indole acrylic acid), which is an analog substance of tryptophan, is added to the medium or a medium containing no tryptophan is added as a medium condition to start the expression of the gene. Preference is given to using hNGF or BD
The production yield of NF is further improved.

This is due to the following reason. That is, gene expression is regulated by a gene region called a promoter. In the case of the trp promoter, RNA synthase binds to the trp promoter, and this enzyme moves downstream of the promoter and transcribes a DNA base sequence to synthesize RNA. And the RNA synthesized here
The polypeptide is synthesized by the translation of the information of ribosome by the ribosome. On the other hand, when the intracellular tryptophan concentration increases, tryptophan binds to the repressor, activates it, and the repressor binds to a region called an operator. As a result, RNA synthase cannot bind to the promoter portion, RNA is not synthesized, and the production of the polypeptide is stopped. In this case tryptophan acts as an inhibitor. Therefore, the present inventors have adopted a method of initiating gene expression by utilizing gene expression regulation in the trp promoter and inactivating the repressor by addition of IA or switching to a medium not containing tryptophan.

In addition to hNGF and BDNF, NT-3, NT-4 and NT-5 (protein nucleic acid enzyme, 38, 1603 (1993)) are known as neurotrophic factors, and these are also hNG.
Since it has high homology with the amino acid sequences of F and BDNF, it can be expressed by the same method.

[0030]

In the present invention, the neurotrophic factor gene is preceded by the amino acid sequence of the N-terminal 8 amino acids of the tryptophan operon leader polypeptide and the factor X linked to the sequence.
Since the nucleotide sequence encoding the recognition sequence of a is linked, a fused neurotrophic factor is produced. As a result, insoluble granules are formed in Escherichia coli and the degradation by proteolytic enzymes is prevented, so that it becomes possible to produce a large amount of neurotrophic factor, and the recognition sequence for factor Xa is present. By acting Factor Xa on the neurotrophic factor, a mature neurotrophic factor in which excess polypeptide is cleaved can be obtained.

In the present invention, since the growth of E. coli and the production of neurotrophic factor can be controlled by utilizing the gene regulatory sequence of Escherichia coli, the productivity of neurotrophic factor is improved.

[0032]

EXAMPLES Examples of the present invention will be specifically described below, but the present invention is not limited thereto.

Example 1 The expression vector pTRLXNGF was prepared as shown in FIG.

1 μg of the expression vector pTRLNGF was added to E
After cleaving with coRI 40U, it was treated with alkaline phosphatase to dephosphorylate the terminal base. On the other hand, DN
240 pmol of A linker was treated with T4 kinase to phosphorylate the terminal base. DNA of these two fragments
After ligating 0.1 μg and 1.5 ng with a ligation kit, the Escherichia coli C600 strain was transformed.
Ten thousand colonies were obtained. 12 clones in this D
As a result of cleavage of NA with a restriction enzyme and analysis, it was found that four clones were the target expression vector. In this way, the expression vector pTRLXNGF could be obtained.

Example 2 The expression vector pTRLXBDNF was prepared as shown in FIG.

1 μg of the expression vector pTRLXNGF was cleaved with 50 U each of SacI and BamHI, treated with alkaline phosphatase, and the terminal base was dephosphorylated. Subsequently, 0.8% agarose gel electrophoresis is performed to collect the DNA fragment of the vector, and then T4 DN
A blunt-ended with 1 U of polymerase. On the other hand, pcBDNF / C in which rat BDNF cDNA was inserted
The BDNF gene portion of 360 base pairs was synthesized by the PCR method using the DM8 plasmid as a template. After ligating 0.1 μg and 0.01 μg of these two fragments of DNA with a ligation kit, the E. coli C600 strain was transformed, and about 2,000 colonies were obtained. As a result of digesting the DNA of 12 clones among them with a restriction enzyme and analyzing it, it was found that 4 clones were the desired expression vectors. In this way, the expression vector pTRLX
I was able to obtain BDNF.

Example 3 Production of hNGF or BDNF by Transformed Microorganism (without Tryptophan) Transgenic Escherichia coli HB101 [pTRLXNGF] obtained by transforming the E. coli HB101 strain with the expression vector prepared in Example 1 or 2 (accession number FERM P-14091 deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology) or HB101 [p.
TRLXBDNF] (consignment number FERM P-1409
0 was deposited at the Institute of Biotechnology, Institute of Biotechnology, AIST. Was cultured as follows to produce hNGF or BDNF. One platinum loop of the preserved strain was treated with 10 ml of M9 medium (NH ₄ Cl 1 g, Na ₂ HPO ₄ 6 g, KH ₂ PO).
_{4 3g, NaCl 0.5g, CaCl 2} · 2H 2 O
_{0.015g, MgSO 4 · 7H 2 O} 0.5g, casamino acid 2.5g, glucose 5g, yeast extract 1.
5 g, tryptophan 0.04 g, proline 0.1
g, thiamine 0.1 g, ampicillin 50 mg, water 1 l, pH 7.0), and the mixture was cultured at 37 ° C. overnight. 5 ml of the preculture liquid was added to 25 ml of M2 (-
Trp) medium and 20 ml of water were inoculated into a Sakaguchi flask and cultured at 37 ° C. for 24 hours. The cells after 24 hours of culturing were collected by centrifugation, washed with 10 mM Tris.HCl buffer (pH 7.5), and the absorbance at 550 nm was adjusted to about 25. 10 mM Tris.HCl + 5 mM
Suspended in EDTA buffer (pH 8). This suspension 10
Sample buffer (10 mM Tris.HCl,
pH 6.8; 2% SDS; 40% glycerol; 2
% Β-mercaptoethanol; 0.04% BPB)
10 μl was added and heated at 100 ° C. for 5 minutes. After this solution was subjected to 0.1% SDS-15% polyacrylamide gel electrophoresis, the proteins in the gel were transferred to a nitrocellulose membrane, and hNGF or BDNF was detected with a polyclonal antibody against hNGF or BDNF. Binding of the antibody was found at almost the same position as.
From this, the expression of fused hNGF or BDNF by E. coli could be confirmed. When the amount of hNGF or BDNF produced by Escherichia coli was calculated from the color intensity of the stained band,
About 15 mg or 16 m per liter of culture fluid
g hNGF or BDNF was produced.

The cells thus obtained were washed twice with 30 ml of 50 mM Tris-hydrochloric acid buffer (pH 7.5) and then washed with 5 mM.
It was suspended in 30 ml of 50 mM Tris-HCl buffer containing EDTA. The suspension was sonicated to destroy the cells, and then centrifuged at 2000 × G for 15 minutes to obtain a pellet. The pellet was dissolved in 5 ml of 0.5 M urea solution, 0.1 ml (1 mg / ml) of factor Xa was added, and the mixture was reacted at 20 ° C. for 1 hour. This reaction solution is 10 mM
After dialysis against an acetate buffer (pH 4.5) and freeze-drying, mature hNGF or mature BDNF from which excess polypeptide was removed was obtained.

Example 4 Production of hNGF or BDNF by a transformed microorganism (I
A use) Recombinant Escherichia coli HB101 [pTRLXNGF]
(Deposited under the deposit number FERM P-14091 to the Institute of Biotechnology, Institute of Biotechnology, National Institute of Industrial Science and Technology) or HB10.
1 [pTRLXBDNF] (accession number FERM P-1
4090 deposited at the Institute of Biotechnology, Industrial Technology Institute. ) Is cultured as follows, and hNGF or BDNF is cultured.
Produced.

In the same manner as in Example 3, the cells were treated with 50 ml of M.
It was inoculated into 9 (-Trp) medium and cultured at 37 ° C for 8 hours. At this time, in order to express the gene, IA was added at 15 μg / ml at 1 hour of culture. After completion of the culture, the same treatment as in Example 3 was carried out to calculate the hNGF production amount or BDNF production amount of the genetically modified Escherichia coli. As a result, almost the same amount of hNGF was produced. Furthermore, in the same manner as in Example 3, mature hNGF or mature B
DNF was obtained.

[0041]

INDUSTRIAL APPLICABILITY In the present invention, the amino acid sequence of the N-terminal 8 amino acids of the tryptophan operon leader polypeptide and the recognition sequence of Factor Xa linked to the sequence are encoded before the nucleotide sequence of hNGF gene or BDNF gene. Since the base sequence is bound to the host microorganism, in particular, a base sequence having high expression efficiency in Escherichia coli and a gene transformed with the plasmid by incorporating the gene into a plasmid together with a promoter having excellent expression efficiency. In the converted microorganism, the expression efficiency of the hNGF gene or BDNF gene is extremely good, and in addition to this, the culture conditions are further improved, and as a result, fused hNGF or fused BDNF can be easily and mass-produced. It became possible. Further, the fusion-type hNGF or the fusion-type BDNF obtained here is cleaved to remove excess polypeptide by allowing the protease Xa to act, whereby mature hNGF or mature BDNF can be obtained. It was Therefore, the present invention greatly contributes to the manufacture of pharmaceuticals and the like.

[0042]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 12 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Met Lys Ala Ile Phe Val Glu Phe Ile Glu Gly Arg 1 5 10 SEQ ID NO: 2 Sequence length: 36 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence features Characteristic symbols: peptide Location: 1..36 Method of determining features: S Sequence ATG AAA GCA ATT TTC GTG GAA TTC ATC GAA GGC AGG 36

[Brief description of drawings]

FIG. 1 shows the structure of the expression vector pTRLXNGF and the structure of the expression vector pTRLXBDNF of the present invention.

FIG. 2 shows the structure of the expression vector pTRLNGF used for producing the expression vector pTRLXNGF of the present invention.

FIG. 3 shows a method for producing the expression vector pTRLXNGF of the present invention.

FIG. 4 shows a method for preparing the expression vector pTRLXBDNF of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Hiroko Hanzawa, Inventor Hiroko Hanzawa 2520, Akanuma, Hatoyama-cho, Hiki-gun, Saitama Stock Company Hitachi Research Laboratory (72) Inventor Kenichi Takahashi 2520, Amanuma, Hayama, Hiki-gun, Saitama Stock Company Hitachi Research Laboratory

Claims (11)

[Claims] 1. A DNA encoding a polypeptide chain of a neurotrophic factor, wherein the 5'-terminal upstream side of the DNA is N, which is the leader polypeptide of the tryptophan operon of Escherichia coli.
A vector in which a DNA encoding a recognition sequence for Factor Xa is bound to a DNA encoding the terminal 8 amino acids, and further, an Escherichia coli regulatory gene is provided upstream of the DNA encoding the leader polypeptide. 2. The vector according to claim 1, wherein the N-terminal 8 amino acid sequence of the leader polypeptide of the E. coli tryptophan operon and the recognition sequence of Factor Xa linked to the sequence are the following amino acid sequences. MET LYS ALA ILE PHE VAL G
LU PHE ILEGLU GLY ARG 3. The base sequence encoding the amino acid sequence of the N-terminal 8 amino acids of the E. coli tryptophan operon leader polypeptide and the recognition sequence of Factor Xa linked to the sequence is the following DNA: vector. ATG AAA GCA ATT TTC GTG G
AA TTC ATCGAA GGC AGG 4. The vector according to claim 1, 2 or 3, wherein the Escherichia coli regulatory gene is a tryptophan regulatory gene. 5. The neurotrophic factor is nerve growth factor or brain-derived neurotrophic factor, which is characterized in that
Or the vector according to 4. 6. A transformant obtained by transforming Escherichia coli with the vector according to claim 1, 2, 3, 4 or 5. 7. A method for producing a neurotrophic factor, which comprises culturing the transformant according to claim 6 and producing the neurotrophic factor in the culture. 8. A method for producing human nerve growth factor, which comprises reacting the fusion neurotrophic factor according to claim 7 with a proteolytic enzyme to produce a mature neurotrophic factor. 9. A method for producing a mature neurotrophic factor, which comprises using Factor Xa as the proteolytic enzyme according to claim 8. 10. The method for producing a neurotrophic factor according to claim 7, wherein IA (3-β-indoleacrylic acid) is added as an inducer and the culture is performed. 11. The method for producing a neurotrophic factor according to claim 7, wherein the culture is performed using a medium that does not contain tryptophan.