JP3063982B2 - Method for producing protein - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for producing a protein by a genetic engineering technique. More specifically, the present invention relates to a method for producing an objective gene product in large quantities by introducing an expression vector having the same structural gene of interest into an animal cell twice or more.

(Problems to be Solved by Conventional Techniques and Inventions) In recent years, various kinds of production of useful trace substances contained in living bodies have been performed by genetic engineering techniques. Biologically active substances include the addition of sugar chains,
Although various modifications such as formation of an SS bond and amidation of C-terminal have been made, such modifications cannot be obtained even when these substances are produced in lower cells such as Escherichia coli. Therefore, it is preferable to produce these substances in animal cells, because it is possible to obtain substances that are more natural.

However, when using animal cells as hosts,
Generally, the expression efficiency is low, and it is necessary to improve the expression efficiency for industrial application.

In order to improve the expression efficiency, the copy number of the gene is increased (DNA level), and the transcription efficiency is increased (R
(NA level) or increase the efficiency of translation. Therefore, the present inventors have focused on improving expression efficiency in animal cells by the above method.

When an animal cell is used as a host, the introduced gene is integrated into the main chromosome of the host cell, so that it is difficult to increase the number of copies of the gene as in the case of using Escherichia coli or the like as a host.

Conventionally, a method known as a method for improving the expression efficiency in animal cells by increasing the number of copies of a gene includes:
This is an intracellular gene amplification method by a co-overlap method using a dhfr (dihydrofolate reductase) gene and MTX (methotrexate). However, this method is difficult to apply (a) unless dhfr-deficient cells are used, and the host cells are limited.

(B) MTX processing takes time.

(C) Unpredictable genetic recombination may occur on a chromosome during gene amplification in a cell, and a high-producing strain may not be obtained. Even if obtained, the gene may not be stable and high productivity may not be maintained.

There was such a problem.

On the other hand, in order to increase the number of copies of the gene, a method of introducing the target gene into animal cells twice or more can be considered.
Although the above-mentioned problems (a) and (b) may be overcome by such a method, the homology between the target gene already existing in the cell and the target gene to be further introduced from the outside during the second and subsequent gene transfer may be improved. Due to the nature, it was considered that genetic recombination could occur between them and a high-producing strain could not be obtained, and no such method was hitherto attempted.

(Means for Solving the Problems) In view of the above problems, the present inventors have recently obtained a transformant by introducing an expression vector having the same structural gene into animal cells twice or more, and obtaining a transformant. When the gene was produced,
The present inventors have found that the expression efficiency can be improved contrary to conventional expectations, and have completed the present invention.

That is, the gist of the present invention is characterized in that animal cells are transformed twice or more with an expression vector having the same structural gene, and the resulting transformant is cultured to produce a protein encoded by the structural gene. The first and second and subsequent transformations are performed using an expression vector containing at least a different number of expression units containing at least a promoter, a translation initiation codon and a structural gene. And a method for producing a protein.

Hereinafter, the present invention will be described. Any known animal cells can be used as host cells in the present invention. Specifically, for example, an ovarian cell line (CHO) derived from Chinese hamster [Proc. Natl. Sc
i.USA (Puroshi - Proceedings Of The National Academy - of Sciences of The User - Esue -), 77, 4261,1980], mouse ascites derived from FM3A cell line [Tohoku J.Exp.Med (door -. Hoku manager - Naru of
Expermental Medicine), 88 , 69, 1966], C127 cell line derived from mouse breast cancer [J. Virol. (Journal of Violology), 26 , 291, 1978], HeLa cell line derived from human cervical cancer (Flow -Commercially available from Sharp Corporation). Of these, the CHO cell line d
When an hfr gene-deficient strain is used, MTX treatment can be used in combination and is useful.

The expression vector used in the present invention comprises at least a transcription initiation site (promoter) such as an SV40 promoter, a mouse metallothionein gene promoter, and a human heat shock protein gene promoter. Promoters, translation initiation codons, structural genes such as various interferons, various interleukins, human tissue plasminogen activator, apolipoprotein A or hepatitis E or B virus Surface antigen, cardionatrine, etc., preferably with a molecular weight of 20,000 to 50,000
Exon-intron junction sites, for example, splice sites such as those derived from SV40 and rabbit β-globin, a transcription termination site (terminator), and a polyA addition region It is preferable to have a DNA fragment containing

The expression vector of the present invention can be introduced into the above-mentioned animal cells according to a conventional method, but a selective marker gene is also introduced simultaneously to detect the introduction of the expression vector. Such a selection marker gene may be incorporated into an expression vector having the structural gene of interest, or may be present on a different expression vector, and introduced into animal cells by double transformation (cotransduction). You may.

Selection marker genes include dihydrofolate reductase (dhfr), aminoglycoside 3'-phosphotransferase (neo), thymidine kinase (tk), xanthine guanine phosphoribosyltransferase (gpt),
Genes encoding adenosine deaminase (ada) and the like can be mentioned.

In the present invention, the expression vector is used to transform animal cells into two.
Transformation is performed more than once, but it is preferable to transform with an expression vector containing at least different numbers of expression units including a promoter, a translation initiation codon and a structural gene. For example, the first transformation was performed by
It is preferable to perform the second transformation by introducing an expression vector containing a plurality of expression units, and to perform the second transformation by introducing an expression vector containing a plurality of expression units, because the expression efficiency is further improved. Construction of such an expression vector containing a plurality of expression units can be performed by a method as described in Reference Examples below.

(Effect of the Invention) The transformant of the present invention obtained as described above contains a plurality of structural genes of interest, and is used in a conventional manner, for example, by using a roller bottle and 10% fetal bovine serum. 10mM HEPES
(N-2-hydroxylethylpiperazine-N'-2-
By culturing at 37 ° C. in an α-MEM medium containing (ethanesulfonic acid), the target protein can be produced in large quantities.

〔Example〕

Hereinafter, the present invention will be described more specifically with reference to Examples. However, the present invention is not limited to the following Examples as long as the gist of the present invention is not exceeded. Reference Example 1 <Construction of Expression Vector Having Multiple Expression Units Containing Human Apolipoprotein E Gene> 5 μg of the plasmid pKCRHAPE shown in FIG. 1 obtained according to the method described in JP-A-61-285990 was used.
10 mM Tris-HCl (pH 7.5), 100 mM sodium chloride and
Aat in 100 μμ of buffer containing 6 mM magnesium chloride
II The mixture was reacted with 10 µm at 37 ° C for 2 hours to digest. Then 75
After heating at 15 ° C. for 15 minutes to inactivate the enzyme, the DNA was precipitated with ethanol and sodium acetate and dried. 33 mM Tris-acetic acid (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate and 0.5 mM dithiosate-
Buffer containing 2 mM 4-deoxytriphosphate and a 40 μl system, and 3 U of 4 U of T4 DNA polymerase.
The reaction was carried out at 7 ° C. for 35 minutes to blunt the 3 ′ protruding end. After heating at 75 ° C. for 15 minutes to inactivate the enzyme, the DNA was precipitated with ethanol and sodium acetate and dried. To the obtained DNA (1.2 pmol) was added 66 mM Tris-HCl (pH 7.
5) a buffer containing 1 mM ATP, 1 mM spermidine, 10 mM magnesium chloride and 15 mM dithiothreitol;
Xho I linker phosphorylated at 5 'end (Takara Shuzo) 0.8
μg (120 pmol) and 600 U of T4 DNA ligase, and add a total of 50
μ, and reacted at 4 ° C. for 15 hours. After heating at 75 ° C for 15 minutes to inactivate the enzyme, 10 mM Tris-HCl (pH 7.
5) Xho I 100 U in a buffer containing 10 mM magnesium chloride, 1 mM dithiothreitol and 100 mM sodium chloride.
Was added to make the total amount 200 μ, and reacted at 37 ° C. for 3 hours. Subsequently, 10 U of Sal I was added, and the mixture was further reacted at 37 ° C. for 2 hours. After precipitating and drying the DNA with ethanol and sodium acetate, 89 mM Tris-borate and 10 mM EDT
DNA was separated by 5% acrylamide electrophoresis in a buffer containing A, and the gel was stained with a 0.05% aqueous ethidium bromide solution to cut out a larger molecular weight fragment (about 3200 bp) under 340 mm ultraviolet light. . The gel was placed in a cellulose dialysis tube and electroeluted in a buffer containing 89 mM Tris-borate and 10 mM EDTA. Ethanol and sodium acetate were added to the eluate to precipitate DNA, which was then dried and stored as a 25 μ aqueous solution <Fragment I>.

5 μg of pKCRHAPE was added to 10 mM Tris-HCl (pH 7.5),
The reaction was carried out for 2 hours at 37 ° C. with 10 U of SalI in 100 μl of a buffer containing 100 mM sodium chloride and 6 mM magnesium chloride.

After heating at 75 ° C. for 15 minutes to inactivate the enzyme, the DNA was recovered by ethanol precipitation and dried. Obtained DNA
Was reacted with 1 U of Escherichia coli alkaline phosphatase in 100 μM of 100 mM Tris-hydrochloric acid (pH 8.0) buffer at 65 ° C. for 1 hour, and DNA was recovered by phenol extraction and ethanol precipitation, followed by drying. . The obtained DNA was subjected to 5% acrylamide electrophoresis in a buffer containing 89 mM Tris-boric acid and 10 mM EDTA, and after separating the DNA, the gel was treated with 0.05% gel.
Staining with a 5% aqueous solution of ethidium bromide was performed to cut out a DNA fragment of about 5,800 bp under ultraviolet light of 340 nm. The gel was placed in a cellulose dialysis tube and electroeluted in a buffer containing 89 mM Tris-borate and 10 mM EDTA. Add ethanol and sodium acetate to the eluate
After the DNA was precipitated and dried, it was stored as a 25 μ aqueous solution <Fragment II>.

6 for Fragment I 2μ and Fragment II 1μ
A buffer containing 6 mM Tris-hydrochloric acid (pH 7.5), 1 mM ATP, 1 mM spermidine, 10 mM magnesium chloride and 15 mM dithiothreyl and 300 U of T4 DNA ligase were added to make a total amount of 20 mM.
μ, and reacted at 4 ° C. for 15 hours. The mixture was further heated at 75 ° C. for 15 minutes to inactivate the enzyme. Escherichia coli HB101 was transformed and grown according to a conventional method, and plasmid pKCR in which two expression units containing the human apolipoprotein E gene were linked in tandem.
HAPE-T2 was obtained.

5 μg of pKCRHAPE-T2 is added to 10 mM Tris-HCl (pH 7.
5), 10 U of Sal I was reacted at 37 ° C. for 2 hours in 100 μl of a buffer solution containing 100 mM sodium chloride and 6 mM magnesium chloride. After heating at 75 ° C. for 15 minutes to inactivate the enzyme, the DNA was recovered by ethanol precipitation and dried.
The obtained DNA was diluted with 100 mM Tris-hydrochloric acid (pH 8.0) buffer 100 μl.
After reacting with 1U of Escherichia coli alkaline phosphatase at 65 ° C for 1 hour in phenol, extraction with phenol and precipitation with ethanol
The DNA was recovered and dried. The obtained DNA was subjected to 5% acrylamide electrophoresis in a buffer containing 89 mM Tris-borate and 10 mM EDTA to separate the DNA. Gel
After staining with a 0.05% ethidium bromide aqueous solution, a DNA fragment of about 7,300 bp was cut out under ultraviolet light of 340 nm. The gel was placed in a cellulose dialysis tube and electroeluted in a buffer containing 89 mM Tris-borate and 10 mM EDTA. Ethanol and sodium acetate were added to the eluate to precipitate the DNA, which was dried and then stored as a 25 μ aqueous solution <Fragment III>.

For fragment I 2μ and fragment III 1μ
66 mM Tris-HCl (pH 7.5), / mM ATP, 1 mM ATP, 1 mM
A buffer containing spermidine, 10 mM magnesium chloride and 15 mM dithiothreyl and 300 U of T4 DNA ligase were added to make a total volume of 20 μm, and reacted at 4 ° C. for 15 hours. Another 75
Heat at 15 ° C for 15 minutes to inactivate the enzyme.
101 was transformed and grown to obtain a plasmid pKCRHAPE-T3 in which three expression units containing the human apolipoprotein E gene were linked in tandem.

The same operation was repeated, and the promoter of SV40, the human apolipoprotein E gene, the exon intron junction site of rabbit β-globin, rabbit β-globin and SV
A plasmid in which four and five expression units each comprising 40 terminators and a poly-A additional region are tandemly linked, respectively.
pKCRHAPE-T4 and pKCRHAPE-T5 were obtained.

Example 1 (1) Plasmid PKCRHAPE (FIG. 1) 10 [mu] g and mouse dihydrofolate reductase (dhfr) containing gene plasmid PMTV dhfr [nature (Neicha -), 294, 228,198
1] 1 μg of ham containing 10% fetal calf serum (FCS) in a 9 cmφ cell culture dish by forming a calcium phosphate precipitate using a Cellfect Kit (Pharmacia) according to the method described in the instructions. COH ahfr grown in 10 ml of F-12 medium (manufactured by Gibco) in a 5% carbon dioxide atmosphere at 37 ° C. until it becomes semi-confluent.
^- cells (5 × 10 ⁶ cells) [Proc, 77, 4
216, 1980] by the method described in the kit instructions, followed by transformation. To increase the efficiency of gene transfer, Hepes-containing 15% glycerol after 6 hours.
Glycerol treatment was performed with 3 ml of a hydrochloric acid (pH 7.5) buffer, and the cells were cultured in a Ham F-12 medium (manufactured by Gibco) containing 10% FCS at 37 ° C. for 48 hours in a 5% carbon dioxide gas atmosphere.

After removing the culture solution, the cells were detached with a 0.05% trypsin solution, diluted 7-fold, and α-MEM medium (manufactured by Gibco) further containing 10% FCS in seven 9 cmφ cell culture dishes.
At 37 ° C. in a 5% carbon dioxide atmosphere. After culturing for about 4 weeks while replacing the medium with a new one every three days, about 200 colonies were formed. The obtained colonies were cultured on a 24-well cell culture plate, and the presence or absence of apoE production was determined according to “Enzyme Immunoassay 3rd Edition” (Medical Shoin).
Confirmed by ELISA method.

Of the colonies formed, 20 colonies expressing apolipoprotein E were grown in a 9 cmφ cell culture dish at 37 ° C. and 5% carbon dioxide atmosphere until they became semi-confluent. Further, 5 ml of FCS-free Ham F-12 medium was added, and the mixture was added under a 5% carbon dioxide atmosphere.
The cells were cultured at 7 ° C for 24 hours. Human apolipoprotein E produced by the transformant was quantified by ELISA method ("Enzyme immunoassay method 3rd edition" Medical Shoin) using apolipoprotein E derived from human serum as a standard. . At that time, the number of cells per dish was measured with a cell counter (manufactured by Toa Medical Electronics Co., Ltd., Sysmex F-300). The results are shown in Table 1.

(2) Plasmid pKCRHAPE-T410 constructed in Reference Example 1
plasmid pS containing .mu.g and the aminoglycoside 3'-phosphotransferase gene from transposon 5
V2neo [J.Mole.Appl.Genet,. (Ja - Naru of Molecular - and Applied Genetics) 1, 3
27,1982] 1 μg was introduced in the same manner as in the above (1) into the colony in which the amount of apolipoprotein E produced was the largest among the colonies obtained in the above (1), followed by transformation. .

Similarly, after culturing in a ham F-12 medium containing 10% FCS and adding trypsin to dilute the cells to 7 cultures,
Further, the cells were cultured at 37 ° C. in a ham F-12 medium containing 200 μg / ml of G-418 (manufactured by Gibco) and 10% FCS in a 5% carbon dioxide atmosphere. After culturing for about 2 weeks while replacing the medium with a fresh same medium every 3 to 4 days, about 200 colonies were formed.

Among them, 37 colonies expressing apolipoprotein E at least as much as the original clone were cultured in the same manner as in (1) above, and the produced human apolipoprotein E was quantified. . The results are shown in Table 1.

(3) Plasmid pKCRHAPE-T5 constructed in Reference Example 1
10 μg of the plasmid pMSG (manufactured by Pharmacia) containing the xanthine guanine phosphoribosyltransferase gene of Escherichia coli was added to the colony having the highest production of apolipoprotein E among the colonies obtained in the above (2). -Was introduced in the same manner as in the above (1) and subjected to transformation treatment.

In the same manner as described above, the cells were cultured in a ham F-12 medium containing 10% FCS, and trypsin was added to dilute the cells to 7-fold, and then mycophenolic acid (Sigma) 1.0 μg / ml.
And Ham's F-12 medium containing 10% FCS at 37 ° C. in a 5% carbon dioxide atmosphere. After about 3 weeks of culturing while replacing the medium with a new one every 3 to 4 days, about 200 colonies were formed.

Among them, 41 colonies expressing apolipoprotein E at least as much as the original clone were cultured in the same manner as in (1) above, and the amount of human apolipoprotein E produced was quantified. . The results are shown in Table 1.

[Brief description of the drawings]

FIG. 1 is a drawing showing a schematic diagram of a plasmid pKCRHAPE containing a human apolipoprotein E gene used in Example 1.

──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Yutaka Teranishi 1000 Kamoshita-cho, Midori-ku, Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Research Institute (56) References JP-A-2-190194 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) MEDLINE (STN)

Claims (2)

(57) [Claims] 1. A protein characterized in that an animal cell is transformed twice or more with an expression vector having the same structural gene, and the resulting transformant is cultured to produce a protein encoded by the structural gene. A production method comprising, in the first transformation and the second and subsequent transformations, transforming expression units containing at least a promoter, a translation initiation codon, and a structural gene by different numbers from each other. A method for producing a characteristic protein. 2. An expression unit containing at least a promoter, a translation initiation codon and a structural gene for the first transformation.
The method for producing a protein according to claim 1, wherein an expression vector containing one unit is used, and an expression vector containing a plurality of units of the expression unit is used for the second and subsequent transformations.