JPH0644875B2 - Method for producing recombinant gene product - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a recombinant gene product.

[Prior art]

There are few reports on the improvement of the culture medium and culture medium for improving the productivity of the recombinant gene product, and the pH control is performed by using a medium in which Davis medium is modified with a focus on slightly high-concentration culture of bacteria. However, a culture method has been reported in which glucose is divided and added. (H.Merietal.,
Journal of Chemical Engineering of Japan 12 . Three
13-319 (1979), T. Kobayashi et al. Proc.-
Pac. Chem. Eng. Conger., 3rd 4 147-50 (198).
3)) However, although high-concentration culture of the bacterium has been achieved in these, the ratio of the target product to the total E. coli bacterial protein is not specified.

[Problems to be solved by the invention]

According to the experiments conducted by the inventors of the present invention, the bacterial concentration is certainly increased in the above-mentioned culture method, but the amount of the target product per total Escherichia coli protein is markedly decreased as compared with the usual culture method, and its productivity is improved at all. It turned out not to be.

[Means for solving problems]

Therefore, as a result of various studies on the method for improving the productivity of the recombinant gene product, the present inventors have found that thiosulfate is added to the medium containing casein hydrolyzate, yeast extract, inorganic salt and Escherichia coli assimilating carbon source as essential components. It was found that the productivity of the gene product was significantly improved by adding salt and culturing Escherichia coli having the recombinant gene.

The present invention has been completed based on the above findings.

That is, the present invention is a casein hydrolyzate, yeast extract, inorganic salts and E. coli thiosulfate is added to the medium containing essential carbon sources, cultivated E. coli having a recombinant gene, the culture thereof. The present invention relates to a method for producing a recombinant gene product, which comprises collecting the recombinant gene product.

The casein hydrolyzate used in the present invention is not particularly limited as long as it hydrolyzes casein, but trypsin, pepsin, hydrolyzate of casein with a protease such as papain is preferable, for example, Bactotrypton (manufactured by Difco) or peptone. Are preferred. This usage is 1
The amount is 1 to 100 g, preferably 5 to 50 g, and more preferably about 10 to 30 g.

The amount of yeast extract used is 1 to 100 g, preferably 2 to 30 g, and more preferably about 5 to 15 g per medium.

As the inorganic salt, for example, NaCl, KCl, Na ₂ SO ₄ , CaCl ₂ , CaCO ₃ , Mg
SO ₄ , CuSO ₄ , FeSO ₄ , ZnSO ₄ , MnCl ₂ , phosphate and the like are preferably NaCl, and the amount of these used is 0 per medium.
The amount is 1 to 50 g, preferably 1 to 30 g, and more preferably 2 to 10 g.

Examples of E. coli assimilating carbon sources include glycerol, glucose, arabinose, mannitol, maltose, trehalose and sorbitol, and glycerol, sorbitol and trehalose are preferable.
The amount used is 5% or less per medium, preferably 0.001
Is about 3%, more preferably about 0.01 to 2.5%.

Other components can be added to the medium used in the present invention. Other components include pH, such as calcium carbonate.
Sources of regulatory elements and trace elements incorporated into the gene product produced include, for example, copper sulfate and zinc sulfate when the gene product is Cu-Zn-superoxide dismutase (SOD).

The thiosulfate is not particularly limited, and examples thereof include sodium thiosulfate (Na ₂ S ₂ O ₃ ), potassium thiosulfate (K ₂ S ₂ O ₃ ), ammonium thiosulfate (NH ₄ ) ₂ S ₂ O ₃ , and thiosulfate. Calcium sulfate (CaS
₂ O ₃ ), etc., and it is preferable to use a special grade reagent as much as possible. The concentration used is 0.01% to 1%, preferably
0.05% to 0.35%, more preferably 0.2 to
It is about 0.3%.

The medium used in the present invention may be a solid medium, but a liquid medium in which the above-mentioned medium components are dissolved in distilled water is more practical and preferable.

To carry out the production method of the present invention, Escherichia coli having a recombinant gene may be cultured in the above medium, and the recombinant gene product may be collected from the culture.

The recombinant gene is not particularly limited as long as it can cause E. coli to produce its gene product, and can be applied to, for example, human SOD gene, bovine SOD gene, yeast SOD gene and the like.

Culturing can be performed by a conventional method, for example, at 20 to 50 ° C., preferably 25 to 40 ° C. for 3 hours while blowing air.
It may be carried out by a method such as shaking culture for about 120 hours, preferably about 10 to 36 hours.

Collection of the recombinant gene product from the culture can be performed by a conventional method, for example, the culture is centrifuged to collect the cells,
When the gene product is present in the supernatant, the supernatant can be treated by a treatment such as chromatography. When the gene product is present in the bacterial cells, the collected bacterial cells are suspended in a buffer solution, sterilized by, for example, ultrasonic treatment, and then the supernatant obtained by centrifugation is subjected to chromatography, etc. The gene product can be obtained by treating with.

[Effect]

 Next, the effects of the present invention will be described with reference to examples.

Experimental Example 1. (1) Experimental method 0.1 mM Cu SO ₄ , 0.1 mM ZnS was added to L medium (containing 1 g of bactotryptone, 5 g of yeast extract and 5 g of salt in Medium 1).
Using the medium supplemented with O ₄ , 20 μg / ml ampicillin and the components shown in Table 1 below, the transformed Escherichia coli described in Example 1 below was cultivated in the same manner as in Example 1 to obtain a culture. After that, centrifugation was performed to obtain a supernatant. SOD for this supernatant
Production was measured by Fridoviclr method, and total protein was measured by Lowry
-Foline method (OHLowry, et al. J. Biol. Chem. 193 , 2
65 (1951)) and the SOD for all bacterial proteins
The protein ratio was calculated.

(2) The results are shown in Table 1.

As can be seen from this table, addition of thiosulfate resulted in SOD
The production amount of SOD protein increases, and the ratio of SOD protein to total bacterial protein also increases.

Example 1. The pRTac SOD8-13 obtained in 7) of Reference Example described below was added to E. coli W3110 strain (ATCC27325) by the method of Haniatis et al.
lar Cloning ； cold spring harbor laboratory254-
255 (1982)) and transformed E. coli with 20 μg / ml ampicillin and 0.1 mM CuSO ₄ and 0.
L medium containing 1 mM ZnSO ₄ , 5 g / g of glycerol and separately sterilized soda thiosulfate added to a final concentration of 0.2% (in addition to 10 g of bactotryptone in Medium 1,
5 g of yeast extract and 5 g of salt) were inoculated and cultured at 30 ° C. with shaking, and the culture temperature was raised to 37 ° C. when the absorbance at 550 nm reached 0.2. Shaking culture was further continued for about 24 hours.

The culture solution 19 thus obtained was subjected to centrifugal sedimentation at 6000 rpm for 10 minutes to collect the cells. The bacterium is 1/10 volume of the culture medium, 50
The cells were suspended in mMTris-HCl (7.5) -mMCuSO ₄ -1 mM ZnSO ₄ buffer. This was sonicated under ice cooling to crush the bacteria. The absorbance of the treated solution at 550 nm is 1 /
The process was terminated when the number decreased to 10.

Finally, this treated solution was subjected to ultracentrifugation for 30 minutes at 30,000 rpm to obtain a supernatant. SOD is extracted in this supernatant.

SOD was purified using 715 ml of the obtained lysis supernatant (total activity: 5815 Ku, specific activity 188.2 u / mg · p).

HP-20 column chromatography Diaion HP-2 preliminarily equilibrated with 50 mM saline.
0 is packed in a column of 5.8φ × 39 cm ^H and fully equilibrated. After 560 ml of 50 mM saline was added to 715 ml of the lysate supernatant, this mixture was adsorbed on the column and immediately washed with 50 mM sodium acetate buffer, pH 5, about 9 times the column volume. Then, the fractions showing SOD activity were collected by elution with a 60% methanol solution of 0.1 M glycine-caustic soda buffer, pH 10.0. (Fraction A, 382 ml) After adjusting this pH to 7.0 with about 0.5 N hydrochloric acid,
Concentrate to dryness by evaporation on a water bath at ℃. After dissolving the dried material in 100 ml of water, dialysis is performed using a dialysis tube against a 5 mM phosphate buffer containing 40 mM sodium chloride and pH 7.5.

DEAE-Toyopearl column chromatography A solution containing dialyzed SOD was previously added to 5 mM containing 40 mM sodium chloride.
To Tsushirube the packed column equilibrated DEAE- Toyopearl phosphate buffer ^{pH7.5 (3 φ × 28cm H)} . Then, it is eluted with the same buffer to obtain SOD as a flow-through without adsorbing SOD. (Fraction B, 176 ml) Sephadex G-100 Gel Chromatography Fraction B, 176 ml, 8 ml using ultrafiltration membrane (YM-5)
The concentrated solution was sephadex G-100 equilibrated with 5 mM phosphate buffer, pH 7.0, containing 1% sodium chloride in advance.
It was adsorbed on a ( ^2φ × 159 cm ^H ) column and eluted with an equilibration buffer to obtain a fraction showing SOD activity (fraction C, 80 ml). This was filtered with a Millifor filter, concentrated by ultrafiltration, and then freeze-dried. As a result, the specific activity is 38
A titer of 1.07 g of 10 Unit / mg · p SOD powder was obtained.

Reference Example (1) Isolation of mRNA from human placenta and identification of SOD mRNA: About 300 g of fresh placenta within 1 hour after birth of newborn baby is washed with phosphate physiological saline (PBS solution), and granidine thiocyanate method [Chirgwins. Biochem, 18, 5294-52
99 (1979)], and total cytoplasmic RNA was extracted. This extracted total RNA was added to a high salt buffer (Tris, 0.5M
Dissolve it in NaCl, pH 7.4) and pass it through an oligo (dT) cellulose (Pharmacia) column to obtain poly ARN.
After adsorbing A (mRNA), a low salt buffer (Tris, Na
Elution was performed at pH 7.4 without Cl and ethanol precipitation was performed. 1.7 mg of mRNA was obtained from 150 mg of total RNA. The precipitate was dissolved in 200 μm of sterilized water, heated at 80 ° C. for 2 minutes and then rapidly cooled, and then separated by molecular weight in the order of 5 to 20% sucrose density gradient centrifugation.

Actually, using Hitachi RPN40T rotor, 35Krpm, 17
Centrifuge at 0 ° C for hours.

Then, a part of each of the separated fractions (0.5 ml) was translated by a rabbit reticulocyte lysate (Amersham) system, and the synthesized protein was subjected to an immunological method (enzyme immunoassay method) (J. Pharm). .Dyn., 5 394-402
(1982)). In this way
The presence of SOD mRNA was confirmed in the 12S fraction.

(2) mRNA annealing and cDNA synthesis: Using the fraction obtained in (1), the method of Okayama-Berg [Mol. Cel.
l.Biol., 2, 161-170 (1982)], and synthesized as follows.

50 mM Tris (pH 8.3), 30 mM KC, 0.
3 mM dithiosuitol (DTT), 8 mM MgCl ₂ , 40 μg / ml
Actinomycin D, 2 mM each of dATP, dCTP, dGTP, TT
P, 30 μCl [α- ³² P] dCTP (600 Ci / mmol) (manufactured by NEN), 280 units of ribonuclease inhibitor (manufactured by Wako Pure Chemical Industries, Ltd.), and 2.8 μg of plasmid primer [Escherichia coli plasmid pSV7186 (manufactured by Pharmacia). Was used to prepare a solution containing 10 μm of a T-tailing primer of about 60 bases synthesized according to the Okayama-Berg method],
Keep at 37 ° C. Next, 10 μm of a solution containing 10 mM Tris (pH 8), 1 mM EDTA and 3 μg of mRNA was prepared, and the solution was mixed at 65 ° C. for 5 minutes.
Immediately after heating for 30 minutes, transfer to 37 ° C, then
And warmed for an additional 5 minutes. Subsequently, 5 units of reverse transcriptase (manufactured by Life Science) was added, and the mixture was heated at 37 ° C for 20 minutes. 2 μ of 250 mM EDTA (pH 8.
0) and 1 μ of 10% SDS solution were added to stop the reaction, followed by phenol / chloroform extraction and ethanol precipitation twice each to proceed to the next step.

(3) Synthesis of plasmid containing nucleotide sequence of formula (1): The precipitate obtained in (2) was mixed with 140 mM sodium cacodylate-30 mM Tris (pH 6.8), 1 mM CoCl ₂ , 0.1 mM DTT,
The solution was dissolved in a solution containing 1 mM dCTP and 50 μCi [α- ³² P] dCTP, heated at 37 ° C. for 2 to 3 minutes, and 18 units of terminal deoxynucleotidyl transferase (Pharmacia) was added to make the whole 15 μm. 37 ° C
After heating for 3 minutes, the same post-treatment as in the reverse transcription reaction was performed to obtain an ethanol precipitate.

Next, the precipitate was treated with 50 mM NaCl, 50 mM Tris (pH 8.0),
It was dissolved in a solution containing 10 mM MgCl ₂ , 100 μg bovine serum albumin (BSA), and 12 units of HindIII (manufactured by Nippon Gene) and heated at 37 ° C. for 2 to 4 hours. After phenol / chloroform extraction and ethanol precipitation,
Dissolve μ in 10 mM Tris (pH 7.3), 1 mM EDTA solution and add 3 μ of ethanol to make 1
3 μ. 0.04 pmol of oligo (dG) linker [E. coli plasmid pSV1932 (manufactured by Pharmacia) was used in 1 μm of this solution, and dG- was synthesized according to the Okayama-Berg method.
Tailing about 12 bases of linker] 10 mM Tris (pH 7
5), 0.1 M NaCl, 1 mM EDTA 10 times concentrated solution 1 μ and distilled water 8 μ were added to make the whole 10 μ, and the solution was adjusted to 65 μm.
The temperature was kept at 0 ° C after aging for 5 minutes at 42 ° C for 30 minutes at 42 ° C.
To this, 20 mM Tris (pH 7.5), 4-mMMgCl ₂ , 10 mM ammonium sulfate, 0.1 M KCl, 50 μg / ml BSA, 0.
1 mM β-nicotinamide adenosine dinucleotide (NA
D) and 0.6 μg of Escherichia coli DNA lyase (Pharmacia) were added to finally add the concentrated solution 10
The solution was heated to 0 μ and heated at 12 ° C. Then, each 20mM
0.4 μ of dATP, dCTP, dGTP and TTP containing
1.5 mM β-NAD 1 μ, E. coli DNA ligase 0.4
μg, 0.3 μg of E. coli DNA polymerase, and 1 unit of E. coli liponuclease H (total 104 μ), and further at 12 ° C. for 1 hour at 25 ° C.
It was heated for 1 hour.

(4) Transformation into Escherichia coli: x1776 (ATCC31244) was used as Escherichia coli. Competent cells include Maniatis et al. (Molecular Cloning, cold spr
ng harbor laboratory, 254-255 (1982)]
It was prepared by the same method as described above and dispensed in 0.2 ml aliquots. The
Five DNA solutions each having 20 μm were transformed, and 1.5% agar containing bactotryptone 10 g /, yeast extract 5 g /, diaminopimelic acid 0.01%, thymidine 0.004% and ampicillin (Ap) 50 μg / ml. About 30,000 colonies were obtained on the medium.

(5) Colony hybridization: Approximately 10,000 of the obtained colonies were transferred onto an agar medium having the same composition (512 cells / 14 × 10 cm plate; 2 sheets 1
One set was stored as a master plate. ),
It was cultured until it grew to a diameter of about 3 mm. Place Whatman 541 paper slowly on this to completely transfer colonies to paper, and then add chloramphenicol 250 μg / ml.
The paper was adhered onto an agar medium of the same composition containing, and cultured overnight. The DNA was immobilized on the paper as follows.

After culturing, the paper is treated with 0.5M NaOH for 5 minutes twice,
Return to neutral with 0.5M Tris (pH 7.4), and add 2 × SSC (p
H7) (1 × SSC: 0.15M NaCl, 0.015M sodium citrate) treatment, lightly washed with 95% ethanol aqueous solution, and then air dried. (A) 17 nucleotides as probe: AA (Tor C) TT (Tor C) GA (Aor G) CA (Aor G) AA (Aor
32 types of G) GA (B) 14 nucleotides: GA (Tor C) CA (To
Twenty-four kinds of rC) TG (TorC) AT (T, CorA) AT were chemically synthesized by the triester method, and used for the high priidization described below.

(B) Pre-hybridization paper 6 x SET (1 x SET: 0.15MB NaCl, 0.01
A solution containing 5M Tris (pH 7.5), 1 mM EDTA), 0.5% Sonidet P40 (manufactured by Hanai Chemical Co., Ltd.) and 100 μg / ml of denatured E. coli DNA (E. coli DNA manufactured by Pharmacia was boiled for 5 minutes and then rapidly cooled). It was heated at 55 ° C. for 2 hours.

(B) High pridization Next, 100 μg / ml yeast tRNA was used instead of denatured E. coli DNA.
(Manufactured by BRL) and [γ- ³² P] ATP (5000 Ci / mmol NEN
Hybridization was carried out at 29 ° C. for 2 hours using a probe and a polynucleotide kinase (manufactured by NEB) and 0.2 ng / ml of a probe labeled with [ ³² P] at the 5 ′ position.

(C) Isolation and washing of the plasmid containing the nucleotide sequence of the formula (1) is carried out by (A) treating at 39 ° C. for 5 minutes, (B) treating at 29 ° C. for 20 minutes, and then treating at room temperature for 10 minutes with a 6 × SSC solution. Each step was repeated 3 times. After air-drying the paper, the X-ray film (Kodak XA
Autoradiography using R ₅ ), (A),
(B) One positive colony was selected for both, the plasmid was taken out from the cells, and the plasmid was used for pHS3.
It was named 237.

(6) Construction of expression vector After cutting the Hae II site closest to the lactose promoter on E. coli plasmid pUC ₁₃ (Pharmacia), about 100 bp at both ends was deleted with exonuclease Bal31 (NEB), and T ₄ A plasmid pΔUC ₁₃ reclosed with DNA ligase (Takara Shuzo) was prepared (this plasmid has lost the function as a lactose promoter). Then, a TrpA terminator (manufactured by Pharmacia) was inserted into the HincII cleavage site of this plasmid to obtain a plasmid pΔUCT ₁₃ .

(A) Preparation of DNA encoding SOD The pH 3237 obtained in (5) (c) above was digested with Pvu II,
An XbaI linker (manufactured by NEB) was ligated with T ₄ DNA ligase to provide an XbaI site, and this plasmid was named pHSX3237. pHSX3237 was digested with PstI and subsequently digested with exonuclease Bal 31. After blunting the ends with T ₄ DNA polymerase, ligating a BamHI linker (Takara Shuzo Co., Ltd.) and digesting with BamHI and XbaI (both are Nippon Gene Co., Ltd.), a DNA of about 630 to 700 bp was 2-16.
Recovered on a% gradient polyacrylamide gel.

(B) Tac promoter and SOD DNA the inserted plasmid prepared plasmid pDR ₅₄₀ (manufactured by Pharmacia) to recover the 121bp containing the digested Tac promoter in BamHI EcoRI (manufactured by Nippon Gene) in polyacrylamide gels, p
The plasmid of about 3 Kb obtained by inserting between EcoRI and BamHI of ΔUCT ₁₃ was designated as pTacI (FIG. 3). BamH of pTacI
Approximately 630-700 bp DN obtained in (7) (A) between I-XbaI
The plasmid obtained by inserting A into E. coli DHl (ATCC3384
9). The nucleotide sequences of various obtained plasmids were determined, and a plasmid having a length of 8 to 13 nucleotides from the SD sequence (AGGA) to the start codon ATG was determined by pTac S
It was named OD8-13.

(7) Construction of runaway SOD expression vector Runaway plasmid pMOB45 (AT
CC37106) (M. Bitter and D. Vapnek, Gene 15 , 319-329,
(1981)) was cut with EcoRI and HindIII (Takara Shuzo, all the following products of the same company), and the 6.7 Kb including the runaway replication origin was cut.
The DNA fragment was cut out. This DNA was purified, treated with Bal 31 enzyme, digested to 0.3 Kb at each end, and treated with DNA polymerase to make less than DNA blunt. On the other hand, pBR322 purchased from ATCC was cut with Tth111II to cut out a 1.3 Kb DNA containing an ampicillin resistance gene. This D
NA was also purified and then sequentially treated with Bal 31 enzyme and DNA polymerase in the same manner as in the above method. Two DNAs thus obtained
The fragments were mixed in equimolar amounts, and the HindIII linker and EcoRI were added.
After adding linker (Takara Shuzo) 10 times molar amount, T4DN
The DNA was ligated by treating with A ligase.

Next, transfer this DNA sample to E. coli W3110 (ATCC27325) strain.
Maniatis et al. (Molecular Cloning; cold spring
harbor laboratory 254-255 (1982), transformation was performed and ampicillin resistant strains were selected. Restriction enzyme analysis was carried out on the plasmids contained in 12 strains selected arbitrarily. As a result, the above two DNA fragments are ligated,
And only two linkers (Hind III and Hind III
A plasmid pR4 having EcoRI) inserted therein was obtained. Then pR
4 is cleaved by cutting with EcoRI and HindIII, and pΔ
A 0.4 Kb EcoRI-HindIII derived from UCT ₁₃ (see (6) (A) above) containing a multicloning site and a transcription termination factor.
The fragment was inserted to construct pR3. Furthermore, this pR3 is EcoRI
And cleaved with XbaI, and pTacSOD8-13 between these sites
(See (6) (B) above) and tac promoter and human SOD
Insert the EcoRI-XbaI DNA fragment of about 0.7Kb containing the gene was constructed pRTac SO ₄ 8~13.

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Claims (1)

[Claims] 1. A casein hydrolyzate, a yeast extract, an inorganic salt, and Escherichia coli carrying a recombinant gene in a medium containing an Escherichia coli assimilable carbon source and thiosulfate as essential components. A method for producing a recombinant gene product, which comprises collecting a replacement gene product