JPH0755152B2 - Recombinant gene product manufacturing method and medium - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing a recombinant gene product and a novel medium used in the production thereof.

[Prior art]

In order to improve the productivity of the recombinant DNA product, mainly by improving the vector incorporating the target gene, or by improving the culture medium and culture method. As an example of the former, construction and use of a strong promoter (de Boer, H.etal.Proc.Natl.Aca
d.Sci. 80 , 21 (1983), modification of the region around the SD sequence start codon (N. Warburton et al., Nacl. Acid.Res. 11 , 5837 (198
3)), increase in vector copy number (Brigitte E. Schoner
et al.Proc.Natl.Acad.Sci. 81 , 5403 (1984)).

On the other hand, there are few reports on the latter medium, improvement of the culture method, with a focus on slightly high-concentration culture of the bacterium, using a modified Davis medium, and dividing and adding glucose while controlling the pH. The law has been reported. (H. Meri et al., Journal of Chemical Engineering
of Japan 12 , 313−319 (1979), T. Kobayashi et al. Pro
c.-Pac.Chem.Eng.Congr., 3rd 4 , 147-50 (1983)) However, although high-concentration culture of the bacterium was achieved in these, the ratio of the target product to the total bacterial cell protein of E. coli was high. Not specified.

[Problems to be solved by the invention]

It takes time and technique to improve the expression vector in order to improve the productivity of the target gene product. Further, the improvement of the vector has the property of being carried out each time the target gene product is different, and is not so versatile. Further, according to the experiments of the present inventors, the bacterial concentration surely increases in the above-mentioned culture method,
It was found that the amount of the desired product per total E. coli protein was remarkably reduced as compared with the usual culture method, and its productivity was not improved at all.

[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 the recombinant gene was isolated in a medium containing casein-hydrolyzing yeast extract, inorganic salt and Escherichia coli assimilating carbon source as essential components. It has been found that the culturing of Escherichia coli has significantly improved the productivity of gene products.

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

That is, the present invention cultivates Escherichia coli having a recombinant gene in a medium when casein hydrolyzate, yeast extract, inorganic salt and Escherichia coli assimilatory carbon source are essential components, and collects the recombinant gene product from the culture. The present invention also relates to a method for producing a recombinant gene product characterized by:

The casein hydrolyzate used in the present invention is not particularly limited as long as it hydrolyzes casein, but trypsin, pepsin, those hydrolyzing casein with a protease such as papain are preferable, for example, Bactotryptone (trade name,
Peptone such as Difco) is preferable. The amount used is 1 to 100 g, preferably 5 to 50 g, and more preferably about 10 to 30 g per medium.

The amount of yeast extract used is 1 to 100 g, preferably 2 to 30 g, and more preferably 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 thereof is 0.1 per medium.
~ 50g, preferably 1-30g, more preferably 2-10g
It is a degree.

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
~ 3%, more preferably about 0.01-2.5%.

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 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 causes E. coli to produce its gene product, and examples thereof include SOD, TPA, TNF interferon, growth hormone, interleukin 2,
Examples thereof include genes encoding insulin, somatostatin, endorphin, calcitonin, and genes encoding amino acid synthases such as tryptophan and phenylalanine.

Culturing can be carried out by a conventional method, for example, shaking culture while blowing air at 20 to 50 ° C., preferably 25 to 40 ° C. for 3 to 120 hours, preferably 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 liquid, the liquid can be treated by a treatment such as chromatography. If the gene product is present in the bacterial cells,
The gene product can be obtained by suspending the collected bacterial cells in a buffer solution, sterilizing the cells by, for example, sonicating, and then centrifuging the resulting supernatant, followed by chromatography and the like.

[Effect]

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

Experimental Example 1. (1) Experimental Method As in Example 1 or 2, using a medium having the same composition as that of Example 1 except that 1 g or 5 g / one of the E. coli assimilating carbon sources shown in Table 1 below was used. After culturing, the culture was obtained, the cells were collected and lysed, and then centrifuged to obtain a supernatant. The amount of SOD produced in this supernatant was measured by the Fridoviclr method, and the total protein amount was measured by Lowry-Folin.
e method (OHLowry, etal.J.Biol.Chem. 193, 265 (1951))
And the ratio of SOD protein to total bacterial protein was calculated.

(2) Results Table 1 shows the results.

As is clear from this table, the addition of a carbon source increases the SOD production amount, and also increases the ratio of SOD protein to the total bacterial protein.

Experimental Example 2. (1) Experimental Method A medium obtained by adding glycerol and yeast extract and bactotripton or peptone in the amounts shown in Table 1 to a liquid obtained by removing glycerol, yeast extract and bactotryptone from the medium used in Example 1. Was used. PR as a recombinant gene
When TAc8-containing bacteria were used, 30 jar fermenters were charged to 1900 ml and sterilized at 120 ° C for 10 minutes. OD = 1 for this
200 ml of the seed culture solution which had reached the temperature was inoculated, and aeration stirring culture was started at 30 ° C. When OD = 0.15, culture temperature is 37 ℃
After culturing for 24 hours, the cells were collected. Also, pT
When the bacterium having Ac8 was used, it was cultured by the method of Example 3. On the other hand, the fed-batch culture of glucose has been reported (H.Mo.
ri et al., J. Chem. Eng. Japan 12 , 313 (1979))
After raising the temperature so that the final concentration will be 0.5% using the following medium,
Glucose was added 5 times every 4 hours (total amount of addition: 3%), and the pH was adjusted by an automatic control operation with aqueous ammonia so that the pH did not fall below 6.0.

The obtained culture was treated and measured in the same manner as in Experimental Example 1.

Fed-batch culture medium composition: KH ₂ PO ₄ 4g, K ₂ HPO ₄ 4g, Na ₂ HPC ₄・ 12H ₂
O7g, (NH ₄ ) ₂ SO ₄ 1.2g, NH ₄ Cl 0.2g, yeast extract 9g, MgSO ₄
7H ₂ O 2.4g, FeSO ₄・ 7H ₂ O 40mg, CaCl ₂・ 2H ₂ O 40mg, MnSO ₄・ 10
mg, AlCl ₃・ 6H ₂ O 10mg, CoCl ₂・ 6H ₂ O 4mg, ZnSO ₄・ 7H ₂ O 30mg,
NaMoO ₄ .2H ₂ O ₂ mg, CuSO ₄ .5H ₂ O 25 mg, H ₃ BO ₃ 0.5 mg, glucose 5 g (2) Results The results are shown in Table 2.

As is clear from this table, increasing the amount of glycerol added also increased the amount of yeast extract and peptone added.
SOD productivity increases. In addition, the productivity is improved by about 2 to 4 times as compared with the known glucose flow-down culture method. Therefore, the present invention is an excellent method for producing a recombinant gene product with good productivity.

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

Example 1 pRTacSOD8 to 13 obtained in 7) of Reference Example described below were treated with Haniatis.
Et al. (Holecular Cloning; cold spring harbor lab
E. coli W311 transformed with oratory 254-255 (1982))
0 strain (ATCC27325) with 20 μg / ml ampicillin and 0.1 mM Cu
L medium containing SO ₄ and 0.1 mM MZnSO ₄ , 5 g / glycerol (other than Bactrypton ^R 10 g in medium 1, yeast extract
5g, containing 5g of salt), shake culture at 30 ℃, 550nm
When the absorbance at 0.2 was reached, the culture temperature was adjusted to 37
Rose to ℃. Shaking culture was further continued for about 24 hours.

3) The culture solution 19 was subjected to centrifugal sedimentation at 6000 rpm for 10 minutes to collect the cells. The bacterium is 50 mM Tris-HCl (7.5) of 1/10 volume of the culture solution.
−1 mM CuSO ₄ −1 mM ZnSO ₄ buffer was suspended. This was sonicated under ice cooling to crush the bacteria. The treatment was terminated when the absorbance at 550 nm of the treatment liquid decreased to 1/10 of that before the treatment.

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

4) SOD was purified using 715 ml of the obtained lysis supernatant (totality: 3077 Ku, specific activity 99.6 u / mg · p).

HP-20 column chromatography 5.Diaion HP-20 pre-equilibrated with 50 mM saline solution.
Pack in a column of 8φ x 39 cm ^H and fully equilibrate. After adding 560 ml of 50 mM saline to 715 ml of the lysate supernatant and adsorbing the mixture on the column, the column is washed with 50 mM sodium acetate buffer, pH 5, about 9 times the column volume. Then, it was eluted with 0.1M glycine-caustic soda buffer solution in 60% methanol, pH 10.0, and SOD was applied.
Fractions showing activity were collected. (Fraction A, 382 ml) This fraction was adjusted to pH 7.0 with about 0.5 N hydrochloric acid and then concentrated to dryness by evaporation on a water bath at 40 ° C. After dissolving the dry matter in 100 ml of water, dialysis is performed using a dialysis tube against a 5 mM phosphate buffer solution (pH 7.5) containing 40 mM sodium chloride.

DEAE-Toyopearl Column Chromatography The solution containing dialyzed SOD was passed through a DEAE-Toyopearl packed column (3 ^φ × 28 cm ^H ) that had been equilibrated with 5 mM phosphate buffer pH 7.5 containing 40 mM sodium chloride. Guide. 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 8 ml of a concentrated solution of 176 ml of fraction B using an ultrafiltration membrane (YM-5) was previously added with 5 mM phosphate buffer containing 1% sodium chloride, pH 7.0
Equilibrated with Sephadex G-100 ( ^2φ × 159cm ^H ).
It was adsorbed on a column and eluted with an equilibration buffer to obtain a fraction showing SOD activity (fraction C, 80 ml). This was filtered through a Millifor filter, concentrated by ultrafiltration, and then freeze-dried. As a result, specific activity 3810Un
A titer of 0.57 g of it / mg · p SOD powder was obtained.

Example 2. The medium used in Example 1 was supplemented with 3% calcium carbonate, and other cultures were performed in the same manner. 6N after culture
Hydrochloric acid was added until precipitation of calcium carbonate disappeared, and then extraction and purification were carried out in the same manner as in Example 1. As a result, 0.50 g of SOD powder having a specific activity of 3800 U / mg · p was obtained from 19 cultures.

Example 3. (1) Preparation of medium A medium having the following composition, which is usually used for culturing Escherichia coli, was used as a basic medium.

Bacto tryptone ^R 10g Yeast extract 10g Glycerol 20g Dietary salt (NaCl) 5g After setting the above ingredients to 1 with distilled water, adjust the pH to 7.0 with 2N NaOH.
Adjust to. After preparation of the L medium, 200 ml was dispensed into a 500 ml triangular Kolben and sterilized under pressure at 120 ° C. for 10 minutes. After cooling, this was sterilized with CuSO ₄ and ZnSO ₄ solutions added to a final concentration of 0.1 mM.

(2) Used strain A mutant strain of Escherichia coli K12 strain carrying the SOD gene expression plasmid pTAc8 was used.

(3) Culture Ampicillin was added to the medium prepared in (1) so that the final concentration was 20 μg / ml, and then SOD-producing Escherichia coli was inoculated. 37
After culturing with shaking at 0 ° C., when the absorbance at 600 nm reached 0.1 to 0.3, isopropyl-β-thiogalactopyranoside (manufactured by Sigma) was added so that the final concentration was 1 mM, and further 5
Incubated for hours.

(4) Extraction The culture solution 19 was subjected to centrifugal sedimentation at 6000 rpm for 10 minutes to collect the cells. The bacterium is 50 mM Tris-HCl (7-5) which is 1/10 of the culture solution.
Suspended in -1 mM CuSO ₄ -1 mM ZnSO ₄ -1 mM PMSF (phenyl methane sulfonyl fluoride) buffer. This is sonicated under ice cooling to crush the bacteria. During the ultrasonic treatment, the absorbance of the treatment liquid is measured appropriately, and the absorbance is 1/10 of that before treatment.
The ultrasonic treatment was terminated when the temperature decreased to 0.

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

Then, in the same manner as in Example 1, SOD having a specific activity of 3750 Unit / mgp
0.1 g of 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 saline (PBS solution), and guanidine thiocyanate method [Chirgwins: Biochem , 18,5294-5299 (197
9)] was used to extract cytoplasmic total RNA. This extracted total RNA was treated with a high salt buffer (Tris, containing 0.5M NaCl, pH
7.4), and this is passed through an oligo (dT) cellulose (Pharmacia) column to adsorb poly A RNA (mRNA), and then a low salt buffer (Tris, NaCl-free, p
It was eluted with H7.4) and precipitated with ethanol. 1.7 mg of mRNA was obtained from 150 mg of total RNA. The precipitate was dissolved in 200 μl of sterilized water, heated at 80 ° C. for 2 minutes and then rapidly cooled, and then separated by molecular weight in order of 5 to 20% sucrose density gradient centrifugation.

Actually, using Hitachi RPS40T rotor, 35Krpm, 17 hours 0
It was centrifuged at ℃.

Then, a part of each separated fraction (0.5 ml) was translated by a rabbit reticulocyte lysate (Amersham) system,
The synthesized protein was examined by an immunological method (enzyme immunoassery method) (J. Pharm. Dyn., 5 394-402 (1982)). In this way, SOD mRNA
The existence of

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

50 mM Tris (pH 8.3), 30 mM KC, 0.3 mM dithiothreitol (DTT), 8 mM MgCl ₂ 40 μg / ml actinomycin D, 2 mM each of dATP, dCTP, dGTP, TTP, 30 μCl (α- ³²
P] dCTP (600 Ci / mmol) (NEN), 280 units of ribonuclease inhibitor (Wako Pure Chemical Industries), and 2.8
μg of plasmid primer [E. coli plasmid pSV718
6 (manufactured by Pharmacia) was used to prepare 10 μl of a solution containing a T-tailing primer of about 60 bases synthesized according to the Okayama-Berg method, and kept at 37 ° C. Then 10 mM Tris (pH
8), 10 μl of a solution containing 1 mM EDTA and 3 μg of mRNA was prepared, heated at 65 ° C. for 5 minutes, immediately transferred to 37 ° C., mixed with 10 μl of the above solution, and further heated for 5 minutes. Then add 5 units of reverse transcriptase (Life Science),
Heated at 37 ° C for 20 minutes. 2 μl of 250 mM EDTA (pH8.0)
And 1 μl of 10% SDS solution to stop the reaction,
Phenol / chloroform extraction and ethanol precipitation were performed twice, respectively, and the process proceeded to the next step.

(3) Synthesis of plasmid containing nucleotide sequence of formula (1): The precipitate obtained in (2) was added to 140 mM sodium cacodylate-30 mM Tris (pH 6.8), 1 mM CoCl ₂ , 0.1 mM DTT, 1 mM dC.
Dissolve in a solution containing TP and 50 μCi [α- ³² P] dCTP,
After heating at 7 ° C. for 2 to 3 minutes, 18 units of terminal deoxynucleotidyl transferase (Pharmacia) was added to make the whole 15 μl. After heating at 37 ° C for 3 minutes, the same post-treatment as in the reverse transcription reaction was performed to obtain an ethanol precipitate.

Next, the precipitate was added to 50 mM NaCl, 50 mM Tris (pH 8.0), 1.0 mM
MgCl ₂ , 100 μg bovine serum albumin (BSA), and 12
It was dissolved in a solution containing a unit of Hind III (Nippon Gene Co., Ltd.) and heated at 37 ° C. for 2 to 4 hours. After phenol / chloroform extraction and ethanol precipitation, add 10 μl of 10 mM T
Dissolve in a solution containing ris (pH7.3) and 1 mM EDTA, and add 3 more
μl ethanol was added to bring the total volume to 13 μl. To 1 μl of this solution, 0.04 pmol of oligo (dG) linker [Escherichia coli plasmid pSV1932 (Pharmacia) was used, and Okayama-B
dG-tailing about 12-base linker synthesized according to the erg method], 10 mM Tris (pH 7.5), 0.1 M NaCl, 1 mM EDTA
Add 1 μl of 0x concentrated solution and 8 μl of distilled water to make a total of 10 μl.
After heating the solution at 65 ° C for 5 minutes and 42 ° C for 30 minutes with aging,
It was kept at ℃. Add 20mM Tris (pH7.5), 4mM MgCl ₂ , 1
0 mM ammonium sulfate, 0.1 M KCl, 50 μg / ml BSA, 0.1 mM
β-nicotinamide adenosine dinucleotide (NAD)
And 0.6 μg E. coli DNA ligase (Pharmacia)
Was finally added to 100 μl of the concentrated solution, and the mixture was heated at 12 ° C. overnight. Then dAT containing 20 mM each
0.4 μl of P, dCTP, dGTP and TTP, 1 of 1.5 mM β-NAD
μl, E. coli DNA ligase 0.4 μg, E. coli DNA polymerase 1 0.3 μg, and E. coli liponuclease H
1 unit was added (104 μl in total), and the mixture was further heated at 12 ° C. for 1 hour and at 25 ° C. for 1 hour.

(4) Transformation into Escherichia coli: χ1776 (ATCC31244) was used as Escherichia coli. Competent cells include Maniatis et al. (Molecular Cloning, cold sp
ring harbor harbor laboratory, 254-255 (1982)]
It was prepared by the same method as described above and dispensed in 0.2 ml aliquots. The DNA
Five 20 μl aliquots of each solution were transformed into bactotryptone 10
g / 1, yeast ectotract 5g / 1, diaminopimelic acid 0.01%, thymidine 0.004% and ampicillin (Ap) 5
About 30,000 colonies were obtained on a 1.5% agar medium containing 0 μg / ml.

(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; two sheets were set as one set, and 1
The sheet was kept as a master plate. ), Diameter about 3mm
Cultivated until it grew to. The Whatman 541 paper was slowly placed on the paper, the colonies were completely transferred to the paper, and the paper was adhered to the agar medium of the same composition containing 250 μg / ml of chloramphenicol and cultured overnight. B DN to paper
A fixation was performed as follows.

After culturing, the paper is treated twice with 0.5M NaOH for 5 minutes, 0.5M
Return to neutral with Tris (pH7.4), 2 x SSC (pH7) (1 x S
SC: 0.15M NaCl, 0.015M sodium citrate), lightly washed with 95% ethanol aqueous solution, and air dried.
(A) 17 nucleotides as a probe: AA (TorC) TT (T
32 types of orC) GA (AorG) CA (AorG) AA (AorG) GA (B) 14 nucleotides: GA (TorC) CA (TorC) TG (Tor
Each of 24 types of C) AT (T, CorA) AT was chemically synthesized by the triester method and used for the hybridization described below.

(B) Pre-hybridization paper 6 x SET (1 x SET: 0.15M NaCl, 0.015M 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) at 55 ° C for 2 hours. Heated.

(B) Hybridization Next, 100 μg / ml yeast tRNA (BRL
) And [γ- ³² P] ATP (5000 Ci / mmol NEN) and polynucleotide kinase (NEB) at the 5 ′ position with [ ³² P] -labeled probe 0.2 ng / ml. 29 ° C, 2
Hybridization was performed for a time.

(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 6 × SSC solution. Use each stage 3
Repeated each time. After air-drying the paper, autoradiography was performed using an X-ray film (Kodak XAR ₅ ),
One positive colony was selected for both (A) and (B), the plasmid was taken out from the cells, and the plasmid was named pH S3237.

(6) Construction of expression vector After cleaving the Hae II site closest to the lactose promoter on E. coli plasmid pUC ₁₃ (Pharmacia), exonuclease Bal31 (NEB) was used to cleave both ends to about 10
A plasmid pΔUC ₁₃ was prepared by deleting 0 bp and re-closing with T ₄ DNA ligase (Takara Shuzo) (this plasmid has lost the function as a lactose promoter).
Next, a TrpA terminator (Pharmacia) was inserted into the Hinc II cleavage site of this plasmid, and the plasmid p
ΔUCT ₁₃ was obtained.

(A) Preparation of DNA encoding SOD The pH S3237 obtained in (c) of (5) above was digested with Pvu II and Xba I linker (NEB) was ligated with T ₄ DNA ligase to obtain Xba I. A site was provided and this plasmid was named pH SX3237.

pHSX3237 was digested with Pst I and subsequently digested with exonuclease Bal31. Then, blunt the ends with T ₄ DNA polymerase, ligate with BamHI linker (Takara Shuzo) and
After digestion with nH I and Xba I (both manufactured by Nippon Gene Co., Ltd.), a DNA of about 630 to 700 bp was recovered by 2-16% gradient polyacrylamide gel.

(B) Tac preparation promoter and SOD DNA the inserted plasmid Plasmid pD R ₅₄₀ (manufactured by Nippon Gene Co., Ltd.) (manufactured by Pharmacia) and EcoR I and then digested with BamHI recovered 121bp containing the Tac probe motor polyacrylamide gel Then p
A plasmid of about 3 Kb obtained by inserting between ΔRICT ₁₃ and EcoRI-BamHI was designated as pTacI (Fig. 3). pTac I Ba
Approximately 630-700bp obtained in (7) (A) between mH I and Xba I
E. coli DH1 (ATCC
33849). The nucleotide sequences of various obtained plasmids were determined, and the start codon ATG was determined from the SD sequence (AGGA).
Plasmids with a distance of 8 to 13 nucleotides to pTac
They were named SOD8-13.

(7) Construction of runaway-type SOD expression vector Runaway plasmid pMOB45 (ATCC purchased from ATCC
37106) (M.Bitter and D.Vapnek, Gene 15 , 319−329,
(1981)) was cut with EcoR I and Hind III (Takara Shuzo, all the following products), and 6.7 Kb including the runaway origin of replication.
The DNA fragment of was cut out. This DNA was purified, treated with Bal31 enzyme, digested to about 0.3 Kb on each end, and treated with DNA polymerase to make the DNA ends blunt. On the other hand, pBR322 purchased from ATCC was cut with Tth111 II to cut out a 1.3 Kb DNA containing an ampicillin resistance gene. After purifying this DNA,
Similarly to the above method, the Bal31 enzyme and the DNA polymerase were sequentially treated. The two DNA fragments thus obtained were mixed in equimolar amounts, Hind III linker and EccR I linker (Takara Shuzo) were further added in a 10-fold molar amount, and treated with T4 DNA ligase to ligate the DNA.

Next, this DNA sample was cloned into E. coli W3110 (ATCC27325) strain.
The method of iatis et al. (Molecular Cloning; cold spring har
bor laboratory 254-255 (1982), transformation was performed and ampicillin resistant strains were selected. About 12 stocks selected arbitrarily,
Restriction enzyme analysis of the plasmid possessed was performed. As a result, a plasmid pR4 was obtained in which the above two DAN fragments were ligated and two types of linkers (Hind III and EcoR I) were inserted only in one ligation part. Then replace pR4 with EcoR I and Hind
It was cleaved by III and cleaved, and a 0.4 Kb EcoR I-Hind III fragment derived from pΔUCT ₁₃ (see (6) (A) above) containing the multiple cloning site and transcription termination factor was inserted between these sites. pR3 was constructed. Furthermore, this pR3 is replaced with EcoR I and Xba.
It is cleaved and cleaved with I, and is derived from pTacSOD8 to 13 (see (6) (B) above) between these sites, and contains tac promoter and human S
Insert the EcoR I-Xba I DNA fragment of about 0.7Kb containing the OD gene was constructed pR TacSO ₄ 8~13.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Akiko Naito 1417-26 Sakata, Okegawa City, Saitama Prefecture (72) Inventor Tsuneo Nakamura 2-18-1, 2-205 Yoshino-cho, Omiya City, Saitama Prefecture (56) References Special Kai 61-111690 (JP, A) "Biochemistry Data Book ▲ II ▼ Miniature Edition" edited by The Japanese Biochemical Society (October 1, 1982) Tokyo Kagaku Dojin P. 881

Claims (1)

[Claims] 1. A medium comprising a casein hydrolyzate, a yeast extract, an inorganic salt and 0.5 to 5% of an Escherichia coli assimilable carbon source per medium, (a) glycerol, sorbitol as an E. coli assimilable carbon source, Escherichia coli having a recombinant gene is cultured in a medium containing at least one selected from trehalose as an essential component or (b) calcium carbonate as an inorganic salt as an essential component, and recombinant from the culture. A method for producing a recombinant gene product characterized by collecting a gene product (2) In a medium comprising casein hydrolyzate, yeast extract, inorganic salt and 0.5 to 5% of E. coli assimilable carbon source per medium, ( A) Escherichia coli contains at least one selected from glycerol, sorbitol and trehalose as an assimilating carbon source as an essential component, or (b) inorganic. A new improved medium for Escherichia coli with a recombinant gene containing calcium carbonate as an essential component as a salt