JPH0817703B2 - Method for producing foreign gene product - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a recombinant plasmid having a desired foreign gene inserted therein [Hybrid plasmid (hereinafter referred to as Hi-plasmid)] The present invention relates to a method for producing an exogenous gene product by culturing Hi-plasmid-introduced Escherichia coli, which is efficiently proliferated while effectively controlling the expression of Escherichia coli, and the time of cell growth and expression of the exogenous gene are separated.

[Conventional technology]

In recent years, due to the development of gene recombination technology, an expression vector that enables the phenotypic expression of a foreign gene in a host bacterium, by incorporating a production gene encoding a desired foreign polypeptide obtained from animals, plants, microorganisms, etc., A method for constructing a Hi-plasmid and culturing a host bacterium into which the Hi-plasmid has been introduced to allow the host bacterium to produce a desired foreign polypeptide has been developed.

With this technology, it is becoming possible to mass-produce useful substances such as human insulin and human growth hormone.

As a host bacterium used to produce a foreign gene product using such a gene recombination technique, its biological characteristics have been sufficiently analyzed, and it is a pathogen-free medium having a simple composition. E. coli is widely used because it can be easily cultured.

[Problems to be solved by the invention]

However, in general, the stability of Hi-plasmid in E. coli is not always high, and in the culture of E. coli into which the Hi-plasmid has been introduced, the growth of the bacterium causes the structural change of the Hi-plasmid or the disappearance of the Hi-plasmid itself to cause the foreign body. Hi-plasmid deficient bacteria, which have lost the gene expression ability, appear.

For example, in industrial-scale production with large-scale culture,
Generally, the culture period including seed culture is long, and
-Emergence of plasmid deficient bacteria is unavoidable, and when the proportion of Hi-defective bacteria per total culture is high, it leads to a decrease in the concentration of the desired foreign gene product in the finally obtained culture, which is efficient. Inability to produce foreign genes.

Further, in the culture accompanied by the expression of the foreign gene, it is difficult to obtain a good growth state of the bacterium.

Therefore, as one of means for solving the problem in the culture using such a Hi-plasmid, a method of separating the growth of the bacterium and the expression period of the foreign gene is being studied.

Specifically, first, the plasmid-introduced bacterium is cultured while controlling the expression of the foreign gene of the introduced Hi-plasmid,
When the desired cell mass is obtained, the foreign gene is expressed by easing the control of expression to suppress the occurrence of Hi-plasmid deficient bacteria as much as possible, and to produce the foreign gene product more efficiently. .

Expression control method of a foreign gene for use in such methods, for example, to a Hi- plasmid, P _L promoter (P _L lambda promoter) of E. coli lambda phage
A method that uses an inducible expression promoter that can induce the expression of a foreign gene at any time by adding an inducing agent that inactivates the repressor such as the operator or the E. coli tryptophan operon promoter (trp promoter) and the temperature dependency Using a temperature-controlled vector having a plurality of initiation regions (ie, having a predetermined temperature or temperature range necessary for the initiation of replication),
A method of controlling the expression of a foreign gene by adjusting the culture temperature and applying heat shock is known.

However, in the method using the inducible expression promoter, the induction reagent for expression induction is expensive, which causes an increase in production cost, and there is a problem that the appearance and increase of Hi-plasmid eradication bacterium cannot be sufficiently suppressed. It is difficult to apply for culturing on an industrial scale.

Even when a temperature-controlled vector is used, Hi-
There is also a drawback that the effect of preventing the emergence of plasmid deficient bacteria is not sufficient and that the culture device becomes complicated due to temperature control.

In view of the above-mentioned problems, the inventors of the present invention have adopted Hi- in order to realize more efficient production of a foreign gene product.
Various studies were conducted on the culture engineering characteristics of the plasmid-introduced Escherichia coli. The Hi-plasmid-introduced Escherichia coli was prepared by culturing by culturing by preparing a high-concentration sugar concentration such as glucose that can be used as a carbon source in the medium. It was newly found that the expression of foreign genes in E. coli can be effectively controlled.

Furthermore, when culturing a Hi-plasmid-introduced bacterium to produce a foreign gene product, the expression control method by adjusting the sugar concentration is used to manipulate the expression control timing of the foreign gene to increase the growth of the bacterium and the foreign gene. It was concluded that culturing by separating the gene expression time from each other would allow good cell growth with sufficient suppression of Hi-plasmid eradication and efficient production of foreign gene products. Completed the invention.

An object of the present invention is to provide a method capable of effectively controlling the expression of a foreign gene inserted in a Hi-plasmid by a simple operation in the culture of Escherichia coli into which the Hi-plasmid has been introduced.

Another object of the present invention is to provide a method suitable for producing a foreign gene product on a commercial scale using Hi-plasmid-introduced Escherichia coli capable of efficiently producing a desired foreign gene product at a high concentration in a culture. To provide.

[Means for solving problems]

The expression control of the foreign gene in the first culture step in the method of the present invention is carried out by culturing Escherichia coli capable of expressing the foreign gene by introducing a recombinant plasmid having a desired foreign gene inserted into an expression vector. The expression of the foreign gene is suppressed by maintaining the concentration of the sugar that can be used as a carbon source in the culture medium of Escherichia coli at 0.3% or more.

The Hi-plasmid (recombinant plasmid) used in the method of the present invention is obtained by inserting a desired foreign gene into an expression vector having a structure that enables the expression of the foreign gene in Escherichia coli.

Expression vectors constituting the Hi- plasmid, for example, a vector replicable in E. coli, was bound to the vector, P _L lambda promoter, trp promoter, a fusion promoter (tac E. coli lactose operon promoter (lac promoter) Promoter) and P _L
An example is a structure having a promoter linked to a lambda promoter. These promoters are not yet known to undergo catabolite repression.

The foreign gene referred to here is, for example, a structural gene encoding a polypeptide that is not normally produced in wild-type Escherichia coli, such as a non-Escherichia coli-derived polypeptide having an amino acid sequence substantially the same as a eukaryotic-derived polypeptide. Say.

The expression control of the foreign gene in the first culture step in the method of the present invention is carried out by controlling the concentration of a carbon source such as glucose, fructose, mannose, maltose, and sorbitol in the medium of Hi-plasmid-introduced Escherichia coli. By adjusting the expression, the expression of the foreign gene incorporated in the Hi-plasmid is suppressed.

Specifically, when it is desired to suppress the expression of a foreign gene, the concentration of sugar in the medium in which the Hi-plasmid-introduced Escherichia coli is cultured is maintained at 0.3 or higher, and when the foreign gene is expressed, -Adjust the sugar concentration in the medium in which the plasmid-introduced E. coli is cultured to less than 0.3%.

Thus, by culturing the Hi-plasmid-introduced bacterium while maintaining the sugar concentration at 0.3% or more, the expression of the foreign gene can be effectively suppressed, good cell growth can be obtained, and the desired bacterium can be obtained. Through the culture period until the body weight is obtained, the appearance of Hi-defective bacteria can be effectively suppressed.

In addition, the expression control of the foreign gene in the first culture process in the method of the present invention can be controlled by a simple operation of adjusting the concentration of the carbon source. It has an advantage that a special expensive reagent such as a reagent does not have to be used and a special temperature control structure need not be added to the culture device.

When the expression control of the foreign gene in the method of the present invention is applied to a method of culturing Hi-plasmid-introduced Escherichia coli to produce a foreign gene product derived from the foreign gene integrated into the Hi-plasmid, the foreign gene can be efficiently introduced. Allows the production of gene products.

That is, the method for producing a foreign gene product in Escherichia coli into which a Hi-plasmid has been introduced using the above-mentioned expression control method comprises: a) a P _L lambda promoter having no operator capable of incorporating a repressor in the presence of sugar, or
A recombinant plasmid having a configuration in which a foreign gene encoding the foreign gene product is inserted at a position where its expression is controlled by the expression vector having a linked promoter in which the P _L lambda promoter is connected immediately after the tac motor The step of preparing, b) the step of introducing the recombinant plasmid into E. coli, and c) the E. coli having the recombinant plasmid introduced, the concentration of sugar that can be used as a carbon source in the medium is 0.3%. A first culture step of maintaining the above and culturing while suppressing the expression of the foreign gene, and d) the concentration of sugars that can be used as a carbon source for Escherichia coli grown in the first culture step is less than 0.3%. A second culture step of culturing in a certain medium.

In the first culture step in the method of the present invention, the sugar concentration in the medium is maintained at 0.3% or more, and the expression of the foreign gene incorporated into the Hi-plasmid and introduced into E. coli is suppressed. As a result, in the first culture process, good bacterial growth can be obtained, and a desired amount of bacterial cells can be efficiently obtained. Moreover, until the desired amount of cells is obtained, Hi-
The appearance of plasmid deficient bacteria is suppressed to a negligible level. Further, even when subculture for a long period of time while maintaining the growth activity of the bacterium, the occurrence of Hi-plasmid deficient bacterium can be effectively suppressed.

In the second culture step in the method of the present invention, the sugar concentration in the culture is set to less than 0.3%, and the suppression of the expression of the foreign gene is released in the Hi-plasmid-carrying bacteria. Then, the foreign gene is expressed in the Hi-plasmid-carrying bacterium, and the foreign gene product is produced. At that time, since the proportion of Hi-plasmid deficient bacteria in the total cultures obtained in the first culture process is almost negligible, most of the total cultures efficiently express the foreign gene, and Foreign gene products can be obtained in culture at very high concentrations.

The combination of sugar concentrations used in the first culture process and the second culture process may be, for example, a combination of 0.3 to 1.0% and less than 0.3%.

By producing a large amount of culture by culturing a large amount of cells by the method of the present invention, a high concentration of the foreign gene product can be obtained in the culture. Can be increased to

Moreover, in the process of cell growth accompanied by the expression of a foreign gene, the proportion of Hi-plasmid deficient bacteria in the culture increases as the culture period increases, as described above.
Although it was not possible to take a long culture period, in the method of the present invention, in the first culture process, if necessary, a sufficiently long culture period should be taken to obtain the desired amount of cells. Therefore, the method of the present invention is particularly suitable for large-scale culturing on an industrial scale that requires a relatively long culturing period, including inoculum culturing and pre-culturing.

The timing of switching between the first culturing process and the second culturing process depends on the type of Hi-plasmid-introduced bacterium to be used and the culturing method used in the first and second culturing processes. When the method of the present invention is applied to the preculture for obtaining the inoculum and the main culture in a large-capacity batch method, the first stage of the present invention is the first stage of the logarithmic growth phase in the preculture and the main culture. The culture process may be performed, and the subsequent process may be used as the second process, which may be appropriately selected as desired. Also, the first
There may be a time difference between the culturing process and the second culturing process.

The method of the present invention will be described below with reference to Examples, Examples and Comparative Examples using phenylalanine ammonia lyase (PAL).

[Reference example]

As a reference example, an example of constructing a recombinant plasmid for PAL expression in Escherichia coli is shown below.

Reference Example 1 1. Isolation and purification of mRNA (PAL) Rhodosporidium t
oruloides IFO 559, which is also listed as ATCC 10788. ) Was cultured in a synthetic medium containing 2% glucose (Table 1) at 27 ° C with aeration and stirring. Immediately after the glucose added at the initial stage of the culture was completely consumed, the cells were centrifuged to collect the cells, and the wet cells were collected. The body was washed with sterilized 0.85% saline and then centrifuged again to obtain wet washed cells.

The wet-washed cells immediately contained PAL induction medium [containing 2% Lphe.
PAL was induced by suspending the cells in 0.17% Yeast Nitrogen Base (manufactured by Difco, ammonium sulfate-free and amino acid-free type) so as to have a cell concentration of 0.5 to 0.8% and shaking and stirring at 27 ° C.

The cells that had been subjected to the induction treatment for 2 hours were recovered from the PAL induction medium by centrifugation, and the obtained wet cells were suspended in an equal amount of sterile water, and the suspension was added dropwise to liquid nitrogen and frozen. The cells were used.

Frozen cells (10 g) are crushed in liquid nitrogen in a mortar to give 50 ml.
Buffer C (0.1MMNa ₂ HPO ₄ (pH 7.
4), 0.15M sodium chloride, 1% sodium deoxycholate, 1
% Tritonx-100) was added and gently stirred for 30 minutes.

After 30 minutes, 50 ml of 50 ml of a phenol / chloroform mixed solution (phenol: chloroform: isoamyl alcohol mixing volume ratio 25: 24: 1) was added, and the mixture was stirred and mixed for 15 minutes.

The mixed solution is centrifuged to recover an aqueous layer, 50 ml of a phenol / chloroform mixed solution is newly added to the aqueous layer, and the mixture is stirred for 15 minutes and then centrifuged, and the aqueous layer is further recovered and the phenol / chloroform mixed solution is extracted again. Was repeated twice.

Finally, 5M saline sterilized to a final salt concentration of 0.2M was added to the obtained aqueous layer, 2.5 volumes of cold ethanol was further added, and the mixture was stored at -20 ° C or lower to precipitate nucleic acid components. .

The precipitate was collected by centrifugation, washed with cold ethanol, and then dried under reduced pressure.

The dried product was dissolved in 10 ml of sterilized water, heat-treated at 65 ° C. for 5 minutes, and the well-known Maniatis method for mRNA using oligo d (T) cellulose [Maniatis.T. Et al., “Molecula
mRNA was isolated according to “R Cloning” (1982)].

The obtained mRNA was used as a sample buffer (5 M urea, 1 mM EDTA,
After dissolving in 0.05% Bromophenol blue), heat treatment at 65 ° C for 2 minutes to denature the higher order structure of RNA, and then use 8M urea-acrylamide slab gel (acrylic concentration 3%, 8M urea present) for running buffer (89mM Tris, 89mM boric acid, 2m
Electrophoresis in M EDTA) at 100 volts for 1.5 hours.

After electrophoresis, the acrylamide gel was treated with ethidium bromide, and the mRNA band was developed under UV light to divide the size of the mRNA into 2.0 to 3.0 kb in three equal parts and cut out each gel fragment from the slab gel. It was

Each gel fragment was enclosed in a dialysis tube, immersed in a running buffer, and mRNA was electroeluted from the gel.

Phenol / chloroform mixed solution was added to the solution in the dialysis tube, and the extraction operation was repeated twice.The residual phenol was extracted with ether, and 1/10 volume of 3M sodium acetate aqueous solution (pH 5.
2) and then 2.5 volumes of cold ethanol to add −20
It was stored at ℃ and the mRNA was precipitated.

In order to confirm that the mRNA obtained above contained PAL mRNA, a method was performed in which each mRNA fraction was translated into a protein and the produced protein was identified using a PAL-specific antibody.

That is, each fractionated mRNA was subjected to a cell-free translation kit using rabbit reticulocyte lysate [(Pelham.
al., European J. Biochem., 67 , 247-256, (1976)].

Rabbit Reticulocyte In Vitro Translation Kit, Promega
Biotec's one was used, and ³⁵ S-methionine (Amersham) was used as a labeled amino acid.

In order to confirm the protein translated by the rabbit reticulocyte in vitro translation system, buffer C was added to the translation reaction solution to dissolve it, the insoluble matter was removed by centrifugation, and the supernatant was prepared using rabbit rabbit anti-PAL IgG was added and the mixture was reacted on ice for 30 minutes. Sheep anti-rabbit IgG (manufactured by myself) was added to the reaction solution, and the mixture was reacted on ice for 30 minutes to precipitate with rabbit antibody.

The precipitate was collected by centrifugation, washed twice with buffer C, and the precipitate was mixed with 2% SDS, 10% β-mercaptoethanol mixed solution and 0.1 M Tris-phosphate (pH 6.8), 1%. SDS, 50
% Glycerin mixed solution at a volume of 3: 1 and dissolved at 95 ° C for 2 minutes to cleave the protein disulfide bond, and SDS-polyacrylamide slab gel electrophoresis (acrylamide concentration 10%) Method [Laem
mli, Nature, 227,680-685, (1970)], and after drying the gel after electrophoresis, PA by autoradiography.
L was identified.

2. Conversion of PAL mRNA to double-stranded cDNA (ds-cDNA) mRNA obtained from cells 2 hours after PAL induction treatment was purified by the above method, and Awv reverse transcriptase was allowed to act on the obtained mRNA. A single-stranded cDNA molecule was synthesized [Gugger, U., et al., G
ene, 25 , 263-269, (1983)].

RNase H, DNA polymerase I, and ligase were allowed to act on the single-stranded cDNA-mRNA hybrid to construct a double-stranded cDNA (ds-cDNA) excluding mRNA.

3. Construction of ds-cDNA having oligo dC tail at 3 ′ end The terminal doxynucleotidyl transferase (TdT) is allowed to act on the ds-cDNA obtained in the above 2nd section to produce ds-cD.
An oligo dC was added to the 3'end of NA.

That is, 3 μg ds-cDNA was dissolved in a reaction solution containing TdT buffer [100 mM potassium cacodylate (pH 7.2), 2 mM cobalt chloride, 0.2 mM dithiothreitol] and 0.2 mM dCTP, and the mixture was incubated at 37 ° C.
Pretreat for 30 minutes, then add 50 units of TdT and incubate at 37 ° C 15
The reaction was allowed to proceed for a minute, then EDTA was added to a final concentration of 40 mM, placed on ice, and a phenol / chloroform mixture was added to denature and inactivate TdT, denatured and insolubilized protein was removed by centrifugation, and the supernatant was extracted with phenol. After cold ethanol precipitation, the precipitate was washed with 70% ethanol and dried under reduced pressure to obtain oligo dC-added ds-cDNA at the 3'end.

4. Construction of hybrid plasmid [Binding of pUC9 (having oligo dc tail) molecule to ds-cDNA (having oligo dc tail) molecule] Oligo dC-added ds-cDNA obtained in the above item 3 and plasmid pUC9 [ Add oligo dG tail. Easily available from Pharmacia (Sweden)] Molecules were bound by a method similar to the Maniatis method known as the dC-dG homopolymer method.

5. Transformation and selection of clones The hybrid plasmid (oligo dG
C consisting of the added pUC9 molecule and the oligo-added ds-cDNA molecule)
It was introduced into E. coli treated with aCl ₂ by the competent method.

After obtaining about 40,000 transformant colonies, α- ³² P-dcTP was used in place of dcTP in the reaction solution when synthesizing single-stranded cDNA from PAL mRNA in the above-mentioned item 2, ^{Using 32} P-labeled single-stranded cDNA as a probe, the method of Grunstein et al. [Grunstein, M. et al., Proc. Natl. A
cad.Sci.USA., 72 , 3961 (1971)], the cells were selected by the colony hybridization method.

As a result, the plasmid was extracted from the positive colonies, purified, cleaved with various restriction enzymes, and the size of the DNA fragment was examined by agarose gel electrophoresis.

6. Construction of ds-cDNA containing complete PAL structural gene From the transformant obtained in the above item 5, plasmid pSW2
And pSW11 were obtained.

That is, it was revealed that the complete cDNA of PAL mRNA can be constructed by combining pSW2 and pSW11.Therefore, plasmids were extracted from the transformed cells containing each, purified, and cleaved with restriction enzyme BanIII, and then pSW2 In the above, the DNA was cleaved with restriction enzyme HindIII and fractionated by agarose gel electrophoresis to recover a DNA fragment of 4.2 kb in size, which was repeated several times for phenol / chloroform mixed solution treatment and cold ethanol precipitation. The fragment was purified.

On the other hand, pSW11 was digested with restriction enzymes BanIII and HindIII, and then a 0.9 kb DNA fragment was recovered by electrophoresis and purified.

The 4.2 kb and 0.9 kb DNA fragments were circularized with ligase, and E. coli was transformed with the product.

A plasmid was extracted from the ampicillin-resistant transformant used as a marker, various restriction enzymes were allowed to act on it to create a cleavage map, and pSW13 having the correct PAL structure having the structure of the restriction enzyme cleavage map shown in Fig. 1 was selected. .

7. Determination of nucleotide sequence of cloned DNA Plasmid pSW13 was isolated from the clone containing plasmid pSW13 described above, the cloned DNA fragment was digested with various restriction enzymes, and appropriate restriction enzyme fragments were respectively isolated.
Nucleotide sequence analysis of DNA is performed by the Maxam-Gilbert method (chemical decomposition method) and the Mart method [Maat, J. et al.,
Nucleic Acids Research, 5, 4537-4545, (1978)] by the Dideoxy method. The results of the nucleotide sequences of the respective DNA fragments thus obtained were subjected to DNA editing by the GENAS program manufactured by Mitsui Information Development Co., Ltd., and the nucleotide sequences were as shown in FIGS.

Up to 2151 bp of this base sequence was a PAL structural gene containing a start codon and an end codon.

8. Construction of pSW101 (see FIG. 3) 0.9 μg of plasmid pUC13 (Pharmacia) was added with 14 μl of a reaction solution of 10 units of restriction enzyme Sal I [7 mM Tris-HCl (pH
7.5), 0.7 mM EDTA, 7 mM MgCl ₂ , 175 mM NaCl, 7 mM 2-
It was allowed to act in mercaptoethanol, 0.01% bovine serum albumin (hereinafter abbreviated as BSA)] at 37 ° C. for 16 hours, treated with a mixed solution of phenol / chloroform, and subjected to ethanol precipitation to obtain a ring-opened linear DNA.

The linear DNA was added to Nick translation buffer [50 m
M Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 0.1 mM dithiothreitol, 2% BSA, 80 μM dATP, 80 μM dGTP, 80 μM dT
DNA polymerase Klenov fragment (Takara Shuzo Co., Ltd.) is allowed to act for 30 minutes at room temperature in the presence of TP, 80 μmM dCTP] to make the sticky ends blunt ends, and then deproteinize with phenol, and then DNA with cold ethanol. The precipitate was recovered. Calf spleen-derived phosphate diesterase (CIP: manufactured by Boehringa) was allowed to act on this DNA fragment to remove the phosphate group at the 5'end, and linear pU
Prevented self-closing of C13.

On the other hand, this plasmid was extracted and purified from cells containing pSW13, and the restriction enzyme Dral was added to reaction solution A (4 mM Tris-HCl).
(PH 7.5), 0.4 mM EDTA, 50 mM NaCl) It was made to act at 37 ° C. for 28 hours, and then a saline solution was added to adjust the salt concentration to 100 mM, and the restriction enzymes EcoRI and HindIII were allowed to act at 37 ° C. for 16 hours.

After the reaction was completed, the reaction solution was subjected to agarose gel electrophoresis, and a 2.3 kb-sized DNA fragment was recovered from the gel, and phenol extraction, phenol-chloroform mixed solution treatment, and cold ethanol precipitation were repeated 3 times each, and then the PAL cDNA fragment. Got

The above-mentioned nick translation buffer was added to the cDNA fragment, the DNA polymerase Klenov fragment was allowed to act at room temperature for 45 minutes, the phenol / chloroform mixed solution treatment and the cold ethanol precipitation operation were each repeated 3 times, and blunt ends were provided at both ends. A cDNA fragment was obtained.

PUC13 fragment with blunt ends and cDNA with blunt ends
The fragment was ligated with ligase to construct circular plasmid pSW101.

Escherichia coli was transformed with this hybrid plasmid DNA by a known method, cells (MT-10410, FERM P-8834) were selected from ampicillin-resistant colonies, and PAL activity was measured.

9. Construction and transformation of pYtrp6 pSW101 constructed by the method described in the above item 8 was digested with PstI and BamHI and subjected to agarose gel electrophoresis to give a 370 bp fragment.
The DNA fragment was recovered, split in two, and digested with BanI and BbeI, respectively.

After digestion, a 70 bp fragment was recovered from the BanI digested product and a 280 bp fragment from the BbeI digested product by acrylamide gel electrophoresis.

The 70 bp fragment was blunt-ended with DNA polymerase, and the ClaI (BanIII) linker was ligated to these by ligase.

DNA with ClaI linkers obtained at both ends
The fragment was digested with Ban III and BbeI, the previously prepared BbeI fragment (280 bp) and pBR322 were digested with Ban III and BamHI, and the DNA fragment of 4.0 kb recovered by agarose gel electrophoresis was ligated with ligase. Then, pSYA1 was obtained, and Escherichia coli was transformed with the known calcium method.

The pSW13 constructed in the above section 6 was digested with XbaI, the sticky end was filled with DNA polymerase to make a blunt end, and the HindIII linker was ligated to construct pSW13H, which was used to transform E. coli by a known method. did.

Extracting pSYA1 from E. coli containing pSYA1 by a known method,
After digestion with BamHI and BanIII, a DNA fragment having a size of 350 bp was recovered.

pSW13H was extracted from E. coli containing pSW13H by a known method, the extracted pSW13H was digested with BamHI and HindIII, and a DNA fragment of 1.0 kb was recovered by agarose gel electrophoresis.

Next, plasmid pVV1 [Brian P. Nicols & Charles Yanofsky, Metho containing a part of the trp operon of E. coli.
ds in Enzymology, 101 , 155, (1983)]
The plasmid pVV1 was digested with I.

The digested plasmid DNA fragment was separated by agarose gel electrophoresis, and a DNA fragment having a size of 0.9 kb was recovered from the gel by the method described above.

The 0.9 kb DNA fragment containing the Hinf I cohesive ends was first
After making a blunt end by the method described in the section 1, EcorRI linker (GGAATTCC) was ligated to the 5'end of the blunt end with ligase.

A restriction enzyme, EcoRI, was allowed to act on the EcoRI linker-bonded DNA fragment to create an EcoRI-cut cohesive end-added DMA fragment [Brian
P. Nicols & Charles Yanofsky, Methods in Enzymolog
y, 101 , 155, (1983)].

The EcoRI sticky end-added DNA fragment and the EcoRI digestion product of pBR322 which had been subjected to CIP treatment by the method described in the above item 8 were ligated with ligase, and the ligation product cormorant was used as a restriction enzyme EcoRI.
And Bg1II, and the digested products were separated by agarose gel electrophoresis to recover a DNA fragment having a size of 0.4 kb.

The DNA fragment contained three cleavage sites of the restriction enzyme TaqI, and the DNA fragment was partially digested with TaqI to recover a DNA fragment having a size of 345 bp.

The 345bP DNA fragment was ligated with a 4.3 kb DNA fragment obtained by digesting pBR322 with EcoRI and ClaI to obtain a plasmid pFtpr2 containing a trp promoter.

The pFtrp2 constructed in this way was transformed into Ban III and HindI
It was digested with II and the 4.7 kb fragment was recovered by agarose gel electrophoresis. This 4.7 kb fragment and the previously obtained 350 bP BamHI + B
The amIII fragment and the 1.9 kb BamHI + HindIII fragment were ligated with ligase to construct pSYA2.

The cohesive ends generated by partial digestion of pSYA2 with Bam III
It was filled in with a polymerase and opened as a blunt end with ligase to construct pYtrp6 having an Nru I cleavage point.

Escherichia coli was transformed with this pYtrp6 by a known method, cells were selected from ampicillin-resistant colonies, and PAL activity was measured. Figure 4 shows the flow sheet for the construction of pYtrp6.
The details are shown in FIGS. Was taken here
An E. coli transformant showing PAL activity was MT-10414 (FERM P-8
876).

Reference Example 2 A schematic diagram of the construction process of each plasmid in this Reference Example is shown in FIG.

[Construction of plasmid pSW115] (1) Plasmid pTac11 according to the process shown in FIG.
Construction of Escherichia coli MT 1041 having plasmid pYtrp 6 in which the PAL structural gene of Rhodosporidium toruloides obtained according to the method described in Reference Example 1 was cloned.
Plasmids extracted from strain 4 (FERMP-8876) were treated with restriction enzyme N
From the DNA fragment mixture obtained by digestion with ruI and HindIII, a DNA fragment having a size of 2.4 kbp was separated and collected by electrophoresis.

Separately, the plasmid p with the tac promoter
KK223-3 (Pharmacia) was digested with restriction enzyme EcoRI to obtain DNA fragments, and the sticky ends of these DNA fragments were treated with DN.
It was smoothed using A polymerase.

Next, the blunt-ended DNA thus obtained
After the fragment and the previously obtained 2.4 kbp DNA fragment were reacted in the presence of ligase, the resulting reaction product was transformed into E. coli.
Introduced by the method of N. Cohen et al.

 Subsequently, the E. coli into which this reaction product was introduced was
Picilin plate {LB medium [Bactrypton (commodity
First name; Bacto-tryptone , Difco) 10g; Bacteast
Extract (Bacto-yeast extract , Manufactured by Difco)
5g; glucose 1g; distilled water 1 (pH adjusted to 7.5 with NaOH)]
To the medium containing ampicillin at a concentration of 50 μg / ml.
Was added at a concentration of 1.5%}. culture
After that, each ampicillin-resistant colony that appeared on the plate
Individual plasmids from each of the
A restriction enzyme map of the mid was prepared and shown in Fig. 9 which is the target.
Colonies containing the plasmid pTac11 with the composition
Identified and further isolated plasmid pTac11 from the colony
Was.

(2) Plasmid pP _L -PAL-hea according to FIG. 10 step
d of the construction plasmid pP _L -λ (manufactured by Pharmacia) the restriction enzyme E
After digestion with coRI and HpaI, a 470 bp DNA fragment was separated and recovered from the resulting DNA fragment mixture by electrophoresis. This 47
The 0 bp DNA fragment was then partially digested with the restriction enzyme HinfI, and a 370 bp DNA fragment was separated and recovered from the resulting DNA fragment mixture by electrophoresis.

Further, the ends of the 370 bp DNA fragment were blunt-ended with DNA polymerase, and then reacted with Cla I linker (Takara Shuzo) in the presence of ligase. After the reaction was completed, the obtained reaction product was digested with restriction enzymes Eco I and Cla I to obtain a mixture containing EcoRI-Cla I DNA fragment.

Separately from this, the plasmid pSYA 2 constructed in the cloning process of the structural gene of Rhodosporidium toruloides in Reference Example 1 described above was digested with the restriction enzyme EcoRI, and the obtained DNA fragment was further digested with the restriction enzyme Cla. Partial digestion with I was performed, and a large DNA fragment was extracted and separated by electrophoresis from the obtained mixture of two kinds of large and small DNA fragments.

Next, the large DNA fragment derived from the plasmid pSYA 2 thus obtained was reacted with the mixture containing the EcoRI-Cla I fragment obtained above in the presence of T ₄ ligase, and the reaction product obtained Was introduced into E. coli and this was cultured on an ampicillin plate. A plasmid was prepared from each colony that appeared on the ampicillin plate, its restriction enzyme map was prepared, and the target plasmid pSYP _{L- having the} configuration shown in FIG. 10 was constructed.
A colony having 3 was identified and the plasmid pSYP _L- 3 was isolated from the colony.

Furthermore, the plasmid pSYP _L- 3 thus obtained was
Was digested with EcoRI and BamHI, and small DNA fragments were separated and recovered from the obtained two kinds of large and small DNA fragments by electrophoresis.

Next, plasmid pBR322 (Pharmacia) was digested with restriction enzymes EcoRI and BamHI to obtain two large and small DNAs.
After the large DNA fragment was separated and recovered from the fragment by electrophoresis, the large DNA fragment was reacted with the small fragment derived from the plasmid pSYPL-3 previously obtained in the presence of ligase to obtain the plasmid having the structure shown in FIG. It was obtained pP _L -PAL-head. Whether or not the desired plasmid was obtained here was determined by introducing the reaction product produced by the reaction using ligase into Escherichia coli, culturing it on an ampicillin plate, and recovering the plasmid from each colony appearing on the ampicillin plate. And prepare
It was confirmed by creating the restriction enzyme map.

(3) Plasmid pSW115 according to the process shown in FIG.
First, the plasmid pTac11 obtained in the above item (1) was digested with restriction enzymes EcoRI and AatI, and a large DNA fragment was separated and recovered from the mixture of the obtained two kinds of large and small DNA fragments by electrophoresis.

Next, the plasmid pP _L -PAL-head obtained in the item (2)
Digested with restriction enzymes EcoRI and AatI to obtain large and small D
Small DNA fragments were separated and collected from the NA fragment mixture by electrophoresis.

Finally, in this way the large DNA fragment and the plasmid pP _L -PAL-head from a small DNA fragment derived from the plasmid pTac11 was coupled by reaction in the presence of T ₄ ligase was obtained, resulting in plasmid PSW115.

In addition, whether the desired plasmid was obtained,
The resulting plasmid was introduced into Escherichia coli, the transformant strain was selected on an ampicillin plate, a plasmid was prepared from each transformant strain having ampicillin resistance, and a restriction enzyme map thereof was prepared, and at the same time, The PAL activity was measured and confirmed by the method described below. The transformed E. coli strain having PAL activity obtained here was designated as MT-10423 strain (FERM P-9023).

(4) Expression of PAL by plasmid pSW115 Transformed Escherichia coli transformed with the plasmid pSW115 obtained in (3) above was used in the composition of the ampicillin plate in LB medium (pH 7.5) to obtain 50 μg / ml of ampicillin. The medium was inoculated into the medium at a concentration, and this was cultured at 30 ° C. with shaking.

Since OD at 660 nm by culturing for 20 hours gave a cultured cell concentration showing 5.40, the cells were collected from the culture solution by centrifugation, and the PAL activity of the obtained cells was measured according to the method described below, The specific activity per dry cell weight was calculated to be 630 U / dry cell weight (g).

The dry cell weight was measured by drying the washed cells.

The introduction of the recombinant plasmid into E. coli in Reference Example 2 described above was carried out by the method of SN Cohen et al. [SN Cohen et al .; P
roc.Natl.Acad.Sci.USA, 69 , 2110 (1972)]. In addition, restriction enzymes, ligase, T ₄ ligase, DN
Treatment of plasmids and DNA fragments with A polymerase and preparation of plasmids from bacterial cells were carried out by commonly used methods unless otherwise specified.
Furthermore, as the host E. coli, MC1061 strain [Δ (lacIPOZY
^{A) F -, araD139, Δ} (ara leu) 7697 × 74gal U gal K s
trA] [MJCasadaban, J. Mol. Biol., 138 , 179, (198
0)] was used. The restriction enzymes, ligase, T ₄ ligase, and DNA polymerase used were those manufactured by Takara Shuzo, unless otherwise specified.

〔Example〕

 Hereinafter, examples and comparative examples of the present invention will be described.

The medium used in the following Examples and Comparative Examples was prepared as follows.

LB medium; tryptone 10 g yeast extract 5 g NaCl 5 g water 1 (adjusted to pH 7.5 with KOH) LB-AP agar medium; 1 g glucose and 15 g agar were added to 1 LB medium having the above composition, and sterilized by an autoclave, Ampicillin (AP) was added aseptically at a concentration of 100 μg / ml,
A plate poured into a dish.

Synthetic medium; monopotassium phosphate 3g dipotassium phosphate 7g magnesium sulfate heptahydrate 0.5g ammonium sulfate 1.5g calcium chloride dihydrate 0.02g ferrous sulfate heptahydrate 0.02g sodium citrate 1g casamino Acid 12 g L-tryptophan 0.1 g Distilled water 1 Example 1 A vector containing the gene encoding AP resistance obtained in Reference Example 2 and a P _L lambda promoter operator bound to the expression vector downstream of the promoter operator.
Hi-plasmid pSYP _L constructed with PAL structural gene inserted
Escherichia coli MT-10424 strain (FERM P-9024), which retains -3,
It was spread on an LB-AP agar plate and incubated at 37 ° C.

On the other hand, 100 ml of LB medium containing AP at a concentration of 50 μg / ml and LB medium containing glucose at various concentrations as shown in Table 2 were separately prepared in a Sakaguchi flask with a cotton plug, 100 ml each, and autoclaved. After sterilization by
AP was added aseptically to a concentration of 50 μg / ml to prepare 5 types of medium having different glucose concentrations.

Next, from the colonies appearing on the LB-AP agar medium plate, the same amount of cells was inoculated into the medium in each Sakaguchi flask, and the cells were shake-cultured at 30 ° C for 110 hours / min for 25 hours.

After completion of the culture, a cell extract was prepared from each of the cultures as follows, and the PAL specific activity of each extract was determined.

Table 2 shows the concentration of cultured cells (OD ₆₀₀ ) at the end of the shaking culture and the value of the obtained PAL specific activity.

Preparation of bacterial cell extract: The cultured bacterial cells are collected from the culture solution by centrifugation, the collected bacterial cells are suspended in 0.85% saline solution, washed, and then centrifuged again to collect the bacterial cells. The washed cells were suspended in 0.05 mM Tris-HCl buffer solution (pH 8.8) at a cell concentration of 1% wet cell concentration, sonicated to disrupt the cells, and the solution was further removed from the solution. Cell debris and the like were removed by centrifugation to prepare a cell extract.

Measurement of PAL activity; The PAL activity of each extract was determined according to the following procedure using an enzymatic reaction that produces cinnamic acid from L-phenylalanine.

First, the bacterial cell extract was diluted with 25 mM Tris-HCl buffer (pH 8.8) to a wet bacterial cell concentration of about 1.0%, and 1.0 ml thereof was diluted with 31.25 mM.
4.0 ml 31.25mM Tris-HCl containing L-phenylalanine
The mixture was added to the buffer solution and reacted at 30 ° C for 20 minutes. 1 ml of 1N-HCl
Was added to terminate the reaction, and the amount of cinnamic acid produced in the reaction solution was analyzed by liquid chromatography under the following conditions to measure its activity.

In addition, 1 U (unit) here corresponds to the amount of enzyme that produces 1 micromol of cinnamic acid per minute.

Liquid chromatography operating conditions; Separation column YMC back A-312 (manufactured by Yamamura Chemical Co., Ltd.) was used, and the mobile phase was methanol: water: phosphoric acid = 50: 41: 0.08 v / v.
Using an ultraviolet spectrophotometer (detection wavelength 26
0 nm).

The amount of cells used to calculate the PAL specific activity was obtained by drying the washed cells.

As shown in Table 2, the glucose concentration in the culture medium was 0.
When the concentration is 3% or more, the growth of E. coli is better than when the glucose concentration is less than 0.3%, and the PAL specific activity is low.
It was shown that AL was only slightly expressed, and it was confirmed that effective control of expression was possible by setting the glycol concentration to 0.3% or more.

Example 2 Hi- having a configuration in which the PAL structural gene was inserted downstream of the trp promoter of the expression vector in which the trp promoter was ligated to the vector containing the gene encoding AP resistance obtained in Reference Example 1.
E. coli MT10414 strain carrying the plasmid pYtrp6 (FERM P
-8876) was applied to LB-AP agar medium and cultured at 37 ° C.
A portion of the resulting colony was inoculated into each of the test tubes with cotton plugs containing AP at a concentration of 50 μg / ml and 5 ml of LB medium containing glucose at various concentrations as shown in Table 2, This was shake-cultured at 30 ° C. for 100 hours / min for 25 hours.

At the same time as in Example 1, the individual body extract was prepared from each culture and the PAL activity of each was measured.

Table 3 shows the concentration of cultured cells (OD ₆₀₀ ) at the end of the shaking culture and the value of the obtained PAL specific activity.

As is clear from the results shown in Table 3, also in the present example, when the glucose concentration in the culture solution was 0.3% or more, the growth of the bacterium was good and the expression of PAL was sufficiently controlled.

EXAMPLE 3 Reference Example 2 in tac promoter vector containing the gene encoding the AP resistance obtained with the coupling promoter downstream PAL structure of the expression vector obtained by combining the coupled promoter and P _L lambda promoter linked downstream thereof Escherichia coli MT10423 strain (FERM P-9023) harboring the Hi-plasmid pSW115 in which the gene was inserted was applied to LB-AP agar medium and cultured at 37 ° C.

Next, glucose, mannose, maltose, and sorbitol were individually added to LB medium containing AP at a concentration of 50 μg / ml so that the respective concentrations were 0.1, 0.3, and 1.0%, and a total of 12 types of sugars were added. Culture solutions with different concentrations were prepared.

Each culture solution (5 ml) thus prepared was inoculated with the same amount of E. coli from the colonies that appeared on the LB-AP agar plate in the previous culture, and shake culture was performed at 30 ° C and 110 times / min for 24 hours. I did. After the culture was completed, the same operation as in Example 1 was performed to determine the PAL activity.

Table 4 shows the concentration of cultured cells (OD ₆₀₀ ) at the end of the shaking culture and the value of the obtained PAL specific activity.

As shown in Table 4, in each culture,
The expression of PAL was effectively controlled at sugar concentrations of 3% or higher.

Example 4 1.5 liters of synthetic medium was placed in a 2 liter mini jar fermenter equipped with a device capable of measuring glucose concentration in the culture medium, and after sterilization, 0.5 g of AP was aseptically added.

Separately, glucose is added to the synthetic medium at a concentration of 1
100 ml of the medium added so that the concentration became 100% was prepared in a Sakaguchi flask, sterilized by an autoclave, and then AP was aseptically added to this medium so that the concentration of AP was 50 μg / ml. Next, this was inoculated with the Escherichia coli MT10423 strain used in Example 3 and shake-cultured at 30 ° C. and 110 strokes / minute for 22 hours to obtain a seed culture solution. 75 ml of the obtained seed culture solution was inoculated into the culture solution in the previously prepared mini jar, glucose was added so that the initial glucose concentration was 1%, and the pH of the culture solution was adjusted to 7.0 by adding aqueous ammonia. Then, the culture was started at 30 ° C. while performing aeration and stirring. The glucose concentration in the culture broth was kept at 1% until the desired amount of cells was obtained. When the target bacterial cell concentration was reached, the glucose supply was stopped and the glucose consumption in the culture solution was used to reduce the glucose concentration to less than 0.3% to express PAL.

The culture solution was collected with time from the start of the culture, and the glucose concentration, the bacterial cell concentration, and the PAL specific activity by the same method as in Example 1 were determined at any culture time. The result is
Shown in the figure.

As is clear from FIG. 12, PAL was expressed at a glucose concentration of less than 0.3%, and finally a sufficiently high PAL specific activity was obtained.

Example 5 The medium used in Example 5 and Comparative Example 1 described later was prepared as follows.

LB medium; 1 g of glucose was added to 1 LB medium containing no glucose shown above, and sterilized by autoclave (12
After 15 minutes at 0 ° C., AP was aseptically added at a concentration of 50 μg / ml.

A medium; 10 g of glycol was added per 1 LB medium containing no glycol shown above, and sterilized in an autoclave (120
AP, was added aseptically at a concentration of 50 μg / ml.

First, the LB-AP agar medium was coated with the E. coli MT10423 strain carrying the Hi-plasmid pSW115 used in Example 3 and cultured at 37 ° C. for 16 hours.

From the colonies that appeared, divide the cells into test tubes with cotton plugs containing A medium (5 ml) and test tubes with LB medium (5 ml), and inoculate them. Culture solution No. 1- shake culture at stroke / min.
1 (A medium culture) and culture solution No. 1-2 (LB medium culture) were obtained.

Next, from culture solution No. 1-1 to culture solution (including bacterial cells)
Using 1 μl of the seed culture as a seed culture, inoculate it into a test tube with cotton plugs containing A medium (5 ml) and a test tube with cotton plugs containing LB medium (5 ml), and perform shaking culture under the same conditions as above to obtain culture medium No. .1-3 (A medium culture) and culture solution No. 1-4 (LB medium culture) were obtained.

Further, the same culture operation as above was repeated in the order shown in FIG. 13 to obtain culture solutions No. 1-5, No. 1-6 and No. 1-7.

In addition, culture solution No. 1-2, No. 1-4, No. 1-6, No. 1-
In Example 7, the cells were collected by centrifugation immediately after the completion of the culture, and the obtained cells were suspended in 0.85% NaCl aqueous solution and washed, and the cells recovered by centrifugation again were stored frozen.

Next, each cryopreserved microbial cell was thawed to obtain a microbial cell suspension, and thereafter, a microbial cell extract was prepared in the same manner as in Example 1, and its PAL was used.
Specific activity was measured. The results are shown in Table 5.

Comparative Example 1 The culture operation was repeated in the same manner as in Example 5 except that the culture in A medium in Example 5 was changed to LB medium as shown in FIG. 14, and the culture solutions No. 2-1 to No. 2-7 was obtained.

Furthermore, culture fluid No. 1-2, No. 1-4, No. 1-6, No. 1-
Cryopreserved cells were prepared from 7 in the same manner as in Example 5, and their PAL specific activities were determined. The results are shown in Table 5.

From the results of Example 5 and Comparative Example 1, it was found that the occurrence of Hi-plasmid deficient bacteria can be effectively suppressed even when subculture for a long period of time.

(Effects of the Invention) According to the present invention, expression of a foreign gene in Escherichia coli introduced with Hi-plasmid can be effectively performed as desired by a simple operation of adjusting the concentration of sugar that can be used as a carbon source in a medium. It is controllable.

Further, by using the expression control method of the present invention, the growth of bacteria in the method of culturing Hi-plasmid-introduced Escherichia coli to produce a foreign gene product and the expression time of the foreign gene are separated, and these are efficiently performed. As a result, it is possible to achieve good cell growth while sufficiently suppressing the generation of Hi-plasmid deficient bacteria, and to obtain the desired foreign gene product at a high concentration in the culture, which enables efficient production of the foreign gene product. became.

In particular, in the method of the present invention, since the expression control is performed by a simple operation of adjusting the concentration of glycol, which is a medium component, it is not necessary to use an expensive induction reagent as in the case of using an inducible plasmid, and It is not necessary to use a special device for temperature control as in the case of using the control type.

Furthermore, since the appearance of Hi-plasmid deficient bacteria can be suppressed more effectively, the method of the present invention can easily improve the productivity of foreign genes by large-scale culture on an industrial scale.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme digestion map of pSW11, pSW2 and pSW13 involved in the PAL structural gene. Fig. 2 shows phenylalanine cloned in the reference example.
FIG. 1 shows the base sequence of one strand of a DNA sequence containing a region encoding ammonia lyase. FIG. 3 is a flowchart of the procedure for constructing pSW101, and FIG.
The figure is a flow chart of the procedure to construct pYtrp6,
FIGS. 5 to 7 show in detail a part of the flow chart shown in FIG. FIG. 8 is a schematic diagram of the construction process of each recombinant plasmid constructed in Reference Example 2, and FIG. 9 is the plasmid pTac11.
Of FIG. 10 of plasmid pP _L -PAL-head, FIG. 11 is a diagram specifically showing the construction process of plasmid PSW115. FIG. 12 is a graph showing changes over time in glucose concentration and bacterial cell concentration in the medium and in PAL specific activity of the cultured bacteria in the culture in Example 4. 13 and 14 are diagrams showing the flow of the culture operation in Example 5 and Comparative Example 1.

Claims (5)

[Claims] 1. A method for producing a foreign gene product in Escherichia coli, comprising: a) a P _L lambda promoter having no operator capable of binding a repressor in the presence of sugar, or
A recombinant plasmid having a configuration in which a foreign gene encoding the foreign gene product is inserted at a position where its expression is controlled by the expression vector having a linked promoter in which the P _L lambda promoter is connected immediately after the tac promoter The step of preparing, b) the step of introducing the recombinant plasmid into E. coli, and c) the E. coli having the recombinant plasmid introduced, the concentration of sugar that can be used as a carbon source in the medium is 0.3%. A first culture step of maintaining the above and culturing while suppressing the expression of the foreign gene, and d) the concentration of sugars that can be used as a carbon source for Escherichia coli grown in the first culture step is less than 0.3%. A second culture step of culturing in a certain medium, and a method for producing a foreign gene product in Escherichia coli. 2. The method for producing a foreign gene product in Escherichia coli according to claim 1, wherein the foreign gene product is L-phenylalanine ammonia lyase. 3. The method for producing a foreign gene product in Escherichia coli according to claim 2, wherein the L-phenylalanine ammonia lyase has the amino acid sequence shown below. Wherein said E. coli recombinant plasmid pSYP _L -
The method for producing an exogenous gene product in Escherichia coli according to claim 1, which is Escherichia coli MT 10424 strain harboring 3. 5. The Escherichia coli recombinant plasmid pSW 115.
Claim 1 which is Escherichia coli MT 10423 strain carrying
The method for producing an exogenous gene product in Escherichia coli according to item 1.