JPS61152278A - Cultivation of microorganism - Google Patents

Abstract

PURPOSE:To enable the cultivation of transformed microorganism in high concentration, by transforming a microorganism to produce the required exogenic protein, culturing the microorganism for the proliferation, and then culturing to induce the protein-synthesis capability. CONSTITUTION:A microorganism is transformed by a chimera plasmid produced by integrating a gene coding exogenic protein under the control of a plasmied having a promoter originated from alkaline phosphatase, and the obtained transformed microorganism is used as the host microorganism. The microorg anism is cultured first in a medium containing inorganic phosphorus of an amount necessary for the proliferation of the microorganism but insufficient to induce the protein synthesizing capability. The cultured microorganism is transferred to a medium free from inorganic phosphorus, and an inorganic phosphorus or a medium containing inorganic phosphorus is added to the medium at a constant rate.

Description

BACKGROUND OF THE INVENTION Technical Field The present invention relates to a method for culturing microorganisms transformed by genetic engineering techniques.

More specifically, the present invention involves the production of a desired foreign protein using recombinant DNA technology. The present invention relates to a method for culturing microorganisms, which comprises culturing with induction of microorganisms.

Prior art Currently, methods for producing useful substances through genetic engineering using recombinant DNA technology are being established, and for specific methods, various books, documents, published patent publications, and patent publications can be referred to. .

This method of producing a substance usually involves transforming a microorganism using a vector created by incorporating a gene encoding the desired substance, culturing the resulting transformant, and then recovering the desired substance. It consists of doing.

By the way, when non-secreted proteins are produced by the above method, the proteins produced in microbial cells are degraded (proc, Nat) by the protease of the host bacteria.
l, Δcad, Sci, USA, 79. −
1830-1833 (1982)), the desired protein could not be obtained stably and in large quantities. In addition, when recovering such non-secreted proteins, the host microorganism is physically destroyed and then the desired protein is isolated and purified, so such recovery operations are generally complicated. Therefore, there are problems in that the yield of the desired protein is low, and that the active protein may be deactivated during the recovery operation.

Therefore, in order to deal with the above problems, we investigated the signal sequences involved in the passage of proteins through membranes [Proteins/Nucleic Acids/Enzymes, 26.38
6-394 (1981)), various methods have been proposed for secreting proteins produced within the host microbial cell to the outside of the cell or the cytoplasmic membrane (Public Patent Publication No. 1981-19092, same). No. 55-45395, same 5
No. 6-137896, No. 56-145221, No. 56
-154999 publications, etc.). Recently, co-researchers of the present inventors have proposed a protein production method that skillfully utilizes such a method (Japanese Patent Application No. 59-159).
No. 703) (hereinafter referred to as the prior invention).

In one embodiment of the method of the prior invention, plasmid pTA1529 is used, which is equipped with a promoter and signal sequence derived from alkaline phosphatase, and into which a gene encoding a desired foreign protein can be ligated immediately after the signal sequence. I am using it. In reality, 1) TA1529 contains a gene encoding a foreign protein [for example, human epidermal growth factor (hUG/EGF)].
(hereinafter simply referred to as hUG)] to create a chimeric plasmid (incorporating the structural gene of hUG, pTAi 52
2) was constructed, and microorganisms are transformed using this plasmid. Therefore, when a microorganism transformed with such a plasmid (hereinafter, a transformed microorganism may be referred to as a "host microorganism" or simply a "bacterium") is cultured, first of all, inside the host cell, A foreign protein is expressed, and then this protein is secreted outside the bacterial cell under the action of a signal sequence. Here, "secreted outside the bacterial cell" means that in a Durham-negative host bacterium such as E. coli, the protein is transferred from the bacterial cell to the periplasm (the space between the cell membrane and the outer membrane). , in Durham-positive bacteria such as Bacillus subtilis, means that it migrates outside the cell. In addition, in the prior invention mentioned above, when the protein is secreted outside the bacterial cell, the signal sequence is hydrolyzed by deenzyme signal veptidase, so the protein bound immediately after the signal sequence is accompanied by extra amino acids. It is secreted outside the bacterial cell as a complete mature protein without any oxidation.

In the method for producing proteins according to the prior invention, the following culture method is adopted for transformed microorganisms that have a promoter derived from alkaline phosphatase. That is, this culture method
The fact that the synthesis of alkaline small sphatase is inhibited in the presence of high concentrations of inorganic phosphorus and induced in the presence of low concentrations of inorganic phosphorus [3iochem, 3iophys, Acta
, , 38.460 (1960), Nature.
183. 1529 (1959)). To give an example of such a member, first, a host microorganism E, coli K12C600 (1) TA1, transformed with pTA1522 into which the structural gene of hUG has been incorporated.
522) in L-medium containing 20 μg/Id ampicillin [1% Bactodributon, 0.5% yeast extract, 0.
5% NaC+, 0.1% glucose) 80#11! Pre-incubate with Next, the pre-cultured bacteria was cultured in a synthetic medium containing 0.64 mM potassium dihydrogen phosphate (the above Bioc
hem, B 1ophys, Acta, T described
G+'20 medium) 2.4.0 and cultured with shaking at 37°C overnight (hereinafter referred to as "first stage" for convenience)
Afterwards, the bacteria were collected, transferred to the above synthetic medium containing 32 μM potassium dihydrogen phosphate, and further cultured with shaking at 37°C for 5 hours (hereinafter referred to as the "second stage" for convenience). be. Here, TG+20 medium refers to glucose 2
'j/fJ.

NaCl 4.68g/, Q, KCl 1.49g/j
, NH4Cl 1.09g/, 11. Na2SO40
．． 43g/, Q, M(IC+295.21rng/
, 11.

CaCl 22.20 my/l, FeCl3324,
lI2ug/R, Zn C12272,56uq/fr
, K1-1. , PO487, 1 my/11 and 0.
It consists of 1M Tris-HCl buffer (1)l-17,2). According to such a culture method, the transformed microorganism can be propagated in the first step, and then culture can be performed with induction of protein synthesis ability in the second step.

By the way, transformed microorganisms are generally loaded with plasmids introduced into the microorganism.
Journal of General Microb
iology.

118.253-261 (1980)), the growth rate is slower than that of non-transformed microorganisms, and the introduced plasmid is likely to be shed, and microorganisms that have shed the plasmid easily become the preferred species. is known (AnnalS of the New Y
ork Academy of 5science, 3
69.

1-14 (1981), Journal of G
eneralMicrobiology). The problem of difficulty in cultivating transformed microorganisms at high concentrations resulting from this fact could be overcome by the culture method of the prior invention. However, when this method is applied to the production of industrial substances, it is necessary to culture at a higher concentration in the first step.

In addition, in the second step of this method, after inoculating the bacteria, the protein synthesis ability is induced once, but as the culture continues, the inorganic phosphorus necessary for the growth of the bacteria runs out, and the bacterial cells themselves disappear. There is a problem that the desired protein cannot be obtained in large quantities because the protein cannot be maintained.

SUMMARY OF THE INVENTION The present invention aims to provide a solution to the above-mentioned problems, and aims to provide a solution to the above-mentioned problems by using a host microorganism transformed with a plasmid having a promoter derived from alkaline phosphatase and into which a gene encoding a desired protein has been integrated. First, the host microorganism is cultured for propagation, and then it is subjected to a culture process that induces protein synthesis ability (actually, the action of the promoter of the plasmid held in the host microorganism is induced). The aim is to achieve this goal using a two-step culture method.

Therefore, the method for culturing a microorganism according to the present invention involves transforming a microorganism with a chimeric plasmid constructed by incorporating a gene encoding a foreign protein under the control of a plasmid having a promoter derived from alkaline phosphatase. First, the microorganism is cultured in a medium that does not induce protein synthesis ability and contains the mother inorganic phosphorus necessary for the growth of the microorganism, and then transferred to a medium that does not contain inorganic phosphorus. It is characterized by being subjected to culture with constant rate feeding of inorganic phosphorus or a medium containing inorganic phosphorus.

Effects As described above, the method for culturing microorganisms of the present invention includes culturing for propagation of transformed microorganisms (hereinafter referred to as the "first step");
Next, the two I
Pi! J: That's what happens. By dividing the cultured culm into two stages in this way, the following effects can be obtained.

(1) High concentration culture of transformed microorganisms is possible.

In general, in recombinant DNA technology, it is known that host bacteria are loaded by the transfer of plasmids, and that plasmids transferred in this way are unstable and easily fall off from the host bacteria [see above]. In the conventional culture method in which a desired protein is produced by culturing a host bacterium in two steps without dividing, the host bacterium is burdened and the frequency of plasmid shedding increases. In such a culture, bacterial cells from which the plasmid has been shed become dominant (see above), making it difficult to culture the bacteria retaining the plasmid at a high concentration. Therefore, in the prior invention of the present inventors, in order to deal with the above problems, the transformant is first cultured for host bacterial growth (first step).
A method was used in which the culture was divided into two stages: culture for inducing protein synthesis ability (second stage); It was difficult to culture the host bacteria at high concentrations.

In the method for culturing microorganisms according to the present invention, in the first step, the composition of the conventionally used medium is changed (for example, the above-mentioned T G -
1-20 in a complex medium (glucose, tribune,
Yeast extract, MU So・71-120゜Kl-I
PO, 2l-IPO4, ampicillin] was used to overcome this problem and furthermore enabled high-density culture of host bacteria to an industrial level.

(2) A desired protein can be obtained efficiently and in large amounts.

Conventionally, in the second stage, there was virtually no inorganic phosphorus in the medium, and although protein synthesis ability was induced by the action of the promoter, it hindered the growth of the host bacteria. However, in the end, it was not possible to obtain a large amount of the desired protein, but the present invention involves periodically supplying inorganic phosphorus in the second step (the specific method will be described later).
As a result, we have established a culture method that efficiently induces protein synthesis ability by overcoming this problem caused by the lack of inorganic phosphorus. The desired substance can be produced in large quantities by subjecting it to the culture process.

In addition, a preferred specific example of the host bacteria used for the culture of the present invention is a plasmid having a signal sequence, which allows a gene encoding a desired foreign protein to be ligated immediately after the gene encoding the signal sequence. This is a microorganism transformed by a microorganism that has been transformed into a microorganism (see the above-mentioned prior invention of the present inventors).

Therefore, when a host bacterium into which the above plasmid has been introduced is cultured, the foreign protein is secreted outside the bacterium by the action of the signal sequence as described above. When the host bacterium is a Durum-positive bacterium, the desired protein is released into the medium, and when the host bacterium is a Durum-negative bacterium, the desired protein is released into the periplasm, and the protein accumulated in the periplasm is released into the periplasm. is the osmotic shock method (J, Biol
.

Chem, 240.3685 (1965)), the protein can be easily recovered from the culture medium or solution in which the protein has been released, since its constituent components are known.

From the above, by culturing a microorganism transformed using the plasmid (for example, pTA529.pTA1529) according to the prior invention of the present inventors according to the method of the present invention, a desired protein can be efficiently produced and can be obtained in large quantities.

Plasmid The plasmid used in the present invention is based on the fact that alkaline phosphatase is affected by the amount of inorganic phosphorus present in the culture method of the present invention [see 31 above.

31ophys, Acta, ), any promoter may be used as long as it has a promoter derived from alkaline phosphatase and can proliferate within the intended host cell. Even more preferred is a plasmid that also utilizes the function of a signal sequence (secretion of protein outside the bacterial body).

Specific examples of such preferable plasmids include plasmids pTA529 (Japanese Patent Application No. 140,748/1982), which was previously proposed by the joint researchers of the present inventors.
There are TA1529 (Japanese Patent Application No. 59-159703), etc. All of these plasmids are equipped with a promoter and signal sequence derived from alkaline phosphatase, and a gene encoding a foreign protein can be ligated immediately after the gene encoding the signal sequence. Here, pTA1529 is pTA529
(1) YK283 (E, coli K12C600
Deposited as (pYK283) at the Microtechnical Research Institute.
No. 56)], this was filed in patent application No. 58-14074.
(produced according to the method described in the specification of No. 8) and p
l-181 (this plasmid is pBR322(E, c
As olt K12C600 (pBR322)
Deposited with the Institute of Microtechnology (Feikoken Article No. 235)] was added to the restriction enzyme Eco
The EcoRT-1-lind11 portion was created from the following synthetic linker (see Japanese Patent Application Laid-open No. 71692/1983). It is something. For details of the procedure for constructing this plasmid, please refer to the specification of Japanese Patent Application No. 59-159703.

EcoRT l-1paI SmaT 1-
ItndI[[Transformant microorganisms can be transformed according to conventional methods known in the field of recombinant DNA technology.

For example, a structural gene of a desired foreign protein is incorporated into the above-mentioned plasmid to create a chimeric plasmid, and this is used to culture microorganisms using known methods (for example, the Kushigona method (Genettc method)).
Engineerin (1, 1978, 17 (19
After transformation according to 78))), the desired transformant is obtained (usually using a plasmid marker)'
? This can be done by any convenient method.

Microorganisms that can be transformed in this way may be any microorganisms as long as the above-mentioned plasmid can propagate within their cells, and specific examples thereof include Escherichia coli, yeast, and Bacillus subtilis. Among them, 12 strains of E. coli are relatively commonly used, and in the examples of the present invention, the mutant strain E, coli K12YK537
(Feikoken Bibori No. 7941) is used.

The structural genes of the desired foreign proteins to be incorporated into the above-mentioned plasmids include hormones, immune-related substances, neuropeptides, enzymes, and the like. Methods for preparing these structural genes include methods for obtaining them from natural chromosomal DNA and methods for artificially synthesizing them.For actual methods for preparing structural genes, refer to various bases and literature. (The above-mentioned prior invention, etc.)

Such a transformant used in the present invention contains hU artificially synthesized in plasmid pTΔ1529 (described above).
Chimeric plasmid pTΔ15 containing the structural gene of G
22 and then use this to infect E and coli K.
12YK537 was transformed by Kushchi method and 4%
It's something like that (".

coli K12YK537 (pTA1522)).

For details of the procedure for creating a transformant, please refer to the reference examples described below.

Cultivation of Microorganisms The cultivation according to the present invention targets microorganisms transformed with a plasmid having a promoter derived from alkaline phosphatase, and the characteristics of the culture method of the present invention are that The properties of the plasmid (the protein synthesis ability may or may not be induced depending on the amount of inorganic phosphorus [see 3 above)
m, B1ophys.

The transformed microorganism is first induced to have protein synthesis ability (Acta, Nature).
Two culture steps: first, culturing for the purpose of propagating the microorganism without inducing the action of the plasmid's promoter, and then culturing for the purpose of inducing protein synthesis ability and maintaining the growth of the microorganism. It means that it becomes more.

Details of such a culture method are as follows.

1) Cultivation method The method for culturing microorganisms according to the present invention involves first culturing a transformed microorganism in single-cell pure isolation, and then subjecting it to preculture (the above is a well-known conventional method for culturing microorganisms; You can refer to the literature and bottom position of
These include Sokoki's ``Microbiology Experimental Methods'' (published by Kodansha), ``Microbial Experimental Methods'' (published by Kyoritsu Shuppan), and ``Bacteriology Practice Recommendations'' (published by Maruzen). ) Following the culture for microbial growth (first step), culture with induction of protein synthesis ability is performed.
(second step). In addition, both the first and second steps belong to the aerated agitation culture, and specifically, after inoculating bacteria in the culture system, the culture medium is filled with sterile air (pure oxygen if necessary). (mixed with)
The microorganism is introduced into the microorganism, and the microorganism is physically stirred while the pH, temperature, dissolved oxygen concentration, etc. are maintained under suitable conditions to suit the growth conditions of the microorganism to be cultured.

Such cultivation is usually carried out using a jar fermenter, and it goes without saying that appropriate scale-up is possible [see "Biochemical Engineering" (top),
(See Volume 2 (University of Tokyo Press)).

2) Culture medium The culture medium used in the first and second steps of the present invention is as described below.

First step: The purpose of this step is to allow microorganisms to grow efficiently without inducing protein synthesis ability. Therefore, in order to achieve this purpose in the method of the present invention, the basic medium is LB medium (tridine, yeast extract, NaCl), and other components are added as necessary. , and further prepared by adding inorganic phosphorus necessary for suppressing induction of protein synthesis ability. Inorganic phosphorus is usually added as a phosphate, and specifically, for example, there is a method in which a suitable amount of 2l-IPO4 is used in combination with KH2PO4 in consideration of the buffering effect.

As a specific example of such a medium, the present invention uses transformant E, coli K12YK537 (pTA
1522) (described below), glucose, tryptone, Ml extract and M (Iso 71-10) to which K+-12PO4 and 2HPO4 were added. In order to stably grow the host bacteria, ampicillin is added to this medium.This is because the plasmid transferred to the host bacteria in the member example of the present invention is equipped with an ampicillin-resistant gene, so the host is also resistant to ampicillin. and
Therefore, the growth of bacteria that shed plasmids during culture (which are sensitive to ampicillin) is inhibited by ampicillin in the medium, so only transformed bacteria that retain the plasmid can grow. This is because you will have to do so. Thus, the addition of antibiotics to the culture medium
This is a means for facilitating the growth of host bacteria. In addition, in the present invention, the amount of inorganic phosphorus added that can achieve the above object is 110 OR, l to 2 g/, 11
A range of possible ranges is possible. In one example of the present invention, inorganic phosphorus is added in an amount of 1.7 g/11.

Second step: This step involves inducing the action of the promoter of the plasmid transferred into the host microorganism (induction of protein synthesis ability), and culturing to maintain the growth of the microorganism grown in the first step. It is. Induction of the action of the plasmid promoter occurs under inorganic phosphorus-deficient conditions, since the promoter is derived from alkaline phosphatase.
Said Biohem, 31ophys, Acta
,, Nature).

Additionally, inorganic phosphorus is necessary to maintain the growth of microorganisms. Therefore, the specific method to satisfy the above conditions is to first inoculate the microorganisms grown in the first step into a medium (usually a synthetic medium) that does not contain inorganic phosphorus, and at this stage, the microorganisms grown in the first step are inoculated into a medium that does not contain inorganic phosphorus. is induced), followed by constant-rate addition of inorganic phosphorus or a medium containing a certain amount of inorganic phosphorus (in one embodiment of the present invention, a complex medium is used as such a medium). (This operation supplies inorganic phosphorus, which maintains the growth of the bacteria).

The medium that does not contain inorganic phosphorus in the second step is M-
, 9 medium, Davis medium, TG+20 medium, etc. (all synthetic media) can be used as a basic medium, and a composition obtained by removing inorganic phosphorus from this medium can be used. One specific example of the inorganic phosphorus-free medium of the present invention is glucose, Na Cl, KCI, N1-I C1
, Na2SO4.

MOCI, CaCl, FeCl3゜ZnC12
and Tris-HCl buffer, to which leucine and thiamin are added for nutritional requirements of host microorganisms, and ampicillin is added for the same purpose as in the first step.

Furthermore, in the present invention, supplementation of the essential inorganic phosphorus necessary for maintaining the transformed microorganism is carried out by constant-rate feeding of a medium containing inorganic phosphorus or a required amount of inorganic phosphorus. Such a medium includes a complex medium consisting of tryptone and yeast extract.

At this time, the constant flow acceleration of inorganic phosphorus is determined by the concentration of bacteria, the amount of culture solution,
It varies depending on the concentration of the onibi yeast egisu,
0D66o is 5 to 50. Culture solution volume 2β, tryptone 50g/, 0. In the case of yeast extract ψ 2511, a constant flow acceleration of between 0.8 me/min and 27/min is practical. In the present invention, it is set to 4 d/min or 8 d/min.

In the present invention, the term "synthetic medium" refers to a medium made of substances with chemically known compositions, and the term "complex medium" (natural medium) refers to natural substances, i.e. chemical compositions obtained from plants and animals. It means something consisting of something that is not clear.

3) Culture conditions (1) Culture temperature The culture temperature in the first step may be any temperature suitable for the growth of the microorganisms used. In the case of commonly used E. coli and E. coli used in the present invention, 37°C is suitable.

The culture temperature in the second step may be any temperature at which growth of the microorganism can be maintained (for example, 25° C. to 40° C.) and at which the product produced by the transformed microorganism is stable. Such a temperature is preferably 30° C. when producing hUG as in the present invention.

(2) Culture time In the first step, culture is usually carried out until the amount of bacterial cells reaches the maximum. In the case of the present invention, the amount of bacterial cells reached the maximum approximately 8.5 hours after inoculating the bacteria in the first step. In this step, it is convenient to determine the culture time by using 0D66o as an index of microbial growth and measuring it over time.

The culturing time in the second step may be determined using the amount of glucose, the number of viable 0D66o1 bacteria, and the amount of inorganic phosphorus in the medium over time, as well as the amount of production of the desired substance within the host microbial cells, as indicators. In one specific example, the preferred culture time is approximately 11 to 13 hours, at which time the amount of hUG produced is at its maximum. In addition, the glucose oxidase method was used to quantify glucose using the euglucostat ``Fujisawa'' kit. ), and the number of viable bacteria was determined using L-medium containing ampicillin (described above).
Inorganic phosphorus was quantified by counting colonies growing on agar according to the molybdenum blue method [industrial wastewater test method: JIS K 0102], and the product (hUG) was quantified by osmodic microorganisms. Shock method (J, Biol.

Chem, 240.3685 (1965)), the solution was centrifuged to obtain a supernatant, which was then subjected to the radioreceptor assay (RRA) method (J, Bi
ol', Chem, , 257.3053 (1
982)) (For details of hUG quantification, refer to the reference examples below and the specification of Japanese Patent Application No. 159703/1983)
This is done by doing this.

(3) Pl-1 of the culture medium 1) H may be within a range in which microorganisms can grow, and a preferable pl-1 may be appropriately set depending on the microorganism used. In the present invention, Escherichia coli is used, and the l)H at which Escherichia coli can grow is usually 4.6 to 8.8, with pl-(7.0 to 8.0 being particularly preferred).

(4) Antifoaming agent If foaming is significant during culturing, use an antifoaming agent (higher alcohol,
It is a common practice to add vegetable oil, etc.). Since the antifoaming agent used in the present invention controls the protein synthesis ability of microorganisms depending on the amount of inorganic phosphorus present, it is important that it does not contain inorganic phosphorus. A specific example of such an antifoaming agent is Antifoam-AF-Emulsion (Gyudon Kagaku).

(5) Dissolved Oxygen (DO> Dissolved oxygen refers to molecular oxygen dissolved in a liquid phase.

It is generally known that in aerated agitation culture, if Do is excessive, the growth of microorganisms is inhibited, while if Do is less than 1 ppm, growth is similarly inhibited. Therefore, it is preferable to control DOffl so that it does not become an inhibitory factor for microbial growth. In the case of a specific example of the present invention, a Dosicon roll device (Oriental Electric (■F
C-4 type] holds DOit at 41) 111II.

Experimental Example Reference Example 1 According to the following method, transformants F, C01i K1
2YK537 (pTA1522) was constructed.

pTA1529 (details of construction are in patent application No. 59-15970)
No. 3 + 7) 5 μg, 50μ
ρ buffer [10mM Tris-HCl solution (hereinafter referred to as T-r)
is -1-ICI) (+)l-17,5) 1.
10mMM! 4 in 7C12, 50mM NaCl)
Unit of restriction enzyme Hind11 [(Tano J) (below!
-1indll) at 37°C for 1 hour. Then, ethanol precipitation was performed, and the resulting precipitate was
Reaction solution (67mM Tris-1-IC
I.

(pH 8,8), 16.6mM ammonium sulfate (<N'H,)2SO4), 6.7mM ethylenediaminetetraacetic acid (EDTA), 0.66mM each d
1 unit of 7 in ATP, dCTP, dGTP, TTP]
The cells were treated with 4-DNA polymerase at 37°C for 15 minutes. Next, the precipitate obtained in J: was added to 50μp of reaction solution (6mM Tris-H) for ethanol precipitation.
Cl(1)H8,0), 6mM MoCI2,15
4 units of restriction enzyme 3alI (0mM NaCl)
Takara] (hereinafter referred to as 5alI) at 37° C. for 1 hour. After the reaction, a 3900 bp DNA fragment (indicated by
) 'tFJ1ko.

Plasmid 1) BR321-hU'G (pBR322
(E, coliK12c600 (ρ BR3
22>, deposited as <Feikoken Article No. 235>).
5 μq of the artificially synthesized hUG structural gene digested with EC0RI and 5al was combined with 50 μq of the reaction solution (100 mM Tris-l-IC'l (pH
7,5), 50mM NaCl.

4 units of restriction enzyme E in 50mM MgCI2)
After hydrolysis at 37°C for 1 hour using coRI (Takara), T4. A DNA fragment (■ in Figure 1) was obtained.

The two DNA fragments prepared above (■ and ■ in Figure 1)
), 30μρ reaction solution (20mM Tris-1-1c
I (1)I-17,5>, 10mM(l CI
, 10mM DTT, 0.5mM ATP).
Unit T, 14℃ using DNA ligase [Takara]
The reaction was carried out for 16 hours.

After the reaction is completed, E. coli is transformed with 12YK537, and the transformed strain CF, coli K, containing the target plasmid [hereinafter referred to as pTA1522] (■ in Figure 1) is obtained.
12YK537 (pTA1522)] was obtained.

Example 1 Transformant F, colt Kl 2YK537 (p
TA 15' 22 ) was cultured according to the following method.

E. coli K12YK537 (pTA1522)
complex medium (bird 11210g/41.yeast extract 5
g/fJ, K toI Po
2 g/J), K 2 HPO47
g/fJ, ampicillin 20~#) 50 containing 100d
28 - Slow culture was performed overnight at 37°C in 0.0 volume of Sakalocolben (preculture).

Next, 10 m of this culture solution was taken and mixed with a complex medium (glucose ICI/, 11. tryptone 10 g/, o, yeast extract 5 g/Jl, Mg80 7H200, !M/,
11 XK1-12PO42w/E, 2 HPO4
'7y/J, ampicillin 20my/fJ) 2'
, I) and then 3. The cells were cultured in a small aerated agitated culture device (mini jar fermentor) with a capacity of 1.5 liters. The culture temperature was 37°C, and the aeration rate was 0 and 5 VVm (l VV
III is 1 volume-volume-minute
1.0 per minute of culture solution. ll
), the pH is 4N
The concentration of dissolved oxygen (DO) was adjusted to 7.2 with Na 0f-1,4Nl-IC'l, and the concentration of dissolved oxygen (DO) was maintained at around 41)l1m by changing the stirring speed using a Do control device. Cultivation was carried out until the amount of bacterial cells reached the maximum (OD66o"-114) (approximately 8.5 hours after inoculating the bacteria) [First step]
. Next, half of this culture solution (1()) was taken out, and after collecting bacteria by centrifugation, the bacterial cells were collected in a solution of about 500mf! (NaC 14.68g/fJ, KCI 1.49q/
41, N1-14CI 3g#, Na2So4
0.43 g/ρ, Ma CI 20.2 g/ρ), and the bacterial cells obtained here were subjected to the next culture step (second step). Note that all of the above operations were performed aseptically.

In the second step, the bacteria grown in the first step are inoculated into a synthetic medium (2, fl) that does not contain inorganic phosphorus, and then this medium is added to a complex medium containing inorganic phosphorus (Top 1 to 50 g/fl).
fJ, yeast extract 25g/, Q) at a constant rate of 4me1 minute at 37°C, aeration rate 0, 5vvm,
Culture was carried out using a mini jar fermenter under the conditions of pl-l 7.2 and D O (/1.ppm). Here, the composition of the synthetic medium that does not contain inorganic phosphorus used in the second step is glucose 15ff/, 1! , NaCl4.6
89/, Q, KCl 1.49! ? /,Q,NHCl
3g/1Na2So40.43y/, 11. MaC
l O,2y/1cac1223mg/fl, Fe
Cl3325μg/, 0, Zn C12275μg
#, leucine 40my/β, thiamine 10my/l ampicillin 20my/D, and Tris-HCl buffer 0.1M. At that time, the culture time OD, number of viable bacteria, amount of inorganic phosphorus, amount of glucose, and amount of hUG over time were as shown in Table 1 and IC.

Table 1 *Number of cells resistant to ampicillin Glucose quantification is by glucose oxidase method (described above)
I followed. The number of viable bacteria is ampicillin 20m'J/
The number of colonies appearing on an agar plate of 1-1 medium containing fJ (Top 1 to 10 g/β, Yeast IL extract 5 g/0, NaCl 15 g/p) was counted for 8 days. 0D66o was measured as an indicator of the amount of bacterial cells, and inorganic phosphorus was determined using the molybdenum blue method. Quantification of hUG was performed by the following method. That is, 10 d of culture solution was taken, centrifuged to collect the bacteria, and the bacteria were mixed with 20% sucrose and 0.03M Tris-H.
The suspension was suspended in a solution 20d consisting of Cl(1)I-18,0) and 0.001M of ethylenediaminetetraacetic acid ([EDTA), and left at room temperature for 10 minutes. Then, the cells were collected by centrifugation again, and the cells were suspended in 20-degree cold water (0°C to 4°C) and left in water for 10 minutes. This suspension was then centrifuged to obtain a supernatant, which was analyzed by radioreceptor assay (RRA).

In the RRA method, first, Ni' B cells (ΔTCCNoCCL17) derived from human pharyngeal cavity carcinoma were cultured in a monolayer in Dulbecco's modified Eagle (DME) medium [Japan] in an 800 d flask. Then remove the medium and add 1.0.05% F.
Cells were detached using phosphate balanced salt solution (PBS) containing DTA to create a cell suspension. After that, 20m
Hank containing M l-1eperS (pH 7,4>
Cells were washed twice with balanced salt solution (HBSS). Cells were treated with 3inding solution (D M E
Medium: 20mM 1epes ('pi-17.4>
-0,3! M# NaHCO3・10'Oμg/d streptomycin], then calculate the number of cells and get 300,000 to 400,000 10.7 B inding so+ut+on
Adjust so that it becomes 0.2 In in the tube! ! , was dispensed in portions. hUG and 125.1-mEG of various 'aqua cucumbers
0.2 of a sample solution containing F (mouse epidermal growth factor (hereinafter referred to as mEGF)) was added to the tube and incubated at 37°C for 1 hour. I cells were ice-cold) - after washing three times with IBSS, 10% trichloroacetic acid C or less, TCA)
and fix the cells using a glass filter. After removing TCA with acetone, the cells were counted using a liquid scintillation counter. And mE hanging hUG
Converted to GF.

From the above results, under the conditions of Example 1, 13
．． Since the amount of hUG reached its maximum at 5 hours and then rapidly decreased, it seems that 13.5 hours is a good culture time.

Example 2 Example 1 except that the culture temperature in the second step was 30°C.
The experiment was conducted in the same way. 0D6 in the second process at that time
The changes over time in the 6o blank count, the amount of inorganic phosphorus, the amount of glucose, and the amount of hUG were as shown in Table 2.

*Number of ampicillin-resistant cells From these results, the hUG production amount reached a maximum of <9.22 m'J/β approximately 11 hours after the start of culture, and even if the culture was continued (up to approximately 19.5 hours). There was no significant change in the production amount of -muG and there was no rapid decrease in the production amount.Therefore, under the conditions as in Example 2, the culture time for the second step may be about 11 hours. Recognize.

Example 3 The amount of bacteria inoculated in the second step was increased (2f of the culture solution in the first step).
Inoculate all the bacterial cells obtained by collecting 1 liter of bacteria), and in the synthetic medium in the second step, increase the amount of glucose to 2! M/41, other components were the same as in the above example, and the same procedure as in Example 1 was performed except that the constant flow acceleration of the complex medium containing inorganic phosphorus was 8 d/min. The time-dependent changes in OD, number of viable bacteria, amount of inorganic phosphorus, amount of glucose, and hUGffi in the second step at that time were as shown in Table 3.

Table 3 *Number of cells resistant to ampicillin ** Could not be detected.

From this table, the culture time is hL under the conditions of Example 3.
It appears that 11 to 13 hours is preferable when the IG production reaches its peak.

Mycological properties and accession numbers of related microorganisms The mycological properties and accession numbers of the microorganisms disclosed in the present invention are as follows.

Contract date (1) June 9, 1981 (2) April 30, 1988 (3) June 9, 1981 (4) November 14, 1980 *Ministry of International Trade and Industry Industrial Technology Accession number of the National Institute of Microbiology and Technology Mycological properties (1) E. coliK 12C600 This bacterium is
It is a Durham-negative bacillus that does not produce spores and has the general attributes of E. coli, such as being facultatively anaerobic. It also does not contain the F factor, lacks the function of the Reaprezer gene E, and has the recBcl gene, which encodes a nuclease involved in genetic recombination. The child has a defect. As auxotrophs, threonine and leucine are required for growth on minimal media. Also,
Taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

The literature related to Honkan is as follows.

(a), Genetics, 39.440(o),
Nal 2 ure, 21 7. 1 1 10 (
2) E. coliKl 2C600 (pYK283) This bacterium is a Durham-negative bacillus, does not produce spores, has general attributes of E. coli such as facultative anaerobic properties, and does not contain F factor.
It lacks the function of the suppressor gene and has a defect in recBcm, which encodes a nuclease involved in genetic recombination.

As an auxotrophy, it requires 1 heleonine and leucine for growth on its minimal medium. It contains plasmid pYK283, which is composed of the replication initiation region derived from the promoter-operator region t*pAcYc177 of the phoA3ff gene and the bla gene of pBR322, and exhibits resistance to ampicillin.

Furthermore, taxonomically, it belongs to the family Enterobacteriaceae and the genus Escherichia coli.

In addition, except for plasmid 1) YK283-derived traits,
The mycological properties of this strain are its parent strain E, coli Kl 2
It is the same as that of C600.

(3) E, coli Kl 2C600 (pBR322)
This bacterium is a Durham-negative bacillus, does not produce spores, has the general attributes of the genus Escherichia coli, such as being facultatively anaerobic, and does not contain the E factor, lacks the function of the suppressor gene E, and does not produce nucleases involved in genetic recombination. It has a defect in the recBC gene it encodes. As an auxotroph, it requires threonine and leucine for growth on its minimal medium. It also contains drug resistance plasmid pBR322. For plasmid I) BR322, refer to Gene, 2°95 (1977), and for E. coli 12C600, refer to the above Nature.

Except for the traits derived from pBR322, E. coli (1
The mycological properties of 2C600 (pBR322) are the same as those of the parent strain.

(4) E. coliK 12YK537 Escherichia coli 1
2YK537 is a known strain of Escherichia coli with 12 strains (Hicrobiological Reviews
.

±4,1-56 (1980)) derivatives of E. coli with 12R
R1 (Gene, 2, 95. (1977), Bioch
em, Biophys, Acta, 655, 2
43 (1981)), and exhibits the following properties, and is a strain that is no different from that of 12RR1 in terms of the properties.

(rec A1, Pho A8, pro”)

[Brief explanation of the drawing]

FIG. 1 is a flowchart for constructing plasmid pTA1522 used to transform E. coli K12Y537. Procedural amendment written March W, 1986 1 Indication of the case 1988 Patent application No. 279586 2 Name of the invention Method for culturing microorganisms 3 Person making the amendment Relationship with the case Patent applicant Yusui Pharmaceutical Co., Ltd. 4
Agent 8 Amend the statement of contents of the amendment as follows. (1) Page 10, line 9, “said” was amended to read “said document.” (2) Delete “However” in the first line of page 11. (3) Page 15, lines 7-9 EcoRI l-1paI SmaI Hi
ndl[[J EcoRI HpaI SmaI 1
lindllI (I) Here, the broken line indicates the restriction enzyme cleavage site, and ECOR■ etc. indicate the name of the restriction enzyme that performs the cleavage. ” he corrected. (4) On page 19, line 7 from the bottom, "rLB medium" was corrected to "L medium." (5) Page 23, lines 4-5 ro, 8mi! / minute” was corrected to “o, 8rd/hour”. (6) Same page, line 5 r20mf! Correct "/minute" to "I'20m/hour". (7) Line 6 of the same page, change “4-7 minutes or 8 hours/minute” to [4d/hour or 8 meters
[time/time] and correction. (8) On page 31, line 10, "minute" was corrected to "hour".

Claims (1)

[Claims] 1. A microorganism transformed with a chimeric plasmid constructed by incorporating a gene encoding a foreign protein under the control of a plasmid containing a promoter derived from alkaline phosphatase is first transformed with a chimeric plasmid that has a promoter derived from alkaline phosphatase, and then transforms the microorganism with a chimeric plasmid that is constructed by incorporating a gene encoding a foreign protein into the plasmid. After culturing in a medium containing the necessary amount of inorganic phosphorus for the growth of the microorganism, the microorganism is then transferred to a medium containing no inorganic phosphorus, and further inorganic phosphorus or a medium containing inorganic phosphorus is added to this medium. A method for culturing microorganisms, characterized by subjecting them to culture with constant-rate feeding.