JP2004321120A - Genetic expression controlling process using co2 responding new promoter derived from marine algae - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find a CO<SB>2</SB>-responding new promoter derived from marine algae and construct a new genetic expression controlling system by using the promoter. <P>SOLUTION: The promoter region of an intracellular β-carbonic anhydrase gene of a marine diatom Phaeodactylum tricornutum has been identified and the new promoter has been found to exhibit CO<SB>2</SB>responding property. A vector having the objective gene connected to the downstream of the promoter is introduced into a host cell and the CO<SB>2</SB>concentration in the cell growing environment is controlled to induce the expression of the objective gene. The genetic expression controlling system is usable for the production of valuable proteins such as enzymes and antibodies. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【図面の簡単な説明】
【図１】本発明の実施例で使用した形質転換ベクターの構造を示す図である。
【図２】本発明の実施例で作製したプロモーター−レポーター遺伝子コンストラクトを示す図である。
【図３】ＧＵＳ活性の測定結果を示すグラフである。
【図４】本発明の実施例で作製したプロモーターの塩基配列を示す図である。
【図５】ＧＦＰ遺伝子の発現誘導を調べた実験結果を示す図であり、Ａは透過光イメージ、Ｂは葉緑体蛍光、ＣはＧＦＰ蛍光イメージ、ＤはＢとＣのイメージを重ね合わせたものである。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to CO2 derived from marine algae. ₂ The present invention relates to a method for controlling gene expression using a responsive novel promoter. The present invention particularly provides a technique that can be used for producing useful proteins having various industrial uses such as pharmaceuticals, foods, and test instruments.
[0002]
[Prior art]
Among marine algae, diatoms and cobblestones are unicellular eukaryotes belonging to a group called so-called yellow plants. These algae are the world's CO ₂ It is estimated that it takes about 20-40% of the fixed amount, ₂ Has been shown to be a crucial group of organisms in the global environment. In addition, these algae are called secondary symbiosis, they belong to a special group of organisms created as a result of multiple symbiotic phenomena, and it is thought that they may have a special molecular mechanism in gene expression. It remains as a research field for the future. In addition, diatoms are organisms that synthesize a large amount of oils and fats useful for humans, such as EPA (eicosapentaenoic acid).
[0003]
A part of the genomic structure of a recently reported diatom, Phaeodactyllum tricornutum, indicates that many of the nuclear genes of this group of organisms are homologs of human and other mammalian genes, and that diatoms Has strong potential as a plant for the expression of mammalian genes. Diatoms, which are originally autotrophs, do not require organic nutrients for their growth. Since marine algae grows as a mineral source using almost inexhaustible seawater on the earth without using freshwater resources, if it can function as a food and substance production factory, construct a very inexpensive production line It is considered possible.
[0004]
By the way, it is known that algae has a function of promoting or suppressing gene expression in response to a change in environmental carbon dioxide concentration. However, although studies on the molecular mechanism have been made on freshwater algae such as prokaryote cyanobacteria and eukaryotic green algae Chlamydomonas, research on marine eukaryotes has not been advanced. For example, as a research result of freshwater algae, Non-Patent Document 1 listed below discloses CO identified in Chlamydomonas, a freshwater single-celled green alga. ₂ A responsive promoter region has been described. This promoter region is relatively long, ₂ In addition, it has the property of responding to light.
[0005]
[Non-patent document 1]
Ken-ichi Kucho, Kanji Ohyama, and Hideya Fukuzawa, Plant Physiology, December 1999, Vol. 121, 1329-1337
[0006]
[Problems to be solved by the invention]
As described above, marine algae grows using seawater as a mineral source, so that if they can function as a substance production plant, a very inexpensive production line can be constructed. Among them, marine diatoms have strong potential as a plant for expressing mammalian genes because of their genome structure. Furthermore, when marine algae is used as a gene expression plant, the gene expression ₂ If a control system can be constructed by using the following, there are the following advantages (1) to (4).
(1) CO ₂ By adjusting the concentration of, the growth phase of the cells can be clearly separated into a period that is exclusively dedicated to division and a period in which the substance is mainly produced.
(2) CO ₂ Can be obtained inexpensively as exhaust gas from power plants and factories.
(3) CO ₂ Is a greenhouse gas, so its use also contributes to environmental issues.
(4) A substrate induction method such as IPTG or a temperature control method has been established as a method for controlling gene expression, but rapid reversibility cannot be expected in the substrate control method. It is thought that it takes. On the other hand, CO ₂ Is a gas molecule, so if this is used as a switching factor, ₂ Can be used quickly by cells and easily removed by normal atmospheric aeration. Therefore, a system having extremely high reversibility in the fission-production phase as described in the above (1) can be constructed.
[0007]
The present invention has been made in view of the above problems, and has as its object to reduce CO ₂ It is an object of the present invention to find a novel responsive promoter and to construct a new gene expression control system using the same.
[0008]
[Means for Solving the Problems]
The present inventors have conducted intensive studies in view of the above problems, and as a result, have identified the promoter region of the intracellular β-type carbonic anhydrase gene of the marine diatom Phaeodactylum tricornutum, and have identified this promoter as CO ₂ The present inventors have found that the present invention exhibits responsiveness, and have completed the present invention.
[0009]
That is, the present invention includes the following inventions A) to N) as industrially useful methods and products.
A) By introducing a promoter sequence of a marine algae-derived carbonic anhydrase gene and a foreign gene subjected to transcriptional regulation into a host cell, the concentration of carbon dioxide or the partial pressure of carbon dioxide in the cell growth environment is regulated. A gene expression control method for inducing the expression of a foreign gene.
B) The method for controlling gene expression according to A) above, wherein a promoter sequence derived from a marine diatom, Phaeodactylum tricornutum, is used as the promoter sequence.
C) The method for controlling gene expression according to B), wherein a DNA sequence containing any one of the following base sequences (1) to (5) is used as the promoter sequence.
{Circle around (1)} In the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence from the 1178th position to the 1353th position
{Circle around (2)} The base sequence from the 1068th position to the 1353th position in the same base sequence
{Circle around (3)} The nucleotide sequence from the 809th to the 1353th nucleotide in the same nucleotide sequence
{Circle around (4)} In the same nucleotide sequence, the nucleotide sequence from position 469 to position 1353
(5) The base sequence shown in SEQ ID NO: 1
D) The method for controlling gene expression according to any one of the above A) to C), wherein a cell derived from a marine algae is used as a host cell.
E) The above-described A) to D), wherein the host cells are aerated and cultured with a gas having a carbon dioxide concentration of 1% or more, and then the expression of a foreign gene is induced by switching the carbon dioxide concentration in the environment to less than 1%. The method for controlling gene expression according to any one of the above.
F) A useful protein comprising a step of inducing expression of a foreign gene using the gene expression control method according to any one of the above A) to E), and then collecting or purifying a protein as a translation product thereof. Production method.
G) A promoter of a carbonic anhydrase gene derived from a marine diatom, Phaeodactyllum tricornutum.
H) The promoter according to the above G), which is a DNA sequence containing any one of the following base sequences (1) to (5).
{Circle around (1)} In the nucleotide sequence shown in SEQ ID NO: 1, the nucleotide sequence from the 1178th position to the 1353th position
{Circle around (2)} The base sequence from the 1068th position to the 1353th position in the same base sequence
{Circle around (3)} The nucleotide sequence from the 809th to the 1353th nucleotide in the same nucleotide sequence
{Circle around (4)} In the same nucleotide sequence, the nucleotide sequence from position 469 to position 1353
(5) The base sequence shown in SEQ ID NO: 1
I) A gene expression vector in which the promoter described in G) or H) above and a gene whose transcription is regulated are integrated.
J) A transformed cell into which the gene expression vector according to I) has been introduced.
K) The transformed cell according to J) above, which is a cell derived from a marine algae.
L) A useful protein production system comprising a growth means for culturing or growing the transformed cell according to J) or K), and a control means for controlling the concentration of carbon dioxide in the growth environment.
M) A useful protein such as an antibody or an enzyme produced by the useful protein production method described in F) or the useful protein production system described in L).
N) The useful protein according to M) above, which is a protein of mammalian type or derived from mammals.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, specific embodiments, technical ranges, and the like of the present invention will be described in detail.
[0011]
(1) The promoter of the present invention
First, as a promoter sequence that can be used in the gene expression control method of the present invention, a promoter sequence derived from a kind of marine diatom, Pheodactylum tricornutum (hereinafter, referred to as “P. tricornutum”) can be exemplified. This promoter sequence has been identified by the present inventors, as shown in the Examples below. That is, P.I. In order to identify the promoter region of the intracellular β-type carbonic anhydrase gene of Tricornutum, the present inventors have proposed five types of promoter candidates consisting of an upstream region of the transcription start site of the gene and a 5′-UTR coding region following the region. Sites were prepared. Then, constructs were prepared in which a reporter gene was arranged downstream of these candidate sites, and each promoter activity was examined. ₂ Highly responsive promoter activity was observed (see FIG. 3).
[0012]
As shown in FIG. 2, the above five types of promoters have different lengths, but have a common DNA sequence from -115 to +61. In addition, these numbers indicate the position of each nucleotide by using the transcription start site as a base point “+1”, the upstream direction as “−”, and the downstream direction as “+”. The shortest promoter is 176 nucleotides in length from -115 to +61, which is shown second from the bottom in the figure, and the remaining four promoters are -225 to +61, respectively, in order of decreasing length. It consists of 286 nucleotides, 545 nucleotides from -484 to +61, 885 nucleotides from -824 to +61, and 1353 nucleotides from -1292 to +61.
[0013]
SEQ ID No. 1 in the sequence listing shows the longest promoter sequence, that is, the nucleotide sequence of 1353 nucleotides from -1292 to +61. In the same nucleotide sequence, the sequence at positions 1178 to 1353 corresponds to the shortest promoter sequence. Hereinafter, the sequence at positions 1068 to 1353 is the second shortest promoter sequence, and the sequence at positions 809 to 1353 is the third. The sequences from the 469th position to the 1353th position correspond to the shortest promoter sequence, respectively. Each of the five types of promoters was obtained as a double-stranded DNA consisting of these base sequences and their complementary sequences.
[0014]
FIG. 4 shows the promoter sequence of the shortest promoter. In the figure, the region indicated by uppercase letters corresponds to the upstream region of the transcription start site, and the region indicated by lowercase letters corresponds to the 5'-UTR region. As mentioned above, CO ₂ Since the five types of promoters in which the responsive promoter activity was recognized have the base sequence of 176 nucleotides in common, this sequence region is considered to be an essential region for the promoter activity.
[0015]
As described above, the above five types of promoters can be used in the gene expression control method of the present invention, but the promoter of the present invention is not limited to these five types. For example, the length of the promoter is not limited to these five types, but may be longer. If the region essential for promoter activity can be further narrowed down to 176 nucleotides, a shorter promoter sequence can be used.
[0016]
In addition, one or more bases that are non-essential to the promoter activity in the base sequences of the above five types of promoters are modified by a known genetic engineering technique (ie, substitution, deletion, insertion, or addition of bases). Is similar to CO ₂ It is thought to show responsive promoter activity. Such a modified promoter sequence is also included in the promoter of the present invention.
[0017]
The above five types of promoters are It was obtained by the present inventors from the promoter region of the intracellular β-type carbonic anhydrase gene of tricornutum, but has the same activity from the promoter region of the carbonic anhydrase gene of other marine diatoms. It is highly likely that a promoter will be obtained. Furthermore, there is a high possibility that a promoter having the same activity can be obtained from the promoter region of the same gene of marine algae, particularly a marine algae belonging to a phylum, which is closely related to diatoms, or a phylum belonging to a haptophyte. Such a promoter may be used in the gene expression control method of the present invention.
[0018]
Although the above five types of promoters were double-stranded DNAs comprising the above-described base sequences and their complementary sequences, the promoter of the present invention ₂ It may be single-stranded or RNA as long as it exhibits a responsive promoter activity.
[0019]
As a method for controlling the expression of a target gene (foreign gene) using the promoter of the present invention described above, a method for preparing a transformation vector (gene expression vector) and introducing it into a host cell is simple. Hereinafter, this method will be briefly described.
[0020]
(2) Preparation of gene expression vector and introduction into host cells
In order for the target gene to be regulated by the promoter of the present invention, transcription of the target gene downstream of the promoter of the present invention is inserted into a multicloning site of an expression vector, and the expression vector is transferred to a host cell. It can be introduced inside.
[0021]
Since there are various expression vectors such as plasmids, phages, and cosmids, an appropriate vector may be selected according to the host cell to be used, the size of the target gene, and the like. In the examples described later, a plasmid (pPtGus vector) having the structure shown in FIG. 1 was used, and various promoter-reporter gene constructs shown in FIG. 2 were ligated to the NdeI and EcoRI sites to prepare a vector.
[0022]
The host cell is not particularly limited, and various plant-derived cells, animal-derived cells, mammal-derived cells, and the like may be used in addition to algae-derived cells. In addition, any cells such as somatic cells, germ cells, stem cells, tumor cells, and hybridomas may be used. However, in order to construct an inexpensive production system, it is preferable to use cells of marine algae (or their origin) that are autotrophic organisms and can basically grow only with light and seawater. Furthermore, among marine algae, yellow plants such as diatoms have similarities in genome structure to mammals, and thus are considered to be suitable for production of mammalian types and proteins derived from mammals.
[0023]
In the examples described below, the marine diatoms P. Tricornutum UTEX642 strain was used.
Various methods for introducing a target gene into a host cell include a molecular gun method (particle gun method), a calcium phosphate method, a lipofection method, a microinjection method, a protoplast fusion method, a DEAE dextran method, a virus method, and an electroporation method. Since a gene transfer method is known, an appropriate method may be selected from known gene transfer methods.
[0024]
After the host cell is transformed in this manner, the method for controlling gene expression of the present invention provides a method for controlling CO in the growth environment of the obtained transformed cell. ₂ Concentration (in other words, carbon dioxide partial pressure pCO ₂ ) To induce the expression of the gene of interest. The target gene is CO ₂ Because of transcriptional regulation by a responsive promoter, CO ₂ By changing the concentration, promotion / suppression of gene expression can be easily switched. Hereinafter, a useful protein production system using this gene expression control system will be described.
[0025]
(3) Useful protein production system
For example, when a cell derived from a marine algae is used as a host cell and transformed therewith, the transformed cell can basically grow (culture) only with light and seawater. CO in this growing environment ₂ To adjust the concentration, for example, high concentrations of CO ₂ After a certain period of time, the supply of gas is stopped, or the supplied gas is changed to low-concentration CO. ₂ Or CO ₂ May be switched to one that does not include. The promoter of the present invention has a high CO concentration of about 5%. ₂ Under the conditions, while suppressing gene expression, low concentration of CO ₂ Under the conditions, it promotes gene expression (see FIG. 3). Therefore, the transformed cells should have at least ₂ After aeration culture with gas with a concentration of 1% or more, CO in the growth environment ₂ By switching the concentration to less than 1%, the expression of the target gene can be induced. For example, during the cell growth period, the concentration of CO ₂ The concentration is set and maintained at 1% or more (preferably 2% or more, more preferably 3% or more and 10% or less). ₂ The concentration is switched to less than 1% (preferably 0.5% or less, more preferably about 0.030% to 0.040%, which usually corresponds to the vicinity of atmospheric partial pressure). Thereby, gene expression can be induced and useful substances can be efficiently produced.
[0026]
As described above, the useful protein production system of the present invention comprises a growing means for growing transformed cells in culture, ₂ What is necessary is just to have the adjustment means which adjusts density. The adjusting means can be easily realized by using a known ventilation system having functions of supplying gas, stopping supply of gas, switching of supplied gas, and removing supplied gas.
[0027]
For example, when culturing transformed cells on a cell culture plate usually used in a laboratory or the like, CO ₂ CO as concentration control means ₂ An incubator or the like may be used. When animal cells or the like are used as host cells, amino acids, vitamins, glucose, salts, serum, and the like may be added to the medium as needed.
[0028]
When marine algal cells are used, they can basically grow only with light and seawater. Seawater may be used by filtering artificial seawater or natural seawater as necessary. The light may be natural light or artificial light such as a fluorescent light. For example, when cultivating and growing marine algal cells by placing seawater in a large-capacity culture tank, CO ₂ It is preferable to design the density adjusting means so that the density can be controlled uniformly. In addition, in order to achieve uniform light irradiation, a light-transmitting material (light-transmitting plastic or the like) is preferably used for the tank container. Further, a stirring means or the like may be provided inside the tank as needed.
[0029]
After the expression of the target gene is induced by the useful protein production system, the protein as a translation product is recovered and purified by a known recovery and purification method. The protein may be expressed in an epitope-tagged manner to facilitate purification, or may be produced using the system of the present invention to produce a protein that has been artificially modified or improved, such as a fusion protein or a mutant protein. Good.
[0030]
As described above, the production system of the present invention can be used to produce various antibodies used in antibody drugs and the like, or useful proteins such as enzymes used in test drugs and the like at low cost. When the present system is used to produce a mammalian type or a protein derived from a mammal, it is preferable to use mammalian type cells as host cells, or to use yellow plant cells such as diatoms. As described above, marine diatoms and the like have a genome structure similar to that of mammals, and are considered to be suitable for production of mammalian types and proteins derived from mammals.
[0031]
In Examples described later, GUS (β-glucuronidase) gene, which is a hydrolase, was used as a reporter gene, and the expression of the enzyme was actually confirmed by the method of the present invention. Therefore, the present invention can be used for producing useful enzymes. One example is utilization of the drug metabolizing enzyme P450 for production. P450 is an enzyme used for safety evaluation of drugs, toxicity evaluation of toxic substances, etc. However, various human P450 molecular species are known, and the production system is not yet established by the existing method using E. coli etc. Some types have not been established. Similarly, there are many useful proteins (for example, antibody proteins) that cannot be appropriately modified with sugar chains when expressed in bacteria or yeast. In addition, by combining human hormone receptors and reporter genes, it is possible to develop a system that can be used for monitoring endocrine disrupting substances that are seriously contaminated in coastal marine areas. The present invention can be used for production of such types of enzymes and proteins.
[0032]
It is also possible to produce secondary useful substances using the system of the present invention. For example, diatoms synthesize EPA (eicosapentaenoic acid), and the expression of enzymes involved in this synthesis pathway is induced by the method of the present invention to obtain a transformant that produces EPA, a useful substance, in large quantities. is there. Such a marine diatom transformant is also included in the present invention.
[0033]
【Example】
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0034]
[Example 1: CO ₂ GUS gene expression induction by concentration control]
[A] Cells and growth method
The host cells include marine diatom P. Tricornutum UTEX642 strain was used and grown in artificial seawater adjusted to pH 7-8. 5% or 0.034% CO in culture ₂ And the light intensity is about 100 μE m ^-2 And
[0035]
[B] P. transformation of tricornutum
a) Preparation of cells: Cells were grown in artificial seawater from the early to mid-logarithmic growth phase to about 5 × 10 ⁷ The cells were collected by centrifugation, suspended in 200 μL of artificial seawater, and then spread on an agar medium so as to have a diameter of about 3 cm. Thereafter, the surface was dried and left for about one hour.
b) Preparation of transformation vector: CO ₂ P. as a responsive promoter candidate About 1.3 kbp of the promoter site of the intracellular β-type carbonic anhydrase gene (PtCA) of tricornutum was amplified by PCR and ligated to the NdeI and EcoRI sites of the transformation vector shown in FIG. As shown in FIG. 2, five types of promoter regions, ie, -1292 to +61, -824 to +61, -484 to +61, -225 to +61, and -115 to +61, were prepared, and a construct having no promoter region was used as a negative control. Was prepared. These constructs were passed on to the plasmid.
c) Coating of DNA on tungsten particles: The gene transfer in this experiment adopted the molecular gun method and followed the protocol of Bio-Rad Biolistic ™ PDS-1000 / He Particle Delivery System. The conditions for bombarding the cells were set as follows.
Target distance: 6 cm
Chamber vacuum: 27 "Hg
Helium pressure: 1550 psi
d) Selection of transformant: The cells after the bombardment were suspended in 600 μL of artificial seawater, and 300 μL of each was spread on an artificial seawater agar medium containing 100 μg / mL Zeocin (trademark) for 3 to 4 weeks under normal light and normal atmosphere. After culturing, Zeocin resistant clones were selected.
[0036]
[C] Measurement of GUS (β-glucuronidase) activity
Transformants were transformed with 5% and 0.034% CO ₂ Aeration culture was performed at a concentration of about mid-logarithmic growth phase, and 10 mL was centrifuged to collect alga. The suspension was suspended in 1 mL of extraction buffer (50 mM NaPO4, pH 7.0, 10 mM beta-Mercaptoethanol, 0.1% Sodium Lauryl Sarcosine, 0.1% Triton X-100), and crushed for 1 minute with an ultrasonic crusher. . 20 μL of the above cell extract was added to 980 μL of Assay buffer (extracted buffer + 10 mM p-nitrophenyl β-D-gluconide), and reacted at 37 ° C., and the total protein amount was 1 mg, and the amount of PNP produced per minute was GUS activity. Calculated.
[0037]
FIG. 3 shows the experimental results. As shown in the figure, high promoters were obtained for all promoter regions of each length tested. ₂ During growth, GUS activity was almost completely suppressed (open bars in the figure), and 0.034% CO ₂ It was shown that GUS activity was induced under the environment (black bars in the figure). In the graph, “1” and “2” are the results of experiments using cells obtained from different colonies after transformation, and in each case, CO ₂ Highly responsive promoter activity was observed.
[0038]
From the above experimental results, CO ₂ It was found that the sequence from -115 to +61 (see FIG. 4) has a sufficient function as a promoter region that responds to. This sequence is the previously known CO of the carbonic anhydrase gene in the freshwater green alga Chlamydomonas. ₂ It was found to be much shorter than the response region and to have no clear homology sequence at this time.
[0039]
[Example 2: CO ₂ Induction of GFP gene expression by concentration control]
Next, the same experiment was performed using the GFP gene instead of the GUS gene, and the induction of GFP gene expression was examined. The GFP gene used was modified in consideration of human codon bias called egfp. As shown in FIG. 5, it was shown that the transformant expressing the construct in which egpt was inherited in the ptCA promoter region was specifically expressed in a site without chloroplast fluorescence. This indicated that GFP was expressed with a function in the cytoplasm. This further suggests that the marine diatom P. cerevisiae may serve as an expression host for genes with human-type codon bias. This indicates that tricornutum is suitable, and the genomic structure of this cell was consistent with the result that the homology was high with humans. It is also thought that this also suggests that a sequence such as the ptCA promoter may function in mammalian type cells other than marine diatoms.
[0040]
【The invention's effect】
As described above, the present invention relates to CO ₂ The present invention relates to a method for controlling gene expression using a responsive novel promoter, and as described above, it can be used for producing useful proteins such as enzymes and antibodies, and has various other usefulness.
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing the structure of a transformation vector used in Examples of the present invention.
FIG. 2 is a diagram showing a promoter-reporter gene construct prepared in an example of the present invention.
FIG. 3 is a graph showing the results of measuring GUS activity.
FIG. 4 is a diagram showing a base sequence of a promoter prepared in an example of the present invention.
FIG. 5 is a view showing the results of an experiment for examining the induction of GFP gene expression, where A is a transmitted light image, B is chloroplast fluorescence, C is a GFP fluorescence image, and D is a superimposition of B and C images. Things.

Claims (14)

After introducing a promoter sequence of a marine algae-derived carbonic anhydrase gene and a foreign gene that is transcriptionally regulated into a host cell, the foreign gene is regulated by controlling the carbon dioxide concentration or carbon dioxide partial pressure in the cell growth environment. A method for controlling gene expression that induces the expression of a gene. The gene expression control method according to claim 1, wherein a promoter sequence derived from a marine diatom, Phaeodactyllum tricornutum, is used as the promoter sequence. 3. The method according to claim 2, wherein a DNA sequence containing any one of the following base sequences (1) to (5) is used as the promoter sequence.
{Circle around (1)} The nucleotide sequence from the 1178th to the 1353th nucleotide in the nucleotide sequence shown in SEQ ID NO: 1 (2) The nucleotide sequence from the 1068th to the 1353th nucleotide in the nucleotide sequence , The nucleotide sequence from the 809th to the 1353th; (4) the nucleotide sequence from the 469th to the 1353th in the same nucleotide sequence; (5) the nucleotide sequence shown in SEQ ID NO: 1 The method for controlling gene expression according to any one of claims 1 to 3, wherein a cell derived from marine algae is used as the host cell. The expression of a foreign gene is induced by switching the concentration of carbon dioxide in the environment to less than 1% after aeration culture of host cells with a gas having a carbon dioxide concentration of 1% or more. 2. The method for controlling gene expression according to claim 1. A method for producing a useful protein, comprising the step of inducing expression of a foreign gene using the method for controlling gene expression according to any one of claims 1 to 5, and then collecting or purifying a protein as a translation product thereof. Method. A promoter of a carbonic anhydrase gene derived from Phaeodactyllum tricornutum, a kind of marine diatom. The promoter according to claim 7, which is a DNA sequence containing any one of the following base sequences (1) to (5).
{Circle around (1)} The nucleotide sequence from the 1178th to the 1353th nucleotide in the nucleotide sequence shown in SEQ ID NO: 1 (2) The nucleotide sequence from the 1068th to the 1353th nucleotide in the nucleotide sequence , The nucleotide sequence from the 809th to the 1353th; (4) the nucleotide sequence from the 469th to the 1353th in the same nucleotide sequence; (5) the nucleotide sequence shown in SEQ ID NO: 1 A gene expression vector into which the promoter according to claim 7 or 8 and a gene whose transcription is regulated are integrated. A transformed cell into which the gene expression vector according to claim 9 has been introduced. The transformed cell according to claim 10, which is a cell derived from a marine algae. A useful protein production system, comprising: a growth means for culturing or growing the transformed cell according to claim 10 or 11; and a control means for adjusting a carbon dioxide concentration in a growth environment. A useful protein such as an antibody or an enzyme produced by the method for producing a useful protein according to claim 6, or the useful protein production system according to claim 12. 14. The useful protein according to claim 13, which is a protein of a mammalian type or derived from a mammal.

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