JPH07203949A - Production of genetic product of escherichia coli using blue-green alga - Google Patents

Abstract

PURPOSE:To obtain a new transformant from a blue-green alga capable of extracellularly releasing a genetic product which is a useful substance and readily separating and purifying the genetic product due to a culture medium which is simple inorganic salts. CONSTITUTION:This method for producing a genetic product of Escherichia coli is to insert a signal sequence (or a partial sequence capable of coding the signal sequence and several amino acids following thereto) in a gene of a beta-lactamase, an alkaline phosphatase, a maltose-binding protein or an arabinose-binding protein which is present in the periplasm of the Escherichia coli into the N-terminal of a structural gene of the objective product, prepare a vector for recombining a blue-green alga, transduce the resultant vector into the blue-green alga, afford a transformant capable of the performing the photosynthesis and readily carrying out the mass culture, grow the resultant transformant in a usual culture medium, transfer the grown transformant to a hypotonic liquid and thereby release the genetic product.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a gene product from cyanobacteria by releasing it to the outside of the cell by gene recombination, and a transformant of cyanobacteria.

[0002]

2. Description of the Related Art Production of useful substances such as growth hormone and insulin by gene recombination using Escherichia coli, Bacillus subtilis, etc. as a host has become widespread. In order to release these gene products extracellularly, so-called signal sequences of secretory proteins, proteins existing in the outer membrane and periplasm have been used.

However, in such a heterotrophic microorganism, since the medium composition is complicated such as bactotryptone and yeast extract, even if the gene product can be released to the outside of the cell, even if these genes are released. The process of separating and purifying the product could not be sufficiently simplified.

Further, biosensors using immobilized microorganisms are expected as a major application field of genetically modified microorganisms, but it is also said that the complicated medium composition destabilizes the enzyme reaction which is the key of the sensor. It was an obstacle.

[0005]

An object of the present invention is to provide a method for producing a gene product which can be easily separated and purified, and a transformant which produces the gene product.

[0006]

Means for Solving the Problems The present inventor has found that cyanobacteria are autotrophic organisms that carry out photosynthesis similar to that of higher plants.
Since large-scale culture is also easy, I am interested in producing useful substances using cyanobacteria and have been developing gene recombination technology from the past.

[0007] In such studies, when cyanobacteria were used as a host, the medium consisted of simple inorganic salts, so that the gene product could be easily separated and purified, and the inhibition of the enzymatic reaction was less likely to occur. I focused on something.

[0008] As a result of intensive studies on a method of releasing the gene product outside the cell by external manipulation, it was found that the gene product is released when osmotic shock is given,
The present invention has been completed.

The present invention relates to a protein existing in the periplasm of Escherichia coli , β-lactamase, alkaline phosphatase, maltose binding protein,
Alternatively, a cyanobacterial recombination vector in which the signal sequence of the gene for the arabinose-binding protein, or a partial sequence encoding the signal sequence and several amino acids following it is inserted at the N-terminus of the structural gene of the desired product, is introduced into the cyanobacteria. It is the transformed transformant.

Further, the present invention provides a signal sequence of a gene of β-lactamase, alkaline phosphatase, maltose binding protein, or arabinose binding protein, which is a protein existing in the periplasm of Escherichia coli , or a signal sequence followed by several amino acids. A transformant in which a cyanobacterial recombination vector, in which a partial sequence coding for the above is inserted into the N-terminal of the structural gene of the desired product, is introduced into cyanobacteria, is grown in a normal medium, and then transferred to a hypotonic solution. A method for producing a characteristic gene product.

The present invention will be described in detail below.

[0012] In the present invention, the periplasm means the space between the outer membrane-peptidoglycan layer on the cell surface and the cytoplasmic membrane of Gram-negative bacteria such as Escherichia coli,
It is also present in the same Gram-negative bacterium, Cyanobacteria, at 20-40% of the total cell volume.

Β-lactamase, alkaline phosphatase, maltose-binding protein, and arabinose-binding protein are present in the periplasm of Escherichia coli, but these proteins are secreted out of the cytoplasmic membrane when secreted into the periplasm after synthesis. It is characterized by having a signal peptide required to be excised, and this signal peptide is excised when secreted out of the cytoplasmic membrane. The present invention also utilizes this signal peptide to enable the secretion of the desired product into the periplasm.

In the present invention, the target product is any protein or polypeptide obtained by introducing a gene, and specific examples thereof include insulin, interferon (α, β, γ), growth hormone, protease (trypsin, Chymotrypsin), chymosin, somatostatin, β-endorphin, invertase and the like.

As the target product, a protein (antibiotic resistance gene) that can also be used as a marker such as β-lactamase can be used.

Next, the vector required for releasing the target product extracellularly will be described. 1. The host cyanobacterial vectors are those derived from Synechococcus and Synechocystis that are available from the American Type and Culture Collection.
Culture Collection) and gene banks. Examples of such vectors include pUC303 and pUC303.
DF30, pSG111, pPUC29 and the like can be mentioned. 2. A DNA sequence (referred to as a signal sequence) encoding an amino acid sequence shown in Table 1 below was inserted into the obtained vector downstream of the transcription initiation point and upstream of the translation initiation. Connect so that the codon frames match. At this time, there may be an intervening sequence bridging between the signal sequence and the structural gene. Table 1. Signal Sequence Alkaline Phosphatase Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala Arg β-Lactamase Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Maltose Binding Protein Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Arabinose Binding Protein Met Lys Thr Lys Leu Val Leu Gly Ala Val Ile Leu Thr Ala Gly Leu Ser Gly Ala Ala Glu If there is no cyanobacterial vector as a host, E. col
The above was prepared using the plasmid vector of i.i., the endogenous plasmid of the cyanobacteria was taken out, and E. E. coli plasmid vector may be ligated or made linear, and then both ends of which are ligated with random fragments of host nuclear DNA. 3. A transcription termination sequence is placed downstream of the structural gene. 4. Usually, in addition to the gene of interest, an antibiotic resistance gene such as chloramphenicol acetyltransferase is placed on the same vector in order to select transformants. 5. As the promoter and transcription termination sequence, those of the vector for E. coli can be used.

In summary, the construction of the vector is as shown in the figure below.

[0018]

[Table 1] Note 1) The promoter contains the following transcription start site. Note 2) The intervening sequence need not be included. Note 3) The transcription termination sequence does not matter as long as it is downstream of the structural gene of the target protein. Note 4) Independently of this, an antibiotic resistance gene such as chloramphenicol acetyltransferase can be inserted.

In the present invention, cyanobacteria used as hosts include Synechocystis spp., Synechococcus spp.
The genus Nostoc, the genus Spirulina and the genus Anabaena are mentioned, and particularly preferable ones are the genera Synechocystis and Synechococcus.

Next, the DNA prepared as described above is introduced into a host as follows for transformation.

The method of introducing DNA in the present invention is as follows:
Although there are various methods such as a natural uptake method, a liposome method, and a particle gun method, the electroporation method is the most general and the transformation efficiency is high, and thus this method is preferably used.

In the case of performing the above electroporation, when the host cyanobacteria are single cells such as Synechocystis and Synechococcus, 1 mM HEPES (N-2-hydrox) is used as it is.
yethylpeperazine-N'-2-ethanesulfonic acid, pH 7.2)
It is suspended in a solution, mixed with vector DNA, and subjected to several 5-10 KV / cm electric pulses between electrodes while cooling with ice.

On the other hand, in the case of filamentous cyanobacteria such as Nostock, Spirulina and Anabena, electroporation is performed in the same manner as described above after the filaments are separated by ultrasonic waves.

The transformant thus obtained is seeded on an agar plate containing an antibiotic such as chloramphenicol acetyltransferase to isolate the transformant.

Next, the method for releasing the gene product from the transformant thus obtained to the outside of the cell will be explained.

The cyanobacterial transformant obtained above is cultivated in 0.1 to 10 liters of culture solution per 1 g of the cyanobacterial transformant at 20 to 40 ° C. for 24 to 120 hours. Then, the cells are precipitated by centrifugation and the supernatant is removed.

The composition of the above-mentioned culture medium which is usually used is such that when NO ₃ ⁻ is used as the N source, NO ₃ ⁻ is added to 2
0 to 3000 mg / L, PO ₄ ^3- to 5 to 1000 mg / L,
K ^{+ 5} to 1500 mg / L, Mg ^{2+ 2} to 1000 mg / L
L, Ca ²⁺ 5-500 mg / L, SO ₄ ^2- 5-250
It contains about 0 mg / L, and Fe, M as trace elements
n, B, Cu, Zn, Cl, Mo, and optionally Co, Ni, EDTA, vitamins and the like. For marine cyanobacteria, NaCl is added up to about 50 g / L as needed. As typical examples of such a medium, BG-11 for freshwater cyanobacteria and MediumA for marine cyanobacteria
Is mentioned.

Next, a medium (hypotonic medium) whose osmotic pressure is reduced by removing salts of the above-mentioned medium components is prepared, and the medium is added to and suspended in the above cells. The hypotonic solution is prepared so as to reduce the osmotic pressure by removing salts,
It is not always necessary to remove all salts, and the total amount of salts may be 0 to 500 mM.

The hypotonic solution in the present invention is not limited to the above-mentioned solution, but includes all culture solutions having a lower salt concentration than the medium used for the culture up to that time, thereby effectively releasing the target product. In order to make it possible, it is desirable that the concentration is half or less of the original culture solution.

When an organic substance such as glucose or sucrose is added to the medium for growth, the one having a reduced concentration of the organic substance can be used in the present invention even if the concentration of the inorganic salt is not reduced. Included in liquid. In this case, the concentration of the organic substance in the hypotonic solution is preferably 0 to 500 mM.

By the above operation, the gene product is released within a few minutes after culturing in a hypotonic solution, the release is almost completed in 1 hour, and the gene product reaches a certain concentration.

The released target product is separated and purified by various protein separation methods, for example, known methods such as gel filtration and ion exchange column.

The protein, which is the above-mentioned target substance, has different production ability depending on the host cyanobacteria and the signal peptide used. Preferable combinations of cyanobacteria and signal peptide include Synechococcus, β-lactamase, and Synechococcus. Examples include cystis and β-lactamase, anabaena and alkaline phosphatase, nostock and alkaline phosphatase, and particularly preferred are synecococcus and β-lactamase.

As another aspect of the present invention, the target substance can be repeatedly collected. That is, once, the used cultured cells are returned to the normal medium again, under the same conditions as above,
The target substance can be produced by culturing again and transferring to a hypotonic solution again.

In this case, the transformant is cultured for 1 to 5 days under the same conditions to recover the vitality, and the hypotonic medium is replaced again to recover the target substance from the extracellular region as described above. Can be released to. By repeating such an operation, the release can be continuously repeated for a long period of time.

It is a very remarkable effect achieved by the present invention that long-life expectancy can be expected in cyanobacteria, as compared with the case where the target gene product stops after repeated culture in, for example, E. coli. .

Many strains of cyanobacteria required for the above are registered in the domestic and foreign culture collections and are available. Because it utilizes changes in osmotic pressure, it is a marine and halophilic strain, for example, Synechococcus sp.PCC 7002, Anabaena sp.ATCC 33047, Osylatoria sp.PCC 6401, Pseudoanabena sp.CCAP 1464/3, Spirulina Sub salsa CCAP 1475/2 etc. is preferable.

Cyanobacteria are E. It belongs to Gram-negative bacteria like E.coli and has a total cell volume of 20 between the outer wall of the cell and the cytoplasmic membrane.
There is up to 40% periplasmic space. The present invention
E. For the first time in the present invention, when a protein accumulated in the periplasm of E. coli is genetically engineered in cyanobacteria It is based on the findings.

Cyanobacteria originally live in an oligotrophic environment and excel in durability. Some species have been discovered that survive for decades in poor environments such as deserts. Therefore, what is expected from the application side is to immobilize it to form a bioreactor or biosensor. In the case of conventional E. coli, for example, the bacteria quickly weaken and the target gene product stops, but in cyanobacteria, a long life can be expected.

[0040]

INDUSTRIAL APPLICABILITY In the present invention, the product of a foreign gene produced by a genetic engineering method can be easily purified by using cyanobacteria, and further, the gene product can be repeatedly released to the outside of the cell. Became.

The present invention will be specifically described below with reference to examples.

[0042]

EXAMPLE An example is shown in which the β-lactamase gene of Escherichia coli is introduced into cyanobacteria Synechococcus for transformation to release β-lactamase extracellularly. Synecococcus used PCC7002 strain preserved in the Pasteur Institute culture collection. (1) Add the strain of Synechococcus sp.PCC7002 to 300 ml of Medium A medium (J.Physol., 9,427 (1973)) and in a 500 ml Erlenmeyer flask while irradiating with a fluorescent lamp and passing 1% CO ₂ air. Incubate to _OD5502 . (2) Isolate the plasmid from this cell by a known method (M
ethods in Enzymology, 153, 199, (1987)) and subjected to agarose gel electrophoresis to obtain the smallest plasmid pAQ1.
Cut out the band from Gene Clean (Bio-Rad)
Purification was performed to obtain about 1 μg of the band. (3) E. coli encoding the β-lactamase gene
2 μg of the plasmid vector pBR322 (Pharmacia) was digested with the restriction enzyme Hind III to give alkaline phosphatase (C
alf Intestine, Takara Shuzo Co., Ltd., and depurination treatment was performed, followed by purification with Gene Clean. Further, 1 μg of pAQ1 plasmid was cleaved with restriction enzyme HindIII and purified with Geneclean. The mixture of both was treated with T4 DNA ligase (Takara Shuzo) for ligation reaction. E.
coli DH5αMCR (Bethesda Research Lab.) was transformed by the calcium method. This solution is ampicillin 50 μg / ml
Transformed strains were selected by plating on agar plates containing. 10 ml of the strain was cultured and a plasmid was prepared by a known method (Muramatsu Tadashi: Lab Manual Genetic Engineering, Maruzen (1988)).
10 μg of pAQE1 was obtained. (4) the Synechococcus Sp.PCC7002 strain was grown to OD ₅₅₀ 1, and harvested the 3ml, was suspended in 1 mM HEPES 3ml of and washed three times with 1 mM HEPES solution. Put 0.8 ml of this into the cell of an electroporation device (electric cell borer, RIKEN), mix 3 μg of plasmid pAQE1 and
While cooling with ice, an electric pulse of 6.5 KV / cm (half-life 2 msec) was applied twice. This was applied to a 1% agar-Medium A plate and left at 30 ° C. under dimming overnight, and then 0.6% agar containing ampicillin (2.5 μg / ml) was overlaid, and 37 ° C., 2000 lx The cells were cultured under fluorescent light to obtain transformant colonies. (5) The thus obtained transformant of Synechococcus sp . PCC7002 was first cultured for one day in Medium A containing no ampicillin, and then ampicillin was added to give a final concentration of 0.5 μg.
/ Ml and allowed to grow. (6) Take 10 ml of the growth medium and centrifuge, and discard the supernatant. Prepare a hypotonic medium obtained by removing NaCl (300 mM) from Medium A, add 10 ml, and let it stand at room temperature for a predetermined time (10 minutes-180 minutes).
I left it. (7) 1 ml of the culture solution was collected, centrifuged, and 0.3 ml of the supernatant was taken. 0.2 ml of 300 mM phosphate buffer (60 mM K
₂ HPO ₄ , 40 mM KH ₂ PO ₄ , 300 mM NaCl, p
H7), 0.1 ml of 0.5 mg / ml nitrocefin (Unipass)
Was added and the mixture was incubated at 30 ° C. for 10 minutes. Transfer the solution to the cell of the spectrometer (Hitachi spectrophotometer U1100),
When the absorbance at 495 nm was measured, there was a remarkable absorption indicating the activity of β-lactamase. (8) Centrifuge the culture of hypotonic medium of (6), remove the supernatant, add 10 ml of normal Medium A medium, culture overnight, and then re-hypotonic medium as in (6). Instead, the activity of β-lactamase by nitrocefin was measured. Almost the same activity as the recent measurement was obtained. In addition, even when the cell densities were compared, there was almost no change, indicating that most of the cells were alive.

Composition of Medium A NaCl 18.0 (g / L) MgSO ₄ .7H ₂ O 5.0 (〃) NaNO ₃ 1.0 (〃) KCl 0.6 (〃) CaCl ₂ .2H ₂ O 0. 37 (〃) Trizma base 1.0 (〃) Na ₂ EDTA 0.03 (〃) H ₃ BO ₃ 34.3 (mg / L) MnCl ₂ .4H ₂ O 4.3 (〃) FeCl ₃・ 6H ₂ O 3.89 (〃) ZnCl ₂ 0.315 _{(〃) CoCl 2 · 6H 2 O 0.0122} ( 〃) MoO ₃ 0.03 _{(〃) CuSO 4 · 5H 2 O 0.003} ( 〃) vitamin B ₁₂ 0.004 (〃)

[0044]

[Sequence listing] SEQ ID NO: 1 Sequence length: 22 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr 1 5 10 15 Pro Val Thr Lys Ala Arg 20 SEQ ID NO: 2 Sequence Length: 24 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Met Ser Ile Gln His Phe Arg Val Ala Leu Ile Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His 20 SEQ ID NO: 3 Sequence length: 27 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys 20 25 SEQ ID NO: 4 Sequence Length: 21 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Met Lys Thr Lys Leu Val Leu Gly Ala Val Ile Leu Thr Ala Gly Leu 1 5 10 15 Ser Gly Ala Ala Glu 20

[Brief description of drawings]

FIG. 1. Plasmid vector pAQE1 expressing β-lactamase in Synechococcus sp. PCC7002.

Fig. 2: β by Synechococcus sp. PCC7002 transformants
-Extracellular release of lactamase Transformed strain (○), wild strain (△)

[Fig. 3] β by Synechococcus sp. PCC7002 transformant
-Repeated extracellular release of lactamase and cell density

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12N 15/09 ZNA C12P 21/02 C 9282-4B // (C12N 1/13 C12R 1:89) (C12N 9/16 C12R 1:89) (C12N 9/18 C12R 1:89) (C12N 15/09 ZNA C12R 1:19) (C12P 21/02 C12R 1:89) C12R 1:19)

Claims (2)

[Claims] 1. A signal sequence of a gene of β-lactamase, alkaline phosphatase, maltose binding protein, or arabinose binding protein, which is a protein existing in the periplasm of Escherichia coli , or a portion encoding the signal sequence followed by several amino acids. A transformant in which a vector for recombination of cyanobacteria in which a sequence is inserted at the N-terminal of the structural gene of the desired product is introduced into cyanobacteria. 2. A signal sequence of a gene of β-lactamase, alkaline phosphatase, maltose binding protein, or arabinose binding protein, which is a protein existing in the periplasm of Escherichia coli , or a portion encoding the signal sequence and several amino acids following it. A gene characterized by transforming a transformant obtained by introducing a cyanobacterial recombination vector into the cyanobacteria, which has a sequence inserted into the N-terminus of the structural gene of the desired product, and then transferring it to a hypotonic solution. Product manufacturing method.