JPH09224677A - New hs gene and expression plasmid in which the gene is connected to downstream of structural gene coding different kind of genetic product and production of different kind of genetic product with transformant having the expression plasmid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new HS gene having a specific base sequence and a homogeneous base sequence through an arbitrary base sequence and capable of transforming the bacteria of the genus Bacillus to massively secrete and produce various kinds of genetic products outside the cells. SOLUTION: This new HS gene has a base sequence of formula I and a base sequence of formula II homologous to the base sequence of formula I through a sequence comprising an arbitrary 3-20bp base sequence and disposed between both the sequences. The new HS gene can massively secrete and produce various kinds of genetic products outside cells by ligating the HS gene to the downstream of a structural gene coding the genetic products, transforming a Bacillus bacterium with the obtained expression plasmid and subsequently culturing the transformant. The HS gene is obtained by extracting the chromosomal DNA of the Bacillus bacterium, cleaving the DNA with a restriction enzyme, inserting the obtained fragment into a plasmid together with a foreign gene, expressing in host cells, selecting a cell capable of highly secreting and producing the products, and subsequently recovering the DNA placed on the downstream of the foreign gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to biotechnology. More specifically, the present invention provides a novel gene, an expression plasmid in which the gene is linked downstream of a structural gene encoding a heterologous gene product, and a Bacillus bacterium transformed with the plasmid, thereby culturing the heterologous gene product. The present invention relates to a method for producing a heterologous gene product, which comprises producing and accumulating in a culture, and collecting the product.

[0002]

2. Description of the Related Art Currently, the production of heterologous gene products using recombinants is widely used in the food, pharmaceutical, cosmetics and other industries. Bacteria such as Escherichia coli and Bacillus subtilis, yeasts, filamentous fungi and the like are used as host bacteria for gene recombination.

[0003] Gene recombination technology is based on Escherichia
coli) system for which has been developed into a center of, E. coli is often used as a host bacterium. However, in a system using Escherichia coli as a host, the produced heterologous gene products such as peptides and proteins remain in the cytoplasm or in the periplasmic space surrounded by the outer and cytoplasmic membranes of cells, and it is difficult to secrete and produce them in the medium. there were. There is a quantitative limit to the accumulation of heterologous gene products in cells, and it is necessary to crush the cells to recover the heterologous gene product. Isolate heterologous gene products,
It was necessary to purify. In addition, some of the produced peptides and proteins form an inclusion body, and when the inclusion body is formed, there is a drawback that it is necessary to perform a regeneration operation to make it an active form.

Many Bacillus bacteria secrete and produce enzyme proteins in a large amount, and development of host vector systems utilizing this property is being actively conducted. Bacillus bacteria, especially Bacillus subtilis , are well studied both genetically and biochemically, and many studies have been conducted on secretory production of heterologous gene products. However, in a system using Bacillus subtilis as a host, there is a problem that heterologous gene products such as peptides and proteins produced by strong proteases inside and outside the cells are decomposed.

In order to eliminate these drawbacks, diligent research has been conducted.
When I went there, Utaka and his colleagues Bacillus Brevis (Bacillus
 brevis), There are many strains that do not produce protease.
I found out. The 1 strain Bacillus brevis 47
(S. Udaka and H. Yamagata, Methods in Enzymology,
217 23-33 (1993)) major extracellular protein (H. Yamaga
ta et al, J. Bacteriol.,169, 1239 (1987); Nori Tsukakoshi
Hiroshi, Journal of Japan Society for Agricultural Chemistry, 61, 68 (1987) to “oute”
r wall protein and middle wall protein ”,“ major bacteria
Extracorporeal protein ").
Lomotor and one of the major extracellular proteins
MW protein (middle wall protein) signal
Construction of secretion vector using the region encoding peptide
Then, using this strain as a host, α-amylase (Japanese Unexamined Patent Publication No.
201583, H. Yamagata et al, J. Bacterio
l.,169, 1239 (1987)) and Butapepsinogen (Ugatakashige)
Proceedings of the 1987 Annual Meeting of the Japanese Society of Agricultural Chemistry, p8
37-838; Norihiro Tsukakoshi, Journal of the Japanese Society of Agricultural Chemistry,61, 68 (1
987)) was secreted and produced successfully.

In addition, Takagi et al., A strain Bacillus brevis HPD31 that does not produce Bacillus brevis protease extracellularly (this strain is Bacillus brevis H1
02 (FERM BP-1087).
Separated. And this Bacillus Brevis HPD31
High-secretion production of thermostable α-amylase (H.
kagi et al, Agric. Biol. Chem., 53 , 2279-2280 (198
9)) and high secretory production of human EGF by Yamagata et al. (H. Yamag
ata et al, Proc. Nati. Acad. Sci. USA, 86 , 3589-359
3 (1989)) has been successful.

[0007]

As described above, the productivity of a heterologous gene product using Bacillus bacterium, particularly Bacillus brevis as a host bacterium is remarkably improved as compared with other host bacterium. However, including Bacillus Brevis,
For production of a heterologous gene product in a system in which a bacterium belonging to the genus Bacillus is used, an expression vector replicable in the bacterium belonging to the genus Bacillus is used, and a suitable promoter, downstream of which is an SD sequence, and a secretion signal sequence starting from a translation initiation codon. , Then the heterologous gene must be connected. However, in such a conventional technique, a case where the production amount of a heterologous gene product is very small is recognized, and further technical development is required for industrial application.

[0008]

In view of the current state of the art, the present inventors have paid attention to an excellent technology in which a Bacillus bacterium secretes and produces a protein extracellularly, and further its productivity. For the purpose of improving the secretory productivity of Bacillus brevis with the aim of improving the production rate from the laboratory scale to the industrial production scale, the present inventors have conducted extensive studies on the existence of a gene. Firstly, the present inventors are investigating the secretory production of human salivary gland amylase by Bacillus brevis, and are carrying out plasmid pTS7 (H. Konishi, Appl. Micr) carrying a structural gene encoding human salivary gland amylase.
obiol. Biotechnol., 34 , 297-302 (1990)) was digested with a restriction enzyme that cuts at one site, and then transformed with a plasmid ligated with a Bacillus brevis chromosome fragment digested with the same enzyme. Among them, a strain which produced a remarkably large amount of human salivary gland amylase was found.

A plasmid was extracted from this transformant,
Genetic analysis revealed that a gene of about 150 bp was inserted downstream of the structural gene encoding human salivary gland amylase. Therefore, when this gene was further extracted and the nucleotide sequence was determined and structural analysis was performed, there was a sequence in which this gene had complementarity with each other via a sequence consisting of 15 bp base pairs, and was transcribed into mRNA. It seemed to have a distinct stem-loop structure.

Therefore, when this gene is ligated downstream of a structural gene encoding a heterologous gene product other than human salivary gland amylase and incorporated into a host bacterium to produce the heterologous gene product, the production amount of the heterologous gene product increases. I found out. Furthermore, as a result of detailed examination, in order to increase the production amount of the heterologous gene product, the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 which have complementarity with each other are inserted through a sequence consisting of arbitrary base pairs of 3 to 20 bp. I have gained new useful knowledge that it is all right. The present invention was finally completed as a result of further research based on these useful new findings.

[0011] In the following, SEQ ID NO: 1 (Table 1 below) and SEQ ID NO: 2 (Table 2 below) in the sequence listing showing complementarity with each other through the sequence consisting of any base pair of 3 to 20 bp.
The gene having the sequence of is referred to as HS gene.

[0012]

[Table 1]

[0013]

[Table 2]

That is, the present invention provides a novel HS gene, an expression plasmid in which the HS gene is linked downstream of a structural gene encoding a heterologous gene product, and a Bacillus bacterium transformed with the plasmid, thereby culturing the heterologous gene product. The present invention relates to a method for producing a heterologous gene product, which comprises producing and accumulating in a culture, and collecting this. Hereinafter, the present invention will be described in detail.

[0015]

BEST MODE FOR CARRYING OUT THE INVENTION The HS gene of the present invention comprises 3 to 20 genes.
It is a gene having the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 which are complementary to each other via a sequence consisting of any base pair of bp, and 3 to 20b via this complementary sequence.
The sequence of p may be any base sequence selected from adenine, guanine, cytosine and thymine. Specifically 1
Examples include the gene of SEQ ID NO: 3 (Table 3 below) in which the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 are linked via a 5 bp base pair. In addition to the HS gene, a gene in which a base pair is added to the 5'end or 3'end of the HS gene can also be used for production of a heterologous gene product. Specifically, 86 bp at the 5'end of the HS gene shown in SEQ ID NO: 3 and 3 '
The gene shown in SEQ ID NO: 4 (Table 4 below) having a base pair of 24 bp added to the end can be used.

[0016]

[Table 3]

[0017]

[Table 4]

The HS gene is linked downstream of the structural gene encoding the desired heterologous gene product. In this case, it may be directly linked to the structural gene, or may be linked via a base pair of several to several tens of bp like the gene shown in SEQ ID NO: 4 above.

The heterologous gene product secreted and produced in the present invention may be a gene derived from any eukaryotic or prokaryotic organism, and gene products derived from humans, animals, birds, fish, microorganisms and various other organisms (enzymes, hormones, interferons, immunity). Globulin, other bioactive peptides, proteins, etc.). For example, it can be applied to the production of gene products such as human epidermal growth factor (hEGF).

The structural gene encoding the heterologous gene product secreted and produced by the present invention and the HS of the present invention linked downstream thereof
As a vector for introducing and retaining the gene in the host bacterium, a plasmid that can be replicated in the host can be used. For example, in the Bacillus subtilis system, pUB1
10 and their derivatives can be used, and in the system using Bacillus brevis as a host, pNU200 (Ushige Shigezo, Journal of the Japan Society for Agricultural Chemistry, 61 , 669 (1987)), pHY700 (S. Ebisu
et al, Biosci. Biotech. Biochem., 56 , 812-813 (199
2)), pHT110 (Japanese Patent Laid-Open No. 6-133782) and plasmids such as derivatives thereof can be used.

As a method for constructing these plasmids, known methods are appropriately used.
Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory (Molecu
lar Cloning 2nd ed., A Laboratory Manual, Cold Spr
ing Harbor Laboratory, 1989) and the like.

The bacterium used as a host bacterium in the present invention may be any bacterium belonging to the genus Bacillus, but Bacillus subtilis, Bacillus brevis, Bacillus choshinensis ( Ba
Cillus choshinensis ) and the like can be suitably used for transforming a host bacterium by a known method, for example, Takaha
Shi's method (Takahashi et al, J. Bacteriol., 156 ,
1130 (1983)) or the method of Takagi et al. (H. Takagi et al,
Agric. Biol. Chem., 53 , 3099-3100 (1989)) and the like.

The medium used for culturing the obtained transformant may be any medium as long as the transformant can grow and produce the desired heterologous gene product. Examples of carbon sources contained in the medium include glucose, sucrose, glycerol, starch, dextrin, molasses, urea, organic acids and the like. As the nitrogen source, casein, polypeptone, meat extract, yeast extract,
Organic nitrogen sources such as casamino acid and glycine, inorganic nitrogen sources such as ammonium sulfate, etc. are used. In addition, inorganic salts such as potassium chloride, monopotassium phosphate, dipotassium phosphate, sodium chloride, and magnesium sulfate are added to the medium as needed. For auxotrophic bacteria, nutrient substances necessary for their growth may be added to the medium. Examples of the nutritional substance include amino acids, vitamins, nucleic acids and the like.

If necessary for the culture, antibiotics such as penicillin, erythromycin, chloramphenicol, bacitracin, D-cycloserine, may be added to the medium.
Add ampicillin, neomycin, etc. Further, if necessary, a defoaming agent such as soybean oil, lard oil, and various surfactants may be added. The initial pH of the medium is 5.0-9.
0, and more preferably 6.5 to 7.5. The culture temperature is usually 15 ° C to 42 ° C, more preferably 24 ° C to 37 ° C.
C., and the culture time is usually 16 to 166 hours, more preferably 24 to 96 hours.

In the present invention, a heterologous gene product is produced in the culture by culturing the transformant under the above conditions,
Stored. The heterologous gene product thus obtained can be purified by known methods, for example, by membrane treatment, ammonium sulfate fractionation method, chromatography and the like (basic experimental method for proteins and nucleic acids, Nankodo, (1985)). In the present invention, H
By linking the S gene to the downstream of the structural gene encoding the desired heterologous gene product, various heterologous gene products can be stably produced at a high level.

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and the present invention is not limited thereto.

[0027]

Example 1 Cloning and Analysis of Gene Containing HS Gene (HS 'Gene) Bacillus brevis HPD31
The chromosomal DNA of (FERM BP-1087) was designated Saito,
Miura's method (Saito, H. and Miura, K., Biochem. Bi
Ophys. Acta., 72 , 639 (1964)) and then digested with the restriction enzyme HindIII to fragment. Next, a plasmid pTS7 (H. Konishi, Appl. Microbiol. Biotechno) having a structural gene encoding human salivary gland amylase
l., 34 , 297-302 (1990)) was treated with HindIII, and the 5'end was dephosphorylated with alkaline phosphatase, and then subjected to 0.8% hyagarose gel electrophoresis, and 6.
The 1 kb DNA fragment was recovered using Gene clean (Bio 101, USA) and ligated with the previously fragmented chromosomal DNA using T4 ligase to obtain plasmid pTS7HS.

Bacillus brevis HPD31 was electroporated with the obtained plasmid pTS7HS (H. Takagi et al, Agric. Biol. Chem., 53 , 3099-31).
00 (1989)). The selection of the highly secreted human salivary amylase-producing strain from the transformant was carried out by using T2 plate medium containing 1.0% starch (starch 1%, peptone 1%,
Meat extract 0.5%, yeast extract 0.2%, glucose 1
%, Agar 1.5%, pH 7.0) and 0.2% I ₂ after culturing
-The KI solution was sprayed, and it was confirmed whether or not the periphery of the colony became transparent (halo generated when the starch was decomposed).

As a result, colonies producing about 50,000 halos were obtained, from which a strain forming a halo having a size twice or more that of the Bacillus brevis HPD31 strain (pTS7 strain) carrying the plasmid pTS7 was found. The strain was designated as HS strain.

The HS strain and the pTS7 strain obtained here were treated with T2.
After inoculating each to Em liquid medium and culturing with shaking at 30 ° C. for 2 days, the amount of human salivary gland amylase contained in the culture supernatant was determined by the method of Saito using soluble starch as a substrate (Saito et al.
al, Agric. Biol. Chem., 155 , 290 (1973)), and amylase activity was measured and determined. As a result, as shown in Table A shown in Table 13 below, the HS strain produced about 8 times as much human salivary amylase as the pTS7 strain.

[0031]

[Table 13]

Alkali extraction method from HS strain (Birnoboim.
HC and Doly J., Nucleic AcidsRes., 7 , 1513 (197
The plasmid pTS7HS was extracted with 9)) and HindIII
After cutting with, the sample was subjected to 5% acrylamide gel electrophoresis. As a result, pTS7HS contained Hin of plasmid pTS7.
It was confirmed that a gene of about 150 bp was inserted at the dIII site.

Analysis of the base sequence of the gene thus obtained revealed that the gene has 157 base pairs shown in SEQ ID NO: 4. Also, as a result of structural analysis based on this sequence, from the 87th position to the 102nd position counted from the 5'end.
The 2nd region from the 1st position to the 118th position to the 133rd position had a sequence having a complementarity (palindromic structure) which seems to have a stem structure when transcribed into RNA. In addition, an open reading frame encoding a protein having the amino acid sequence shown in SEQ ID NO: 4 was present in this sequence. The gene consisting of 157 base pairs was designated as HS 'gene.

Secretion production of hEGF using the HS 'gene The HS' gene on the plasmid pTS7HS is shown in Table 5 below.
SEQ ID NO: 5 shown in SEQ ID NO: 5 and Primer HSRV of SEQ ID NO: 6 shown in Table 6 below were used to amplify by PCR (Primer HSM1).
As a result, the HindIII site on the 5'side of the HS 'gene is B
Converted to amHI site. This gene was designated as HS'B. ). The amplified HS'B gene was replaced with BamHI, Hin
treated with dIII and subjected to 5% acrylamide gel electrophoresis, and the HS'B gene fragment was electroeluted (Molecular Clon).
ing 2nd ed., A Laboratory Manual, Cold Spring Harb
or Laboratory, 1989).

[0035]

[Table 5]

[0036]

[Table 6]

Next, Bacillus brevis HP926
The plasmid p carried by (FERM BP-5382)
Plasmid pHT110EGF prepared from HT926
(JP-A-6-133782) was treated with BamHI and HindIII, subjected to 0.8% agarose gel electrophoresis, and then subjected to 3.5 k
The fragment of b was recovered using Gene clean (Bio 101, USA) and ligated with the previously obtained HS'B gene fragment using T4 ligase to obtain a plasmid pHT110EGF-HS'B. Bacillus brevis HPD31 / pHT110 carrying pHT110EGF-HS'B was introduced into Bacillus brevis HPD31 in the same manner as in.
An EGF-HS'B strain was obtained.

Bacillus brevis HPD3 obtained here
1 / pHT110EGF-HS'B in 2SLE liquid medium (peptone 4%, yeast extract 0.5%, glucose 2
%, MgSO ₄ 0.01%, FeSO ₄ 0.001
%, MnSO ₄ 0.001%, ZnSO ₄ 0.000
1%, erythromycin 10 μg / ml, pH 7.2)
Was inoculated into a test tube into which 3 ml was dispensed, and after shaking culture at 30 ° C. for 3 days, the amount of hEGF in the culture supernatant was measured by HPLC (column: C18-100A, diameter 4 mm × length 250 mm,
Buffer: 0.1% TFA / H ₂ O, 0.1% TFA
/ 50% acetonitrile, linear gradient, detection:
UV276 nm) was used for analysis, and the amount of production was determined by comparison with the peak area when HPLC was performed under the same conditions using commercially available EGF (manufactured by Funakoshi Co., Ltd.) as a standard product. as a result,
Bacillus brevis HPD31 / pHT110EGF-HS'B is Bacillus brevis HPD31 / pHT110EGF as shown in Table B shown in Table 14 below.
It produced about 1.4 times the amount of hEGF.

[0039]

[Table 14]

[0040]

[Example 2: Effect of structure of HS 'gene on production of heterologous gene product] Primer HSRV of SEQ ID NO: 6 and Primer HSS of SEQ ID NO: 7 shown in Table 7 below
Was used to prepare a mutant HS 'gene HSS by PCR. Similarly, Primer HSRV of SEQ ID NO: 6 and Primer HSFS of SEQ ID NO: 8 shown in Table 8 below.
Using the mutant HS 'gene HSFS, Pr of SEQ ID NO: 6
imager HSRV and SEQ ID NO: 9 shown in Table 9 below.
HS 'gene HS using Primer HS3 of
3, Primer HSRV of SEQ ID NO: 6 and Table 1 below
The mutant HS ′ gene HS20 was prepared using Primer HS20 of SEQ ID NO: 10 shown in 0.

[0041]

[Table 7]

[0042]

[Table 8]

[0043]

[Table 9]

[0044]

[Table 10]

HSS is the HS of SEQ ID NO: 1 from the 5'end.
Up to 10 bp, which is 76 bp shorter than the HS 'gene of SEQ ID NO: 4. In HSFS, a protein consisting of 27 amino acids encoded by the HS 'gene is not translated due to frame shift. HS3 and HS20 are RN
When transcribed into A, the stem loop loops are 3 bp and 20 bp, respectively (the HS 'gene of SEQ ID NO: 4 is 15 bp).
p).

The four kinds of mutant HS 'genes thus prepared were cleaved with HindIII and BamHI, respectively, subjected to 5% acrylamide gel electrophoresis, and the mutant HS' genes were recovered by electroelution. The obtained fragment was inserted into the plasmid pHT110EGF in the same manner as in Example 1,
pHT110EGF-HSS, pHT110EGF-H
SFS, pHT110EGF-HS3, pHT110E
GF-HS20 was constructed.

Bacillus brevis with each plasmid
HPD31 was transformed and cultured in the same manner as in Example 1, and the amount of hEGF in the culture supernatant was measured. The results are shown in Table B. The four mutant HS 'genes produced here produced about the same amount of hEGF as when the HS' gene of SEQ ID NO: 4 was used. From this, it is considered that the improvement of the secretory productivity of the heterologous gene product is not affected by the peptide consisting of 27 amino acid residues, but that the palindromic structure on the HS 'gene is important.
It was found that the loop-forming portion of the stem loop when the S ′ gene was transcribed into mRNA was 3 to 20 bp.

[0048]

[Example 3: Effect of HS gene (palindromic structural region of HS 'gene) on production of heterologous gene product] From the results of the examination of Example 2, the palindrome of HS' gene is shown to be improved in secretory production of a heterologous gene product. The structure was considered important. Therefore, only the palindromic structural region of the HS 'gene (SEQ ID NO: 3) was added to hT of pHT110EGF.
It was inserted downstream of the F gene to confirm hEGF productivity.

P of SEQ ID NO: 11 shown in Table 11 below
The palindromic structural region (HS gene) on the HS ′ gene was amplified using the primer STEM and Primer STEMRV of SEQ ID NO: 12 shown in Table 12 below. The obtained HS gene was treated with PstI to give T4DN.
The HS gene fragment was blunted by reacting with A polymerase and subjected to 10% acrylamide gel to recover a fragment of about 60 bp. Then pHT110EGF was added to BamH
After treatment with I and HindIII, blunting with T4 DNA polymerase, treatment with alkaline phosphatase and subjecting to agarose gel electrophoresis, the 3.5 kb fragment was cleaved with Gene c.
Recovered using lean, ligated with the previously obtained HS gene using T4 ligase, and plasmid pHT110EGF-HS
Was built. The introduction into Bacillus brevis was performed by the same method as in Example 1, and the orientation of the inserted HS gene was confirmed by DNA sequencing.

[0050]

[Table 11]

[0051]

[Table 12]

The resulting transformants were cultured in the same manner as in Example 1 and the hEGF production was measured. As a result, as shown in Table B, pHT110EGF-HS was pHT110EG.
Similar to F-HS 'and compared to pHT110EGF
It produced 1.3 times the amount of hEGF.

[0053]

Industrial Applicability According to the present invention, a novel HS gene has been newly developed. This novel HS gene can be ligated downstream of a structural gene encoding a heterologous gene product, and the novel expression plasmid thus prepared is used to transform Bacillus bacteria to obtain the trait obtained. By culturing the transformant, various heterologous gene products can be secreted and produced extracellularly in a large amount.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 1/21 C12R 1:08) (54) [Title of the invention] New HS gene and its gene Expression plasmid linked to downstream of structural gene encoding heterologous gene product and method for producing heterologous gene product using transformant carrying the expression plasmid

Claims (6)

[Claims] 1. An HS gene having the nucleotide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 which are complementary to each other via an arbitrary nucleotide sequence. 2. An arbitrary nucleotide sequence selected from adenine, guanine, cytosine, and thymine.
H of claim 1, wherein the H sequence is
S gene. 3. The HS gene according to claim 1 or 2, which is represented by the nucleotide sequence of SEQ ID NO: 3. 4. The H according to any one of claims 1 to 3, downstream of a structural gene encoding a heterologous gene product.
An expression plasmid comprising a S gene linked thereto. 5. A heterologous gene characterized by culturing a bacterium belonging to the genus Bacillus carrying the expression plasmid according to claim 4 to produce and accumulate a heterologous gene product in the culture, and to collect the heterologous gene product. Product manufacturing method. 6. The method according to claim 5, wherein the heterologous gene product is human epidermal growth factor.