JP2857730B2 - Method of protein secretion by Bacillus subtilis strain - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to a method for separating desired proteins with high efficiency.
Increase protein production using Bacillus subtilis strains that can be produced
The present invention relates to efficient secretory production technology. The invention more particularly relates to extracellular neutral proteases
Or / and the activity of extracellular alkaline protease
Bacillus-derived protease production in reduced Bacillus subtilis
Proteolytic activity obtained by introducing a promoter gene is
And reduced host strains and protease genes or
Is the DNA sequence of the desired protein
To combine the recombinant DNAs constructed by combining
A more efficient method for secretory production of desired proteins
Thing. [0003] 2. Description of the Related Art
Heterologous gene using microorganism as host by recombinant DNA technology
Can be expressed to produce the desired heterologous gene product
It became. Bacteria such as Escherichia coli and Bacillus subtilis as host bacteria
Alternatively, yeast and the like are generally used. These days, things
Heterologous protein is secreted into the culture supernatant from the viewpoint of quality production.
Attempts have been made to study the host microorganisms in this case.
Originally, protease, amylase, etc.
Bacillus spp.
Is believed to be new. Especially among Bacillus bacteria
Bacillus subtilis is used for genetic, biochemical, molecular biology,
Because a lot of biological knowledge has been obtained and the safety is high
Attention has been drawn up for the extracellular secretion of heterologous proteins
Efforts have been made to create a host-recombinant DNA system. I
An attempt to secrete heterologous proteins using Bacillus subtilis as a host
Means that the heterologous protein is derived, in particular, from higher eukaryotes.
Very low secretion, is it still an industrial practice?
Are not enough. This is caused by Bacillus subtilis
Primarily from genes encoding heterologous proteins from eukaryotes
Despite the large amount of messenger RNA synthesis,
How much protein can be translated by translating messenger RNA
Due to low abundance, eukaryotic heterologous proteins are intracellular
Or by being degraded by the host Bacillus subtilis during secretion.
(Ref. 1). According to previous knowledge,
As a host strain of Bacillus subtilis, cells are used to prevent the degradation of heterologous proteins.
Strains with reduced protease activity
However, even if this is done,
The experimental results show that a sufficient amount of heterologous protein from higher eukaryotes is still present.
No Bacillus subtilis strain capable of secreting white is known
No. Parva et al., For example, reduced extracellular protease activity.
A Bacillus subtilis host was established to secrete α-amylase.
The human interface after the base sequence encoding the signal
And introducing the recombinant DNA linked to the
In a study using the resulting transformant,
Interferon at a rate of 10^FiveUnit secretion
You. This value indicates that interferon-α is 1 / L of medium.
mg-secreted, but α-amylase itself
It is lower than the secretion amount of
Reference 2). Also, Kawamura and Doi provide neutral protection.
Reduces both ase and alkaline extracellular protease activity
The creation of a depleted B. subtilis DB104 strain has been reported.
(Reference 3). On the other hand, the present inventors have
Using a strain with reduced extracellular protease activity as a host
With human interferon-β, human growth hormone
As a result of secretory production in the bacterial system,
Regardless, their secretions are not as large as expected
10 mg, 30-50 mg per liter of medium,
As the culture time elapses, the heterologous proteins produced
I understand that I understand it (Reference 4). This
Low extracellular alkaline and neutral protease activity
It is desirable to use B. subtilis as a host for secreting heterologous proteins
Sufficient accumulation of heterologous proteins from higher eukaryotes
There is no. [0004] SUMMARY OF THE INVENTION In view of this point, the inventors of the present invention have proposed a method for solving the above problems.
As a result of repeated studies, the inventors have already
Inheritance isolated as a zea production-promoting gene (Reference 5)
Bacterial grass with reduced extracellular protease activity
When introduced into bacteria, casei is an indicator of proteolytic activity.
Halo formation on an agar plate is unexpectedly suppressed
It was found that the present invention described in the claims was completed.
Was. The effects of the present invention shown in the embodiments will be summarized and described.
In other words, there is a protease production promoting gene in the chromosome.
And extracellular neutral protease and / or extracellular
To Bacillus subtilis, which has reduced the activity of potassium protease,
Promote human growth hormone gene to protease gene
Derived from protein and pre-propeptide coding region
Recombinant D constructed by binding downstream of the DNA base sequence
Using the transformant obtained by introducing NA and thus obtained,
Secretion production of 200mg human growth hormone per liter of medium
Have been successful. And this amount is the parent stock
Reduced both extracellular alkaline and neutral protease activity
5 to 10 times as high as when only a strain is used as a host
In. Hereinafter, the present invention will be described in detail. Bacillus
Inherited as a gene promoting bacterial extracellular protease production in bacteria
Pap whose existence is known from biological analysis
6), sacQ (Ref. 7), hpr (Ref. 8), etc.
You. Genetics that promote the production of the protease used in the present invention
Even if these children are known, they are newly
Even if isolated by a replacement technique,
When introduced into Bacillus subtilis with reduced Rotase activity
Halo formation on casein-agar medium was determined by the parental pro
What is necessary is just to suppress as low as the casease-deficient strain.
Because of the ease of implementation, it was
That is, it is better to have the DNA sequence in hand.
Good. And as an example of such a thing, the inventor has already
La Bacillus amyloriqifaciens (B. amyloliq
uefaciens) as a protease production promoting gene
DNA base sequences that have been successfully released are particularly suitable for use.
(Ref. 9). [0007] The protease production promoting gene is
A to construct a B. subtilis transformant by introducing it into
DNA sequence of the protease-promoting gene and Bacillus subtilis staining
Normal genetics using homology of body DNA base sequences
A recombination method may be used. Also in other ways
Introduce a protease production promoting gene into chromosomal DNA
Any method is possible as long as it can be tightened. Extracellular neutral and / or alkaline pro
Bacillus subtilis with reduced activity of thease is nitrosoguanidinium
Mutagen treatment by UV irradiation, γ-ray irradiation
Protease residue obtained by the method of genetic or genetic recombination
For deleting the gene in vitro and re-introducing it into B. subtilis
Methods and methods that combine them
You. The method of gene recombination is more suitably
More preferred because it can cause deletions in the gene itself
No. The extracellular neutral protea produced by such a method
Activity of protease and / or extracellular alkaline protease
Bacillus subtilis strains with reduced
The condition that the forward gene is contained in the plasmid or staining
Surprisingly introduced when introduced in the body
Previously remaining protease activity is suppressed by the introduction.
Has been found by the present inventors as a starting point of the present invention.
It was done. The protease production promoter obtained as described above
Bacillus subtilis with gene in chromosomal DNA is desired as host
Bacillus spp.
Area responsible for expression and secretion of extracorporeal protease gene
Promoter, pre-propeptide coating region
Downstream of the gene encoding the desired heterologous protein or
DNA constructed by combining DNA base sequences containing
A may be introduced into the host. Because it is used here
As recombinant DNA that can be cut, the present inventors have already
PPIF25 for terferon-β secretion (Reference 10), etc.
Is building. [0010] Bacillus amyloliquefaciens
Examples of extracellular protease genes include Bacillus
Extracellular neutral protease of amyloliquefaciens,
Extracellular alkaline protease, etc.
You. [0011] The recombinant DNA for expression and secretion described above is
Protease production promoting gene is present in chromosomal DNA
Normal protoplast trait for introduction into Bacillus subtilis hosts
The conversion method can be implemented. Using the transformant thus obtained,
In order to obtain the protein, the strain should be cultured in the usual manner in liquid culture.
Good. Preparation of a desired protein from a culture solution is performed by a conventional method.
The method can be performed by recovering and purifying from the culture supernatant
You. In particular, heterologous proteins derived from eukaryotes can be
Using white as a host, Bacillus subtilis
Lactic acid can be produced, and recovery and purification of the desired protein
It will be easier. [0014] The present invention will be described below with reference to specific examples.
Is not limited by the example. Embodiment 1Extracellular protease production promoting gene in chromosomal DNA
Of Bacillus subtilis transformant Extracellular neutral protease and alkaline protease
Bacillus subtilis MT-400 strain (FERM BP-1078),
Its extracellular proteolytic activity is less than 5% of that of the wild-type strain.
Is what it is. Prototype of chromosomal DNA of MT-400 strain
The introduction of the thease production promoting gene was carried out as follows.
First, the Bacillus amyloliquefaciens protea
Plasmid pNP181 containing zea production-promoting gene (References
Bacillus subtilis MT-0181 strain (FERM BP-343) having 11)
 PNP181 was prepared according to the method of Gryczan et al. For preparation
Incubate for 12 hours at 30 ° C using 1 L of LB medium.
Were used. Part of the obtained plasmid is a restriction enzyme
Check the cleavage site and confirm that this plasmid is pNP181.
And confirmed. Next, the method of Saito et al.
Accordingly, the competent cells of the MT-400 strain were prepared. Obtained
500 μl of the culture solution containing the competent cells
 Add 0.5 μl of pNP181 and incubate at 37 ° C for 1 hour.
Was. One hour later, the entire 500 μl of the culture solution was
To 20 ml of LB medium containing 5 μg / ml
And cultured at 37 ° C. for 12 hours. Then, centrifuge the cells
Separate and collect the resulting cells in 20 ml of Schaeffer spores.
24 hours at 37 ° C by adding to the formation medium (Ref. 12)
Culture was continued. Take 1.5 ml of the culture
After heating at 70 ° C for 10 minutes, 10^FourTimes 10^FiveTimes 10⁶
After diluting 1 fold, add 100 mL of each diluted solution to TBAB medium (Difco).
μl was plated. Incubate this at 37 ° C overnight.
The colonies obtained after the culture were cultured in TBAB medium and kanamycin.
Subculture into TBAB medium containing 5 μg and culture at 37 ° C for 15 hours
Was. The kanamycin-containing TB grows on the TBAB medium.
Select 200 colonies that did not grow on AB medium
Planted in In medium (Ref. 13) and cultured at 37 ° C for 60 hours
did. Halo formed around colonies after 60 hours
As a result of examining the size of
There was a clear halo around some colonies. C
A strain that does not produce rho (named MT-430)
When liquid culture is performed using Ibros (manufactured by Difco), the logarithm is
The cells in the growth phase showed a characteristic of elongation. This feature
 It is commonly found in Bacillus subtilis with pNP181.
You. Similarly, MT-400 strain with pNP181
Growth of the cells without formation of the strain, and the strain MT-430 (micro
Plasmids are present in Kokenjo No. 1079, FERM BP-1079).
PNP181 on the chromosome of the MT-430 strain
There is a protease production promoting gene contained in
Judged that. [0017] In addition, bacterial cells were obtained using the chromosomal DNA of the MT-430 strain.
Transformation of Bacillus subtilis, a wild strain, for exoprotease
A trait that forms a large halo on casein medium when converted
By genetic analysis using the fact that transformants are obtained
The protease production promoting gene is the chromosome DN of the MT-430 strain.
A. Embodiment 2Human growth hormone gene linked to protease gene
Of recombinant DNA for human growth hormone secretion into MT-430 strain
Introduction As recombinant DNA for human growth hormone secretionB. amylol
iquefaciensPromoter for the neutral protease gene
-, Prepeptid coding region and following
Part of the peptide coding region (21 amino acids
Consisting of 63 nucleotides)
Mature human downstream of the A base sequence (SEQ ID NO: 1 in the sequence listing)
DNA containing DNA sequence encoding growth hormone
The phGH427 (FIG. 1) to which the fragments were linked was used. ph
GH427 was already built by the inventorB. amyloli
quefaciensNeutral protease clone pNP150 (reference
Reference 14) Pre-propepter of neutral protease gene
Restriction enzymes present in the idcoding regionPvuI cutting
Exonuclease from siteBalObtained by hydrolysis at 31
DNA fragment containing human growth hormone gene
It is constructed by joining DNA fragments. pN
P150 is Bacillus subtilis MT-0150 strain (Microtechnical Laboratories No. 0150, FERM
 BP-425) according to a conventional method. Human growth hormone
The complete DNA base sequence of the gene has already been reported.
In this example, human growth hormones obtained by chemical synthesis were used.
A DNA fragment containing the mon gene was used. phGH
Introduction of 427 into MT-430 strain according to the protoplast method
did. 50 obtained kanamycin resistant transformants were selected
And the presence or absence of growth hormone secretion into the culture supernatant
Results of assay using immunoassay and immunodiffusion method
It turns out that all of them secrete and produce growth hormone
did. For the above assay, shake culture was performed at 30 ° C for 15 hours in LB medium.
The supernatant of the cultured culture was used. Obtained recombinant DNAph
MT-430 strain including GH427 (Microtechnical Research and Development No. 1080 FERM BP-10
80) to determine the extracellular production of growth hormone.
Examined. Embodiment 3Human growth using MT-430 strain containing recombinant DNAphGH427
Hormone secretory production MT-430 strain containing recombinant DNAphGH427 and as control
PhGH427 was added to the MT-400 strain exactly as described in Example 2.
MT-400 strain containing recombinant DNAphGH427 introduced as above
Using a double concentration LB medium (100 ml).
Shaking culture was performed at 17 ° C. for 17 hours. From the culture solution after 17 hours,
A large amount of cells are collected by high-speed centrifugation, and
Suspend the cells in double concentration LB medium and continue shaking culture at 30 ° C.
Was. Eight hours later, the MT-430 strain containing the recombinant DNAphGH427 and
Concentration of MT-400 strain containing recombinant and recombinant DNAphGH427
(A660) were 9.86 and 10.00, respectively.
Was. The pH of the culture supernatant was 8.06 and 7.98, respectively.
there were. Using this culture supernatant, culture by enzyme immunoassay is performed.
As a result of measuring the growth hormone concentration in the ground, MT-430 strain
Mainly, 205 mg / L of medium, MT-400 strain
The main case was 30 mg per liter of medium. Immunity
MT-430 containing recombinant DNA phGH427 from double diffusion method
Strain and MT-400 strain containing recombinant DNAphGH427
Growth hormone indistinguishable from human pituitary gland?
Was. In addition, 17 MT-430 strains containing recombinant DNAphGH427
Trichloroacetic acid was added to 100 μl of the supernatant
The resulting precipitate was redissolved in 30 μl of sample buffer.
Resolve and test for SDS-polyacrylamide gel electrophoresis
Analysis of the protein in the culture supernatant
20% -30% of the protein present is growth hormone
There was found. [0020] According to the above results, the MT-430 strain and the protea
Of recombinant DNA for the secretion of heterologous proteins using a lyse gene
The resulting host-plasmid system can be achieved to date.
The secretory production of large amounts of heterologous proteins
Was shown. (List of references) 1. Ulmanen.l. (1985) J. Bacteriol.,162176-182, Ya
mane.K. et al. (1985) in "Molecular Biology of Micr
obial Differentiation "Hoch. J. A., andSetlow. P.E
cl 117-123, American Soc. for Microbiol. 2. Palva. L. Et al. (1983) Gene.twenty two229-235. 3. Kawamura. F. and Dol.R.H. (1984) J. Bacterial.,
160 442-444. 4. Honjo. M. et al (1985) J. Biotech.,Three73-84. Hon
jo. M. et al (1986) in "Genetics and Biotechnology o
f Bacilli "TwoGanesan. A., and Hoch, J.A.EdAcademic
 Press. 5. Tomioka. N., et al. (1985) J. Biotechnol.,Three85-9
6. 6. Steinmety. M. et.al. (1976) Mol. Gen. Genet.,14
8281-285, Yoneda. Y. and Maruo. B. (1975) J. Bacte
riol.,12448-54. 7. Kunst. F. et.al. (1974) Biochimie56 1481-148
7. 8. Higerd. T. et al. (1972) J. Bacteriol., 112 1026-
1028. 9. Tomioka. N. et al. (1985) J. Biotechnol., 3 85-9
6. 10. Honjo. M. et al. (1985) J. Biotechnol.,Three73-8
Four. 11. Tomioka. N. et al. (1985) J. Biotechnol., 3 85-
96. 12. Schaeffer, P. and Ionesco, H. (1980) Comt.Re
rd.,251 3125. 13. HOnjo. Et al, (1985) J. BIotechnol.,173-84. 14． Honjo. M. et al. (1984) J. Biotechnol.,1265-
277, Shimada, H. etal. (1985) J. Biotechnol.,Two75-
85. 15. Saito.H. Et al, (1961), J. Gen. Appl. Microbiol,7
, 243-252, [0022]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the structure of phGH427. [Description of Symbols] Shaded area Partial Pm promoter derived from neutral protease gene prepro Partial hGH DNA fragment containing human growth hormone gene derived from prepropeptide coding region [Sequence Table] SEQ ID NO: 1 Sequence length: 144 sequences Type: Number of nucleic acid strands: Double-stranded topology: Type of linear sequence: Genomic DNA Origin organism: Bacillus amyloliquefaciens (Bacillus)
amyloliquefaciens) Location: 1-114 Sequence characteristics 1-81 S sig peptide 82-114 S pepide sequence 1 5 10 15 Met Gly Leu Gly Lys Lys Leu Ser Ser Ala Val Ala Ala Ser Phe Met GTG GGT TTA GGT AAG AAA TTG TCT AGT GCT GTA GCC GCT TCC TTT ATG 48 20 25 30 Ser Leu Thr Ile Ser Leu Pro Gly Val Gln Ala Ala Glu Asn Pro Gln AGT TTA ACC ATC AGT CTG CCG GGT GTT CAG GCC GCT GAG AAT CCT CAG 96 35 40 45 Leu Lys Glu Asn Leu Thr Asn Phe Val Pro Lys His Ser Leu Val Gln CTT AAA GAA AAT CTG ACG AAC TTT GTA CCG AAG CAT TCT TTG GTG CAA 144

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1: 125) Examiner Michiaki Kogure

Claims (1)

(57) [Claims] By introducing a Bacillus amyloliquefaciens-derived extracellular protease production promoting gene into the chromosome of a strain in which the activity of Bacillus subtilis extracellular neutral protease or / and extracellular alkaline protease has been reduced,
To a Bacillus subtilis strain in which the remaining extracellular protease activity is further reduced, a Bacillus amyloliquefaciens-derived extracellular protease gene or DNA derived therefrom
A method in which a recombinant DNA for expression and secretion constructed by binding a gene encoding a desired protein to a base sequence is introduced to secrete and produce the desired protein. 2. The method according to claim 1, wherein the extracellular protease gene derived from Bacillus amyloliquefaciens or a DNA base sequence derived therefrom is a neutral protease gene derived from Bacillus amyloliquefaciens. 3. Bacillus amyloliquefaciens-derived extracellular protease gene or a DNA sequence derived therefrom linked to a gene encoding a desired protein, and a recombinant DNA for expression and secretion constructed using the chromosome of a protease-deficient strain of Bacillus subtilis Characterized in that the Bacillus subtilis strain whose extracellular protease activity has been reduced by introducing a Bacillus amyloliquefaciens-derived extracellular protease production promoting gene is the MT-430 strain containing recombinant DNAphGH427. The method according to claim 1, wherein