JPH07184642A - Production of phospholipase c - Google Patents

Abstract

PURPOSE:To provide the subject production method widely used as a subject of studies on the biosynthesis and processing until the mainfestation of a membrane protein on the cell surface membrane, capable of applying, e.g. to medicines, food materials or reagents for biochemical studies and, therefore, remarkably useful in the fields of industry. CONSTITUTION:Bacillus bravis 47 transformed by a recombination vector containing a phospholipase C gene derived from Bacillus thuringiensis and a method for production of phospholipase C (PIPLC) carried out by culturing this transformed Bacillus brevis 47 in a culture medium and collecting a phosphatidylinositol-specific PIPLC from the cultured material are provided.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to transformed Bacillus brevis 47 and a method for producing phosphatidylinositol-specific phospholipase C using the Bacillus brevis 47.

[0002]

2. Description of the Related Art Phosphatidylinositol-specific phospholipase C (hereinafter referred to as PIPLC) is phosphatidylinositol (hereinafter referred to as PI) which is a functional phospholipid that plays an extremely important role in cell signal transduction system.
Is called an enzyme). The presence of PIPLC is known in animals and bacteria [J. Biol. Chem., 256, 6].
769 (1981), Biochim.Biophys.Acta, 450,154 (1976)],
It is known that bacterial-derived PIPLC has a releasing action of a membrane-bound protein [Biochim. Bioph
ys. Acta, 450, 154 (1976)]. Due to the releasing action, GPI
The existence of a membrane protein called a (glycosylphosphatidylinositol) type anchor has become established [J. Biol. Chem., 260, 14547 (1985)]. Examples of GPI anchor membrane proteins include enzymes such as alkaline phosphatase and G ₂ -type acetylcholinesterase, and Thy.
-1, CD55, folate receptor and other surface membrane proteins of mammalian cells are known [Japanese Journal of Bacteriology, Vol. 47,
477 (1992)], a membrane protein having such an anchor plays an extremely important role in vivo such as activation of T lymphocytes and uptake of folate. Therefore, the significance of the existence of PIPLC having the action of releasing the membrane-bound protein is extremely large.

Isolation and purification of PIPLC have already been carried out in Bacillus thuringiensis and Bacillus cereus derived microorganisms belonging to the genus Bacillus [Biochim.Biophys.Acta]. , 450,154 (1976), Bioc
him.Biophys.Acta, 619,48 (1980)], the nucleotide sequence of the gene has been determined [Mol. Microbiol., 3,621 (1989),
J. Bacteriol., 171, 6077 (1989)].

On the other hand, Bacillus brev
revis) 47 is known to have a large amount of protein secretion ability [Biotech. Genet. Eng. Rev., 7, 113-146 (1
989)] Moreover, since the protease is hardly released into the medium, its usefulness as a highly efficient protein-producing bacterium has been clarified.

[0005]

In the present invention, the PIPL is used.
The C gene was integrated into Bacillus brevis 47 to obtain a transformant, and the transformant was cultured to obtain PIP.
The purpose is to produce a large amount of LC.

[0006]

Means for Solving the Problems As a result of intensive studies on the above problems, the present inventors have found that a gene encoding PIPL production of Bacillus thuringiensis has a large Bacillus brevis 47 gene secretion ability.
Introduced into the above, it succeeded in obtaining a novel and useful transformed Bacillus brevis 47, and further succeeded in mass production of PIPLC by culturing the transformed strain, thus completing the present invention. It was

That is, the present invention is Bacillus brevis 47 transformed with a recombinant vector containing the PIPLC gene derived from Bacillus thuringiensis. Furthermore,
A method for producing PIPLC, which comprises culturing the above-mentioned transformed Bacillus brevis 47 in a medium and collecting PIPLC from the culture. Hereinafter, the present invention will be described in detail.

The chromosomal DNA used in the present invention is Bacillus thuringiensis (IAM12077), which is a microorganism belonging to the genus Bacillus, or chromosome D prepared from Bacillus cereus (IAM1208).
NA fragments can be mentioned. The chromosomal DNA fragment is a DNA that carries the genetic information involved in the extracellular extracellular phospholipase C selective production of Bacillus metabolites.

After extraction of the chromosomal DNA, the gene may be amplified by using the polymerase chain reaction (PCR) method or the like. The plasmid into which the chromosomal DNA fragment is incorporated may be any plasmid (vector DNA) that can be propagated in cultured cells, for example pNU211 and pNU200.
Etc.

In addition, the vector DNA contains the chromosome D.
As a method for incorporating the NA fragment, any known method may be used. For example, a method is used in which an appropriate restriction enzyme (endonuclease) is selected and treated to cut the DNA at a specific site, which is then mixed with the DNA similarly used as a vector and ligated with ligase. At that time, if the vector DNA contains a site that is difficult to be cleaved by a restriction enzyme, it may be inserted into the E. coli vector and then subcloned into the vector DNA.

The chromosomal DNA thus obtained
By introducing the ligated product of the fragment and the vector DNA into the cells of a bacterium of the genus Bacillus, which is a recipient bacterium (host), by the transformation method, and proliferating until stable as a genetic trait, the genetic trait on the desired chromosome and the vector A transformant having a DNA trait can be obtained. For example, as a host, Bacillus brevis 47 strain, which is a bacterium that has a large extracellular extracellular secretory ability of an enzyme protein and scarcely secretes a protease into a medium, is preferable. As a transformation method, known Tris-PEG method [Takahashi et al., J. Bacteriol., 15
6,1130-1134 (1983)], etc. to obtain a transformant efficiently.

The transformants obtained in the examples described below can be obtained as follows. That is, the PIPLC gene prepared from Bacillus thuringiensis (which was amplified by PCR was used) was once inserted into the E. coli vector pUC119, and then the vector DNA (p
NU211 plasmid) and the resulting plasmid is introduced into Bacillus brevis 47 strain for transformation reaction.

This transformed Bacillus brevis 47 strain is
It is called Bacillus brevis 47 PIPLC and has been deposited as FERM P-13912 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry. The restriction enzyme digestion map of the pNU211 plasmid of the Bacillus brevis 47 strain is shown in FIG. As is clear from FIG. 1, this plasmid is B at the restriction site of pNU211 plasmid DNA.
A circular DNA molecule in which a DNA fragment of PIPLC, which carries the genetic information involved in extracellular production (secretion) of PIPLC of the Bacillus thuringiensis strain, is incorporated into the spHI and XbaI sites, that is, pNU211 plasmid DNA
And a circular DNA molecule of 5.4 Kb consisting of DNA of about 1 Kb of base pair.

In the figure, EcoRI, BspHI, PstI, Nco
I, ApaL1, HpaI, Mfl II, SacI, BclI, Hp
aI and ClaI represent respective restriction enzyme cleavage sites,
MCS is a multicloning sit (multicloning sit
e), MWP is a middle wall protein
Represents The restriction enzyme site contained in MCS is Pst
I (illustrated), BamHI, SalI, XbaI, KpnI, Bgl
I, XhoI, SacI, and HindIII.

Next, the transformant is cultured, and PIPLC can be obtained from the culture. As the medium,
What is used for the large-scale expression system of Bacillus brevis 47, for example, PX medium is preferable. The PX medium is a medium containing polypeptone S, yeast extract, glucose, and uracil, respectively. Culturing is usually 96-12 at 30 ° C.
It is carried out by culturing with shaking for 0 hours.

The term "cultured product" means any of a culture solution, cultured bacterial cells / cells or a disrupted product thereof, or a culture supernatant solution. As for the expression of PIPLC, after dialysis of the culture obtained by culturing the transformant, the activity using PI as a substrate and SDS-polyacrylamide gel electrophoresis (hereinafter,
It can be confirmed by SDS-PAGE).

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, there is provided Bacillus brevis 47 transformed with a recombinant vector containing the phospholipase C gene derived from Bacillus thuringiensis, and the transformed Bacillus brevis 47 is cultured,
It is possible to provide a method for producing PIPLC, which comprises collecting PIPLC from the culture.

According to the present invention, by mass production of the PIPLC, it is widely used as a research object of biosynthesis and processing until the membrane protein is expressed on the cell surface membrane. The present invention is extremely useful industrially because it can be used as a food material, a reagent for biochemical research, and the like.

[0019]

EXAMPLES The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to these examples. Example 1 Preparation of Transformant Containing Genetic Information for PIPLC Production Ability (1) Preparation of PIPLC Chromosome DNA Bacillus thuringiensis (IAM12077), which has the ability to produce and accumulate PIPLC extracellularly, was used as a medium ( 1% polypeptone, 1% yeast extract, 0.5% NaCl, 0.0
Shaking culture was performed overnight (15 hours) at 37 ° C. in 4% K ₂ HPO ₄ , pH 7.0. 20 ml of the obtained culture was centrifuged at 3,000 rpm for 5 minutes to remove the supernatant, and TE buffer (10 mM Tris, 1
mM EDTA, pH 7.6) 7.56 ml, 10% SDS 400μ
80 .mu.l each of 10 .mu.l and 10 mg / ml of proteinase K (Proteinase K; manufactured by Sigma) were added, and the mixture was allowed to stand at 37.degree. C. for 60 minutes.
To this, 1333.4 μl of 5 M sodium chloride (final concentration 0.7
M) and 1066.4 μl of 10% CATB (hexadecyltrimethylammonium bromide) -0.7M sodium chloride mixture were added, and the mixture was heated at 65 ° C. for 5 minutes. Then, a solution 1 in which chloroform and isoamyl alcohol were mixed at a ratio of 24: 1
0.4 ml was added and the mixture was centrifuged at 3,000 rpm for 5 minutes.

After centrifugation, the upper layer liquid (aqueous layer) was collected (collected amount 9 ml), and 9 ml of a solution prepared by mixing phenol, chloroform and isoamyl alcohol in a ratio of 25: 24: 1 was added thereto, and the mixture was mixed at 5 rpm at 3,000 rpm. Centrifuge for minutes. After centrifugation, collect the upper layer liquid (collection amount 8.4 ml), add 5.04 ml of isopropanol,
It was left standing for 20 minutes at room temperature. After centrifugation at 3,500 rpm for 20 minutes, the supernatant was discarded, the pellet was rinsed with 70% ethanol, air-dried, and dissolved in 200 μl of TE buffer. The obtained chromosomal DNA was about 0.7 mg.

(2) Amplification of PIPLC gene by PCR method PC using 1 μl of chromosomal DNA obtained in (1) as a template
R was performed. As the primers, Sequence Listing 1 (forward primer) and Sequence Listing 2 (reverse primer)
The base sequence shown in was designed and purified. At that time, restriction enzyme sites (BspHI, XbaI) to be incorporated into the expression vector pNU211 and E. coli vector pUC11
Restriction enzyme site (BamHI, Xba
I) was introduced. The purification method is as follows. That is,
The designed primers were synthesized and purified using an automatic DNA synthesizer manufactured by ABI (synthesis and purification were according to the instructions). The purified DNA was dissolved in TE buffer and stored at -20 ° C until use.

Then, the above-mentioned primer was added to a final concentration of 0.5 μM.
Used in. Reaction system is 100 μl, 8 μl in addition to the above
DNTP (dATP, dCTP, dGTP, dTTP
Deoxynucleoside triphosphates consisting of 4 types; manufactured by Takara Shuzo Co., Ltd.), reaction buffer for DNA polymerase, Taq
1 μl of (registered trademark) polymerase (manufactured by Toyobo Co., Ltd.) was added (these used a PCR Regent kit manufactured by Takara Shuzo Co., Ltd. except for the enzyme). The reaction is 94 ° C for 1 minute,
One cycle consists of 94 ° C for 1 minute, 40 ° C for 1 minute, and 72 ° C for 2 minutes, which is 25 cycles, 72 ° C for 5 minutes, and 4 ° C
It was cooled in order.

As a result, a fragment of about 1 kb was obtained. (3) Subcloning of PIPLC gene The PIPLC gene was inserted into an E. coli vector once and then subcloned into pNU211. That is, the PCR fragment obtained in (2) above and the E. coli vector pUC119 were both digested with restriction enzymes BamHI and XbaI, and ligated using a ligation kit manufactured by Takara Shuzo Co., Ltd. according to the instructions. It was introduced into a competent cell JM109 (manufactured by Takara Shuzo) according to the instructions.

As a result of the introduction of JM109, 11 clones having a DNA fragment considered to be the PIPLC gene were obtained. Of these, # 7 (pPI-1) and # 5 (pPI-2) were obtained.
And PIPL for introduction into Bacillus Brevis 47
It was used for subcloning the C gene. pPI-1 (4.
2Kb) and pPI-2 (7.4Kb) were cleaved with restriction enzymes BspHI and XbaI, and this mixture was further treated with restriction enzymes PvuI and PvuII to obtain a 1,014 bp fragment. After ligating the fragments derived from pPI-1 and pPI-2 with pNU211 (designated pNUPI1 and pNUPI2, respectively), Tris-
PEG method [Takahashi et al., J. Bacteriol. 156, 1130-11
34 (1983) transformed into Bacillus brevis 47.

Next, this cell suspension was separately cultivated with shaking to carry out a transformation reaction, and then the cells were collected and washed to obtain transformed Bacillus brevis 47. Then, for erythromycin resistant colonies, plasmid DNA was prepared by alkaline lysis and phenol extraction, and BspHI and XbaI were prepared.
The presence or absence of the 1 Kb fragment produced by

As a result, 41 erythromycin-resistant colonies were obtained from pPI-1 and 70 from pPI-2. Of these, those containing the 1 Kb insert were 30 from pPI-1 and 21 from pPI-2. It was an individual. In addition, PI
Those expressing PLC have 29 pPI-1 origins,
The number of pPI-2 origins was zero. Here, the PIPL
In a pPI-1-derived colony expressing C,
A plasmid containing the PIPLC gene was hereafter referred to as pNUP
I1-1.

Example 2 Expression of PIPLC The transformed Bacillus brevis 47 obtained in Example 1
By culturing (pNUPI1-1), PIPL
Attempted to produce a large amount of C. Therefore, when culturing the transformed Bacillus brevis 47,
The culturing conditions capable of producing a large amount of L.

(1) Cultivation of transformed Bacillus brevis 47 [Study of medium composition] Transformed Bacillus brevis 47 was shake-cultured in a medium test tube together with 2 ml of each composition shown in Table 1 (30). ℃
48 hours or 72 hours). Further, Bacillus brevis 47 into which the plasmid pNU211 having no PIPLC gene was introduced was used as a control, and shake culture was performed in the same manner.

Here, in the PX medium used in this example, a medium in which the content of polypeptone S was 3% was PX '.
As the medium, it was decided to distinguish it from the PX medium in which the content of polypeptone S was 2%.

[0030]

[Table 1]

Each medium contains 10 μg / ml erythromycin. After culturing, the culture is centrifuged (6,000 rpm, 5
Min) and dialysis (4 ° C., 24 hours) to obtain a culture supernatant, which was used as a sample for SDS-PAGE and PIPLC activity measurement. The following operations were performed to measure the activity of PIPLC contained in the sample. That is, the obtained culture supernatant was mixed with 10 mM Tris-HCl buffer (pH 7.5).
After dialysis using P., the production of PIPLC was confirmed by PI-SDC (phosphatidylinositol-sodium deoxycholate) mixed micelle degrading activity. Then, the amount of the enzyme that decomposes 1 μmol of PI per minute to produce 1 μmol of myo-inositol phosphate was defined as 1 unit (U) of PIPLC.

As a result, the highest PIP was found in the sample obtained by culturing for 72 hours in PX 'medium containing 0.7% of yeast extract.
LC activity was observed (see Figure 2). In the medium name of Fig. 2,
PX'0.3, PX'0.5, PX'0.7, and PX'0.9 refer to PX 'medium containing yeast extract at 0.3, 0.5, 0.7, and 0.9%, respectively. On the other hand, SDS-PAGE for the sample
Then, the expression of PIPLC was confirmed (Fig. 3).

Table 2 shows the retained plasmids and medium composition (content of polypeptone S and yeast extract) corresponding to each lane in FIG.

[0034]

[Table 2]

As shown in FIG. 3, a protein (PIPLC; arrow position) having the same mobility as the purified enzyme of Bacillus thuringiensis (lane indicated by BT) was detected. The strongest band was observed in the sample obtained by culturing in the PX 'medium containing 0.7% yeast extract (lane 8). In addition, M represents a molecular weight marker (kDa). In addition, transformed Bacillus containing pNU211
No band recognized as PIPLC appeared in the sample derived from Brevis 47.

(2) Cultivation of transformed Bacillus brevis 47 [Study of pH of medium] When the composition of the medium of (1) was examined, it was found that the pH of the medium increased with time. That is, in the case of T2ura medium, the second day was 7.8, the third day was 8.4, and PX and P
In the case of X'medium, the second day is 8.0-8.2 and the third day is 8.2-
It was 8.4. Then transform Bacillus brevis 47
The pH at the start of culturing and the PIPLC
The relationship with the expression level was examined. P containing 0.7% yeast extract
The culture system was 2 ml using X'medium. From the obtained culture supernatant, the PIPLC activity was measured in the same manner as in (1), and the absorbance at a wavelength of 660 nm (OD ₆₆₀ ) was measured as an index of the growth of the bacterium.

In addition, in addition, when HEPES was added to the medium [HEPES (+)] and when it was not added [HE
The effect of the culture conditions of PES (−)] on the increase in pH (the effect of relaxing or suppressing the increase in pH, etc.) was examined, and the effect on PIPLC expression was also examined. The HEPES concentration was 10 mM. Pre-sterilization medium,
The results of the relationship between the pH at the start and the end of the culture and the PIPLC activity and the amount of protein are shown in Table 3 for HEPES (+) and Table 4 for HEPES (−).

[0038]

[Table 3]

[0039]

[Table 4]

On the other hand, SDS-PAGE was performed on the samples obtained by culturing at various pHs, and the results shown in FIG. 4 were obtained. In FIG. 4, M represents a molecular weight marker, and arrows represent the positions of the PIPLC bands (hereinafter, SD
The same in the S-PAGE figure). Table 5 shows the retained plasmids corresponding to each lane, the pH of the medium, and the presence / absence of HEPES.

[0041]

[Table 5]

From Table 3, Table 4 and FIG. 4, the influence of pH at the start of culture was hardly seen in the 2 ml system. Also, H
The effect of mitigating and suppressing pH rise by adding EPES is 10 mM
Was not observed in. (3) Culture of transformed Bacillus brevis 47 [Study of aeration conditions of medium] PX 'containing 0.7% yeast extract in 1,000 ml Sakaguchi flask
Transformed Bacillus brevis 47 carrying pNUPI1-1 in 50 to 200 ml of medium for 24 to 120 hours (1 to 5
(Day) The cells were cultured, and the proliferation of the cells after the culture was measured using the absorbance at a wavelength of 660 nm as an index. At the same time, the activity of PIPLC in each culture was also measured. As a control, Bacillus brevis 47 carrying pNU211 was cultured in 50 ml of medium for 1 to 5 days.

The results showing the degree of growth of bacteria and the PIPLC activity are shown in FIGS. 5 and 6, respectively. On the other hand, the expression of PIPLC in each culture was confirmed by SDS-PAGE (FIGS. 7 and 8). The culture conditions corresponding to each lane are shown in Table 6 for FIG. 7 and Table 7 for FIG.

[0044]

[Table 6]

[0045]

[Table 7]

From these results, it is considered that it is suitable to use 100 ml of medium for 1,000 ml Sakaguchi flask. In addition, the ratio of PIPLC to the total protein determined from the results of SDS-PAGE and the specific activity of the purified enzyme derived from Bacillus thuringiensis (600-1,000 U / mg
It is considered that about 400 μg / ml of PIPLC is produced from the protein).

(4) GPI-type membrane protein release test of PIPL The PIPLC thus obtained was subjected to a GPI-type membrane protein release test using alkaline phosphatase release as an index. 1 ml of pig kidney microsomes, p
PIPLC derived from transformed Bacillus brevis 47 harboring NUPI1-1 or pNU211 (100 μ each)
l; 1.0 mU or 0.5 mU), or PIPLC derived from Bacillus thuringiensis (100 μl each; 1.0 mU or 0.5 mU) (0, 10, 20 minutes) and centrifuged at 40,000 × g to collect the supernatant. Then, an enzyme solution containing free alkaline phosphatase was obtained. Then, 100 μl of the enzyme solution was added to 900 μl of the substrate solution of alkaline phosphatase, and the mixture was reacted at 37 ° C. for 10 minutes.
After the reaction, 200 μl of 15% TCA was added to completely inactivate the alkaline phosphatase activity, followed by centrifugation (3,000 rpm).
Later supernatant was obtained. Then, after adding 100 μl of 0.1N sodium hydroxide to 600 μl of the supernatant, the absorbance was measured at a wavelength of 420 nm.

As a result, as shown in FIG. 9, pNUPI1
P derived from transformed Bacillus brevis carrying -1
IPLC is a PI derived from Bacillus thuringiensis
It released alkaline phosphatase to the same extent as PLC.

[0049]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence: GCGGATCCTC ATGAGCAATA AGAAG 25 SEQ ID NO: 2 Sequence length: 23 Sequence type: Nucleic acid Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence: CGTCTAGAAT CGCTTCTTAC TCT 23

[Brief description of drawings]

FIG. 1 is a view showing a restriction enzyme digestion map of pNU211 plasmid.

FIG. 2 is a diagram showing PIPLC activity.

FIG. 3 is a diagram showing a result of SDS-PAGE.

FIG. 4 is a diagram showing a result of SDS-PAGE.

FIG. 5 is a diagram showing a growth curve of transformed Bacillus brevis 47.

FIG. 6 is a diagram showing PIPLC activity.

FIG. 7 is a diagram showing a result of SDS-PAGE.

FIG. 8 is a diagram showing a result of SDS-PAGE.

FIG. 9 is a diagram showing the alkaline phosphatase releasing action of PIPLC.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:06) (72) Inventor Etsumi Tamura 3-15 Otetsuyama Tenpaku-ku Nagoya-shi Aichi 6 (72) Inventor Cormorant ▲ High ▼ Shigezo 1-24-3 Uenoencho, Meito-ku, Nagoya-shi, Aichi (72) Inventor Hiroo Ikezawa 2-44-6 Shimoyama-cho, Mizuho-ku, Nagoya-shi, Aichi

Claims (2)

[Claims] 1. Bacillus thuringiensis
Bacillus brevi transformed with a recombinant vector containing a phosphatidylinositol-specific phospholipase C gene derived from us thuringiensis).
s) 47. 2. A phosphatidylinositol-specific phospholipase C characterized by culturing the transformed Bacillus brevis 47 according to claim 1 in a medium and collecting phosphatidylinositol-specific phospholipase C from the culture. Production method.