JPH0520067B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a recombinant DNA incorporating a staphylokinase producing gene and a recombinant thereof.
This invention relates to Bacillus subtilis containing DNA and a method for producing staphylokinase by culturing the Bacillus subtilis. More specifically, the present invention provides lysogenic phage DNA of Staphylococcus aureus.
A recombinant DNA in which a DNA fragment carrying genetic information for staphylokinase production obtained by cutting with a restriction enzyme was incorporated into a multicopy plasmid (muticopy plasmid) that can be replicated in Bacillus subtilis 168 strain, and this recombinant DNA The present invention provides a method for producing staphylokinase, which comprises culturing Bacillus subtilis and collecting staphylokinase from the culture solution. What is staphylokinase (hereinafter referred to as SAK)?
It is a fibrinolytic enzyme produced by Staphylococcus aureus (hereinafter referred to as S. aureus) and has the function of converting plasminogen in the blood into plasmin. This plasmin acts on fibrin and dissolves it. Urokinase, which is present in human urine, and streptokinase, which is produced by streptococci, are known to have similar effects, and both are used medically as so-called fibrinolytic agents such as blood clotting inhibitors and thrombolytic agents. It's something you get. Therefore, various methods for producing and extracting and purifying these fibrinolytic agents have been studied. Regarding the production of SAK, it has been shown that the ability to produce SAK is a trait that is imparted to the host (this is called lysogen conversion) during lysogenization of the lysogenic phages that host S. aureus. It is known,
Conventionally, we have either selected S. aureus strains that have the ability to produce SAK, or lysogenized S. aureus strains that do not produce SAK using a phage that converts SAK into lysogens, and cultured these strains. Therefore, a method of collecting SAK from the culture solution has been used. However, since S. aureus is a highly pathogenic bacterium and its handling requires extremely careful consideration, this method is not originally suitable for industrial SAK production. In order to increase the yield, long-term cultivation is required, and in this sense as well, it lacks suitability as an industrial production method. As a method for industrially producing SAK, it was previously discovered that the gene responsible for staphylokinase production genetic information (hereinafter referred to as SAK production genetic information) exists on the lysogenic phage DNA of S. aureus. A method has been proposed in which a fragment of S. aureus lysogenic phage DNA is introduced into E. coli via an E. coli vector and the E. coli is cultured (see Japanese Patent Application No. 164064/1982). The present inventors have developed a method using Escherichia coli, in which a fragment of S. aureus lysogenic phage DNA is transferred to Bacillus subtilis 168 strain and strains derived therefrom via a multicopy plasmid that can be replicated in Bacillus subtilis strain 168. It was successfully introduced into a strain derived from Bacillus subtilis 168, and the present invention enables production with high production efficiency.
This led to the provision of a method for manufacturing SAK.
Therefore, the present invention carries genetic information for SAK production.
The present invention provides a recombinant DNA incorporating a DNA fragment, a Bacillus subtilis containing this recombinant DNA, and a method for producing SAK by culturing this Bacillus subtilis. The present invention will be explained in detail below. Recombinant DNA incorporating the staphylokinase-producing gene according to the present invention, and its recombinant
Bacillus subtilis having the ability to produce SAK containing DNA is obtained in the following steps. Lysogenic phage DNA of S. aureus, which is a DNA donor, is cut with restriction enzymes. Extract DNA fragments containing SAK production genetic information. The vector DNA is cleaved with restriction enzymes. at the cleavage site of the vector DNA or
Incorporate DNA fragments. This recombinant DNA is introduced into a host strain derived from Bacillus subtilis 168. Bacillus subtilis capable of producing SAK is selectively isolated. The above lysogenic phages as DNA donors are
Any lysogen phage isolated from an S. aureus lysogen strain capable of producing SAK and having lysogen conversion ability can be used. Examples are 42D, L42E,
77 (Reference: Mason, RE and Allen, WE (1975)
Can.J.Microbiol. 21 , 1113−1116), pφ1, Pφ2,
Tφ−42D, pφ−406 (Reference: Kono, I.and
Fujise, K. (1977) Infect. Immun. 18 , 266-272).
Rφ19, SφC (Reference: Isamu Kondo et al. (1980) Tokyo Jikei University School of Medicine Journal 95 , 1203-1206). The means used to prepare lysogenic phage DNA are conventional. That is, the lysogenic phage is infected with its host bacterium, S. aureus, and the infected bacterium is cultured.
E. and Williams, REO (1961) Bull. WHO 24 ,
771-784). This causes the phages to come out into the medium (the bacteria lyse),
This phage can be removed by a suitable method (e.g. cesium chloride equilibrium density gradient centrifugation, Reference: Rosenblum, ED).
and Tyrone, S. (1964) J. Bacteriol. 88 , 1737−
1742), and phage DNA can be further extracted from this phage by a known method (for example, phenol extraction method, etc.). Cleavage of lysogenic phage DNA with restriction enzymes in the above step is performed as follows. By adding appropriate restriction enzymes to phage DNA and performing the reaction under appropriate reaction conditions, phage
The DNA is cut into various fragments by added restriction enzymes. The restriction enzyme that can be used to cleave this phage DNA must be 1) capable of cleaving phage DNA, and 2) not cleaving the DNA portion that carries genetic information for producing SAK. Suitable restriction enzymes include Hind, Pst, Acc
,Ava,EcoR,Hpa,Hpa,Hind
, Sst, Cla, BstE, Sst, Xho,
Bcl, Bgl, Pvu, Xor, Kpn, Sma
, Xba, etc. The above step is a step of separating a DNA fragment having SAK production genetic information from a group of fragments generated as a result of cutting phage DNA with restriction enzymes. gel electrophoresis, etc.) are used. In order to determine in advance which fragments of the phage DNA fragments obtained in the process contain SAK production genetic information, it is necessary to
Measuring the molecular weight (i.e. length as DNA) of the DNA fragment by a known method, such as agarose gel electrophoresis, and determining the location and size on the phage DNA of the DNA fragment that carries the genetic information for SAK production. I can do it. Although the above step may be omitted depending on the case, it is desirable not to omit this step in order to operate the process efficiently. The above step, cleavage of vector DNA,
The vector is created by adding an appropriate restriction enzyme to the vector DNA and carrying out the reaction under appropriate reaction conditions.
Can cleave DNA. The vector DNA is a multicopy plasmid that can be replicated in Bacillus subtilis strain 168,
For example pUB110 and its derivatives can be used. The above step is a step of incorporating the above-mentioned DNA fragment into the cleavage site of the vector DNA,
The method used in this step is a conventional one, and depends on the type of phage DNA and vector used.
Appropriate reaction conditions are selected depending on the type of DNA and the type of restriction enzyme. As long as the replication ability of the vector is not impaired, the direction and site for inserting the above-mentioned phage DNA fragments do not matter. The above step is a step in which recombinant DNA is introduced into a host strain derived from Bacillus subtilis 168, but the host Bacillus subtilis is a Bacillus subtilis strain lacking restriction ability among the restriction/modification systems specific to Bacillus subtilis 168 strain. are all available. In addition, recombinant plants that incorporate DNA that carries SAK production genetic information that has undergone modification
DNA can also be used against Bacillus subtilis which has restriction capacity. As the host Bacillus subtilis, Bacillus subtilis
168 strain and strains derived therefrom can be used. In addition, in the display of Bacillus subtilis original strains used below, r ^- ₁₆₈ indicates a strain lacking restriction ability, and
m ^- ₁₆₈ indicates a strain that does not have modification ability. The means of introduction itself is a known method (for example, Spizizen's method: Reference Spizizen, J. (1958) Proc. Natl.
Acad.Sci.USA 44 , 1072-1078) can be used. Even if the above-mentioned step is performed, the DNA fragment carrying the SAK production genetic information of the lysogenic phage may not be incorporated, and if the step is omitted, the SAK production ability In some cases, DNA fragments that are not related to the protein may be incorporated. The process for that purpose, namely SAK
It is necessary to selectively isolate Bacillus subtilis that has the ability to produce. This selective isolation of Bacillus subtilis can be carried out using known methods used for S. aureus (for example, see Kondo, I and Fujise, K. (1977)).
Infect. Immun. 18 , 266-272). That is, by this method, a heated plasma agar medium is prepared, test bacteria are placed on it, and fibrin decomposition ability is determined by culturing for a certain period of time.
Those that have the ability to decompose fibrin comb the area around the colony and create a transparent zone, so only this bacteria can be extracted. Bacillus subtilis having the ability to produce SAK obtained as described above has no different mycological properties compared to the original Bacillus subtilis except that it has the ability to produce SAK. Its general mycological properties are shown in the attached table below (examples are those obtained in the Examples below). In the method for producing SAK according to the present invention, the novel Bacillus subtilis having SAK-producing ability obtained as described above is cultured according to a conventional method, and from the culture solution, SAK
This is done by separating and purifying the The means used for separation and purification are conventional means used in such cases, such as salting out, gel filtration, adsorption chromatography, affinity chromatography, and combinations thereof, and the type thereof is not specified. The present invention has made it possible to industrially produce SAK using Bacillus subtilis. Unlike other methods, it offers extremely superior advantages in terms of safety, growth rate, mass culture (mass production), etc. To explain this point in more detail, Bacillus subtilis, Bacillus subtilis 168 UOT-0734r ^- ₁₆₈ m ^- ₁₆₈ (pBSAK 135) obtained in Example 1 below.
100ml of kanamycin (5mg/) added to L-broth (polypeptone 10g/, yeast extract 5g/, glucose 1g/,
The amount of SAK produced when cultured with shaking at 37°C for 6 hours and 19 hours in NaCl 5g/) was calculated using lysogen phage.
S. aureus RL-TS lysogenized by SφC
Table 1 shows the results of comparing the amount of SAK produced when the strain was cultured in the same amount of L-broth without kanamycin for the same period of time. Bacillus subtilis can produce 10 times more SAK than S. aureus RL-TS strain when cultured for 19 hours (stationary phase), and about 27 times more when cultured for 6 hours (logarithmic phase).
It will be possible to produce SAK. This not only shows that the novel method for producing SAK using Bacillus subtilis of the present invention is suitable for mass production, but also because a sufficient amount of SAK is produced even in the logarithmic phase.
In addition to being able to shorten the culture time, Bacillus subtilis inherently has the ability to produce various other enzymes (e.g. amylase), and most of these are produced during the stationary phase, so the culture can be stopped before that point. This indicates that a product with a low content of other enzymes can be obtained, and therefore the separation and purification of SAK can be extremely efficient. [Table] Examples of the present invention are listed below. Example 1 This example describes the lysogen phage of S. aureus.
E. coli containing SφC-derived SAK production genetic information,
pUBSF-, a derivative of plasmid pUB110 that can be replicated in Bacillus subtilis, contains a fragment containing SAK production genetic information from Escherichia coli K12 WA802 (pSAK361).
This is an example of a recombinant strain introduced into 13ΔH and Bacillus subtilis. a) Preparation of fragment containing SAK production genetic information Escherichia coli, Escherichia coli K12 WA802
(pSAK361) ATCC39178 with ampicillin (40
mg/) in L-broth (polypeptone
After inoculating 100 ml of yeast extract 5 g/), glucose 1 g/, NACl 5 g/) and culturing at 37°C for 16 hours, chloramphenicol was added to the culture solution to a concentration of 150 μg/ml, and further 37 After culturing at ℃ for 15 hours, collect the bacteria by centrifugation. 50ml 10mM Tris−HCl (PH8.0) −15mM
Wash the bacterial cells with NaCl-1.5mM sodium citrate solution and add 3.2ml of 25% sucrose-50mM Tris-
Suspend in HCl (PH8.0). Add 1.6ml to this
250mM EDTA (PH8.0), 1ml of 5mg/ml lysozyme, 6ml of 2% bridge 58-62.5mM
Add EDTA-50mM Tris-HCl (PH8.0),
Incubate at 30°C for 15 minutes to gently lyse the cell wall, then centrifuge at high speed (at 4°C).
30000 rpm, 30 minutes) to precipitate the bacterial cells,
Obtain the supernatant. This was centrifuged using ethidium bromide-cesium chloride equilibrium density gradient centrifugation (at 10°C).
36,000 rpm for 48 hours) to purify the plasmid DNA. 1.0μg of this plasmid DNA
10mM Tris−HCl (PH7.6) −7mM MgCl ₂ −
7mM β-mercaptoethanol - 50mM
Add to the reaction mixture containing NaCl and 4U EcoRI,
Incubate at 37°C for 60 minutes, stop the reaction by keeping at 60°C for 10 minutes, add 4 μl of 5M NaCl, add 50 μl of ethanol chilled at -20°C, and incubate at -20°C.
After standing for 30 minutes, centrifuge at 15,000 rpm for 5 minutes to collect the precipitate, and wash again with cold ethanol. Dry this under reduced pressure and dissolve by adding 20 μl of sterilized water.
Add 3μl of 10mM ATP, 3μl of 100mM dithiothrate, and 660mM Tris-HCl (PH7.6) to this solution.
Add 3 μl of 66 mM MgCl ₂ and 1 μl of 200 U/μl T4 ligase, and react at 15° C. for about 16 hours.
The reaction product is washed with cold ethanol as above. This washed plasmid was added to 10mM Tris−
HCl (PH7.6) - 7mM MgCl2 _- dissolved in 10μl of 7mM β-mercaptoethanol, 4U/μl Psi
Add 1 μl of the solution and react at 37°C for 60 minutes.
After stopping the reaction by keeping at 60°C for 10 minutes, wash with cold ethanol as above. This wash was then diluted with 10mM Tris-HCl (PH7.6)-7mM _MgCl2-
7mM β-mercaptoethanol - 50mM
Dissolve 4U/μl Hind solution in 10μl of NaCl.
Add 1 μl and react at 37°C for 60 minutes. 10 at 60℃
Stop the reaction for a minute and wash with cold ethanol as above. When the size of this fragment was measured by agarose gel electrophoresis, it was found to be 2.5 kb. b) Preparation and cleavage of pUBSF-13ΔH pUB110 was converted into Spores 109-113.
pUBSF-13 as described in (1981).
(6.7kb). 1μg of this pUBSF−13
10mM Tris−HCl (PH7.6) −7mM MgCl ₂ −
7mM β-mercaptoethanol - 50mM
Dissolve 4U/μl Tind solution in 10μl of NaCl.
Add 1 μl and react at 37°C for 60 minutes. Stop the reaction by keeping at 60°C for 10 minutes, add 4 μl of 5M NaCl,
Add 50 μl of ethanol chilled to −20 °C and
Cool at 20°C for 30 minutes. 5 minutes at 0℃
After centrifugation at 15,000 rpm, the supernatant is discarded and washed again with -20°C ethanol. After centrifugation (15,000 rpm for 2 minutes), the ethanol is discarded, and the ethanol is completely evaporated. Now add 20 μl of sterile water and dissolve.
3μl of 10mM ATP, 3μl of 100mM dithiothreitol, 660mM Tris-HCl (PH7.6) -66mM
3 μl of MgCl ₂ and 200 U/μl of T4 ligase.
Add 1 μl and react at 15°C for about 16 hours. The reaction product is pUBSF-13ΔH (6.4kb).
The reaction product is washed with cold ethanol as above and cleaved with Pst and Hind as in a). This cut DNA is washed again with cold ethanol. c) Preparation of recombinant plasmid 1.0μg of DNA fragment obtained in a), 0.5μg of cleaved pUBSF-13ΔH obtained in b), 30mM Tris-
The reaction was carried out at 20℃ for 4 hours in a 20μl reaction solution containing HCl (PH7.6), 10mM MgCl ₂ , 10mM dithiothreitol, 0.1mM ATP and 1μT4 ligase, and 1μl of 0.25M EDTA (PH8.0) was added. In addition, the reaction is stopped. After that, heat at 65℃ for 5 minutes to inactivate the ligase, and add 180μl of 10mM Tris.
-HCl (PH8.0) - Add 1mM EDTA -20℃
Save to. This recombinant plasmid
It was named pBSAK135 (8.5kb). d) Introduction of recombinant plasmid into Bacillus subtilis Bacillus subtilis, Bacillus subtilis 168UT0734r ^- ₁₆₈
m ^- ₁₆₈ lys21metB5 hisA1 (Microtechnology Research Institute No. 6872)
into 5 ml of CI medium for transformation (Ana-
gnostpoulos, C. &. J. Spizizen (1961) J. Bact.
81, 741) and cultured with shaking at 37°C for 5 hours. After culturing, collect the bacteria by centrifugation. This bacterium is suspended in 10 ml of C medium for transformation (see above-mentioned document), and cultured with shaking at 37°C for 30 minutes. To 0.5 ml of this bacterial solution, add 1 μg of the recombinant plasmid solution obtained in step (c) above as DNA, and
Incubate with shaking for 1 hour. Add 4.5 ml of C medium to this, and further culture with shaking at 37°C.
This bacterial solution was applied to an L-broth agar medium (agar concentration 1.5%) containing 5 μg/ml of kanamycin.
Kanamycin-resistant Bacillus subtilis is isolated by culturing at ℃ for 16 hours. e) Selective isolation of Bacillus subtilis capable of producing SAK Dispense 5 ml of human plasma into test tubes and incubate at 56°C.
Heat for 20 minutes, then dispense 15 ml of ordinary agar medium containing 2% agar, which was dissolved separately, and warm to 56°C. Mix both quickly on a shear plate,
Leave it until it hardens. Place the bacterial suspension of Bacillus subtilis test bacteria obtained in step d) on this agar medium, and
Culture at ℃. Bacteria capable of producing SAK decompose fibrin and comb the area around the colony, creating a transparent zone. The bacteria that creates this transparent zone was isolated and Bacillus subtilis UOT-
It was named 0734 (pBSAK135) (Feikoken Bibori No. 6871). f) Production and extraction of SAK Bacillus subtilis UOT- obtained in e)
0734 (pBSAK135) was inoculated into 50 ml of L-broth and cultured with shaking at 37°C for 16 hours. 6 times this bacterial liquid
The cells were inoculated into L-broth and cultured with shaking at 37°C for 12 hours. After the culture, the cells were centrifuged to remove the bacterial cells. Add ammonium sulfate to the culture solution while stirring to achieve 100% saturation, and leave at 4°C for 16 hours. Collect the precipitate by centrifugation (8000 rpm for 20 minutes) and dilute to 10mM
Dissolve in 10 ml of Tris-HCl (PH7.5) and dialyze against the same buffer 2 at 4°C for 16 hours. A crude SAK solution was thus obtained. This crude SK solution is
It had SAK activity of 2 million U. Example 2 This example describes the lysogenic phage of S. aureus.
Directly cut SφC DNA with Pst and Hind.
This is an example of recombining pUBSF-13ΔH and introducing it into Bacillus subtilis. a) Preparation and cleavage of SφC DNA Literature (Blair, JEand Williams, REO
( 1961 ) Bull. 300 ml of molecular weight 7,000-8,000) was added, stirred well, and left at 0°C for several hours. after that
Centrifuge at 10,000 rpm for 15 minutes to collect the precipitate, which is added to 20 ml of buffer (10 mM Tris-HCl, 10 M
MgSO ₄ , 5mM CaCl ₂ , 0.01% gelatin; PH
7.4), DNase and RNase were added thereto at a final concentration of 1 μg/ml, and the mixture was reacted at 37°C for 20 minutes. After the reaction
Centrifuge at 10,000 rpm for 10 minutes and remove the supernatant.
Dissolve the precipitate obtained by centrifugation at 30,000 rpm for 60 minutes in 2.5 ml of the same buffer as above, and add about 1.7 g to this.
of cesium chloride and subjected to equilibrium density gradient centrifugation at 27,000 rpm for 18 hours. The banded phages thus obtained are collected and dialyzed against the same buffer as above in 1. Add 50 μl of 1.5 M NaCl − 0.15 M sodium citrate, 5 μl of 250 mM EDTA (PH7.0) to 0.5 ml of the obtained Phage solution.
After adding 5 μl of 10% SDS and leaving the mixture at 37° C. for 5 minutes, add 0.6 ml of 0.15 M NaCl-15 mM sodium citrate solution saturated phenol and mix gently at room temperature for about 10 minutes. 10 at 3000rpm
Centrifuge for a minute and collect the upper layer (aqueous layer). After repeating this phenol treatment, washing with equal amounts of ether was performed three times, and the resulting aqueous layer was washed with 0.15M
NaCl - 15mM Sodium Citrate - 1mM
Dialyze 3 times against 200 ml of EDTA. Add 4U of Pst to 1μg of this DNA,
10mM Tris−HCl (PH7.6)−7mM _MgCl2 −
10 μl of 7mM β-mercaptoethanol buffer
After the reaction, the reaction was stopped at 60°C for 10 minutes, and the reaction was washed with cold ethanol. 4U of Hind was then added to this wash, and 10mM Tris-HCl (PH7.6) _-7mM Mgcl2
-7mM β-mercaptoethanol -50mM
Incubate in 10 μl of NaCl buffer at 37°C for 60 minutes. After the reaction, wash with cold ethanol in the same manner. The obtained fragment group is subjected to 1% agarose gel electrophoresis to collect only fragments with a size of 2.5 kb. b) A recombinant plasmid was prepared in the same manner as in Example 1, and Bacillus subtilis
When introduced into 168UOT-0734r ^- ₁₆₈ m ^- ₁₆₈ lystB5 hisa1, the same bacteria as obtained in Example 1 were obtained. [Table] [Table]

Claims (1)

[Scope of Claims] 1. A multi-copy plasmid capable of replicating a DNA fragment carrying staphyrokinase-producing genetic information obtained by cleaving Staphylococcus aureus lysogenic phage DNA with a restriction enzyme in Bacillus subtilis strain 168.
Recombinant DNA incorporated into pUB110 or its derivatives. 2. The recombinant DNA according to claim 1, wherein the multi-copy plasmid pUB110 or its derivative is pUBSF-13ΔH. 3. The recombinant DNA according to claim 1, wherein the recombinant DNA is pUBSAK135. 4 A multicopy plasmid that can replicate the DNA fragment carrying the genetic information for staphyrokinase production obtained by cleaving Staphylococcus aureus lysogenic phage DNA with restriction enzymes in Bacillus subtilis strain 168.
A novel Bacillus subtilis having the ability to produce staphylokinase, which is obtained by introducing a recombinant DNA incorporated into pUB110 or a derivative thereof into Bacillus subtilis 168 strain or its derived strain. 5 A multi-copy plasmid that can replicate the DNA fragment carrying the genetic information for producing staphyrokinase obtained by cleaving the lysogenic phage DNA of Staphylococcus aureus with a restriction enzyme in Bacillus subtilis strain 168.
A method for producing staphylokinase, which comprises culturing Bacillus subtilis in which a recombinant DNA incorporated into pUB110 or a derivative thereof is introduced into Bacillus subtilis strain 168 or its derived strain, and collecting staphylokinase from the culture solution. .