JPH0479640B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing staphylokinase by culturing a novel Escherichia coli containing a staphylokinase-producing gene. Staphylokinase (hereinafter abbreviated as SAK)
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, but both are used medically as so-called fibrinolytic agents such as anticoagulants and antithrombotic agents. It is something that can be provided. Therefore, various methods for producing and extracting and purifying these substances are being 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 lysogen phages that host S. aureus. is known,
Conventionally, S. aureus strains with the ability to produce SAK are selected, or S. aureus strains that do not produce SAK are lysogenized using a phage that converts SAK into lysogens, and these strains are cultured. Therefore, a method is being used to collect SAK from the culture fluid. However, S. aureus is a highly pathogenic bacterium, and its handling requires extremely careful consideration, so this method is not originally suitable for industrial SAK production, and it also reduces the yield of SAK. In order to increase the yield, long-term cultivation is required, and from that point of view as well, it is not suitable as an industrial method. In terms of safety and efficiency, the present inventors
As a result of intensive research into an appropriate method for industrially producing SAK, we discovered that a gene responsible for SAK productivity information exists on phage DNA.
We succeeded in introducing a lysogenic phage DAN fragment of S. aureus into E. coli via a vector. The present invention is based on these results, and therefore, the present invention provides a method for producing SAK by culturing a novel E. coli containing a SAK-producing gene. The present invention will be explained in detail below. The novel E. coli used in the method of the present invention is
It is obtained by the following steps. Lysogenic phage DNA of S. aureus, which is a DNA donor, is cut with restriction enzymes. Extract DNA fragments containing SAK biogenetic information. (This step can be omitted.) Cleave the vector DNA with a restriction enzyme. at the cleavage site of the vector DNA or
Incorporate DNA fragments. This recombinant DNA is introduced into host E. coli. E. coli that has the ability to produce 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: Kondo, 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), etc. 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 (this method is described, for example, in the literature: Blair et al.
JEand Williams, REO (1961) Bull.WHO
24, 771–784). This causes the phage to come out into the medium (the bacteria lyse), so the phage can be removed using an appropriate method (e.g., cesium chloride equilibrium density gradient perspective method, Reference: Rosenblum,
EDand 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 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. Restriction enzymes that can be used to cleave phage DNA include: (1) those that can cleave phage DNA;
and (2) it must not cut the DNA portion that carries genetic information related to SAK production. Restriction enzymes suitable for this include Hind, Pst, Acc
,Ava,EcoR,Hpa,Hpa,Hind
, Sst, Cla, BstE, Sst, Xho,
Bcl, Bgl, Pvu, Xor, Kpn, Sma
, Xba, etc. Next, the phage DNA was cut with restriction enzymes.
DNA containing SAK production information from a group of DNA fragments
The fragments are separated by various known methods (eg, agarose gel electrophoresis, etc.). For this purpose, it is necessary to identify which fragments among the phage DNA fragments.
It must be determined in advance whether it has SAK production information. This determination method will be described later. The above DNA fragment separation operation may be omitted depending on the case. On the other hand, cleavage of vector DNA
The vector DNA can be cleaved by adding an appropriate restriction enzyme to the vector and carrying out the reaction under appropriate reaction conditions. Known vector DNAs can be used. For example, Colel, pMB9, pSC101,
p15A and its derivatives (e.g. pBR322,
pACYC184, etc.) and the chiaron vector derived from λ phage. Next, insert the above-mentioned DNA into the cleavage site of the vector DNA.
The fragments are incorporated, but the method itself is a conventional method, and the type of phage DNA used,
Appropriate reaction conditions are selected depending on the type of vector DNA and the type of restriction enzyme. The above phage
The direction and site for inserting the DNA fragment are not restricted. Next, the recombinant DNA is introduced into the host E. coli, and any E. coli strain lacking the restriction ability among the E. coli-specific restriction/modification systems can be used as the host E. coli. Furthermore, a plasmid incorporating a DNA carrying genetic information for producing SAK that has been modified can also be used for E. coli strains that have restriction ability. The host E. coli may be limited depending on the type of vector used. Of the E. coli original strains used, r ^- _K indicates a strain lacking restriction ability, and m ^- _K indicates a strain lacking modification ability. The method of introduction is a known method (for example,
Method of Comeron et al.: Cameron, JRet al.
(1975) Proc.Natl.Acad.Sci.USA 72 , 3416~
3420) 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. For this purpose, it is necessary to selectively isolate E. coli that has the ability to produce SAK. For this selective isolation of E. coli, 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 spotted 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. E. coli having the ability to produce SAK is thus isolated, and the DNA carrying genetic information for producing SAK from the E. coli having the ability to produce SAK is analyzed as follows. That is,
A recombinant plasmid or recombinant phage is extracted from E. coli capable of producing SAK, and the recombinant plasmid or recombinant phage is cleaved with the restriction enzyme used to cleave the phage DNA and the vector DNA. This cut DNA
By measuring the molecular weight (i.e. length as DNA) of the fragment by a known method (e.g. agarose gel electrophoresis, etc.), the position of the DNA fragment carrying SAK production genetic information can be determined on the phage DNA. I can do it. An example of an analytical experiment is shown below. Experimental example: When using SφC as phage DNA (1) When cut with restriction enzyme Pst: 15.8kb (2) When cut with restriction enzyme Hind: 4.9kb (3) When cut with restriction enzymes Hind and Ava
2.0kb (4) When cut with restriction enzymes Ava and Acc
It was found that SAK production genetic information was present in a 1.3kb region. The bacteriological properties of the E. coli having the ability to produce SAK obtained as described above are the same as those of the original E. coli except that it has the ability to produce SAK. Its general mycological properties are shown in the attached table below (examples are given for those obtained in the Examples below). According to the present invention, by using the novel E. coli obtained as described above, culturing the E. coli and collecting SAK accumulated in the cultured cells, industrial
A method for manufacturing SAK is provided. In the method for producing SAK according to the present invention, the E. coli having the ability to produce SAK obtained as described above is cultured by a conventional method, and after harvesting, Talmadge, K. et al.
al (1980) Proc.Natl.Acad.Sci.USA 77 , 3369~
The periplasmic fraction was collected by the method described in 3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SAK was subjected to conventional protein purification methods such as salting out, gel filtration,
It was purified using adsorption chromatography. By the method of the present invention as described above, using Escherichia coli
Although it has become possible to produce SAK in this way, by making E. coli produce SAK, unlike the conventional technology using S. aureus, it has improved safety, growth rate, and mass culture (mass production). ) and other surprising benefits. Moreover, in the case of E. coli, SAK accumulates in the periplasm,
The conventional method provides a concentration more than 100 times higher than when obtained from the supernatant fraction using S. aureus, and there are fewer contaminating proteins than when extracting by disrupting the whole cell. A particular advantage is obtained that the separation and purification can be easily carried out. The above-mentioned phenomenon of accumulation in the periplasm can be seen, for example, in E. coli K12 C600r ^- _K m ^- _K (pSAK 361) obtained in Example 1 listed later.
200ml L-broth (polypeptone 10g/, yeast extract 5g/, glucose 1
The table below shows an example of the distribution and percentage of SAK activity when cultured at 37°C for 5 hours with
was found to be present in the periplasmic fraction. Totality (u) % Supernatant fraction 950 23 Periplasm fraction 2400 58 Cytoplasm fraction 800 19 Examples of the present invention are listed below. Example 1 This example uses SφC as the DNA donor and vector
pBR322 as DNA and Hind as restriction enzyme
and Escherichia coli as host E. coli original strain.
K12C600r ^- _K m ^- _K or Esierhia coli K12
This is an example using WA802r ^- _K m ⁺ _K. (a) Preparation and cleavage of SφCDNA Literature (Blair, JEand Williams, REO
Phage SφC was grown according to the method of ( 1961 ) Bull. 300 ml of average 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 diluted with 20 ml of buffer (10 mM Tris-HCl, 10 mM
MgSO ₄ , 5mM CaCl ₂ , 0.01% gelatin;
PH7.4) and add DNase and RNase to this.
were added at a final concentration of 1 μg/ml, and 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 phage bands thus obtained are collected and dialyzed against 1 g of the same buffer as above. Add 50μ of 1.5M NaCl-0.15M sodium citrate, 5μ of 250mM EDTA (PH7.0) to 0.5ml of the obtained Phage solution.
and 5μ of 10% SDS, and after standing at 37°C for 5 minutes, add 0.6ml of 0.15M NaCl-15mM sodium citrate solution saturated phenol, and mix gently for about 10 minutes at room temperature. This at 3000rpm
Centrifuge for 10 minutes and collect the upper layer (aqueous layer). After repeating this phenol treatment, the resulting aqueous layer was washed three times with equal amounts of ether.
0.15M NaCl-15mM Sodium Citrate-1
Dialyze 3 times against 200 ml of mM EDTA. To cut phage DNA, 1 μg of DNA
Add 3μ Hind to 10mM Tris-
Buffer of HCl-7mM MgCl2-7mM _β- mercaptoethanol-50mM NaCl (PH7.6)
The reaction is carried out in a 20μ solution at 37°C for 3 hours, and after the reaction is completed, 1μ of 250mM EDTA (PH8.0) is added. Add 5μ to the DNA solution treated with this restriction enzyme.
Add 1.5M sodium acetate (PH7.0), add 60μ of ethanol, and incubate at -70℃ for 10 minutes.
After standing for a minute, centrifuge at 12,000 rpm for 5 minutes to collect the precipitate, and wash it again with ethanol. This was dried under reduced pressure, and 10μ of 10mM tris−HCl−
Dissolve in 1mM EDTA (PH8.0, TE buffer). (b) Cleavage of plasmid pBR322 Add 1 μg of Hind to 1 μg of pBR322,
React for 3 hours at 37℃ in 20μ of the same buffer as above, and after the reaction, add 1μ of 250μ
Add M EDTA (PH8.0). Thereafter, the DNA solution is washed in the same manner as the treatment of the DNA solution after the reaction in (a) above. (c) Recombination reaction (Ligation) Ligation reaction (Ligation) is 50ng pBR322
DNA (obtained in (b) above), 1.5μg SφC
DNA (obtained in (a) above), 30mM Tris
-HCl (PH7.6), 10mM _MgCl2 , 10mM dithiothreitol, 0.1mMATP and 0.1μ T4
48 hours at 4°C in a 20μ reaction mixture containing ligase.
Incubate for several hours or at 20°C, and
Stop the reaction by adding 0.25M EDTA (PH8.0).
After that, in order to inactivate the ligase, it was heated to 65℃ for 5 minutes.
Heat for 1 minute, add 180μ TE buffer and -
Store at 20℃. (d) Introduction of recombinant plasmid into E. coli Each E. coli was inoculated into 10 ml of glucose-free L-broth (polypeptone 10 g/, yeast extract 5 g/, NaCl 5 g/) and grown to 5×10 ⁸ /ml. After that, collect the bacteria by centrifugation at 4°C. Add this bacteria to 5ml of chilled 50mM
Suspend in _CaCl2 , leave in an ice water bath for 5 minutes, centrifuge again, and add 0.67ml of chilled 50mM
Suspend in CaCl ₂ and leave in an ice water bath for 5 minutes.
Plasmid recombined in this E. coli suspension
Mix 0.33 ml of DNA solution (obtained in step c above) and keep in ice water bath for 5 minutes. Thereafter, this reaction solution is held at 42°C for 2 minutes to stop the introduction of the recombinant plasmid into E. coli. This suspension was applied as it was or diluted 10 times or 100 times and applied to an L-broth agar medium (agar concentration 1.3%) containing ampicillin (40 μg/ml).
Culture at ℃ overnight, and isolate E. coli recombinants that have become resistant to ampicillin. (e) Selective isolation of E. coli capable of producing SAK Dispense 5 ml of human plasma to multiple testers,
Heat at 56℃ for 20 minutes. Separately, dispense 10ml of ordinary agar medium containing dissolved 2% agar into multiple containers and warm them to 56℃. Mix both quickly on a shear tray and leave until solidified. The bacterial suspension of the E. coli test bacteria obtained in (d) above is spotted on the plurality of agar media and cultured at 37°C. E. coli, which has the ability to produce SAK, breaks down fibrin and combs the area around the colony, creating a transparent zone. Each of the new E. coli obtained at this time was
coli K12 C600r ^- _K m ^- _K (pSAK361) and E. coli K12 WA802r ^- _K m ⁺ _K (pSAK361)
It was named. (f) Analysis of E. coli plasmid Each E. coli obtained in (e) above was incubated at 37°C in 400 ml of L-broth containing ampicillin (40 μg/ml) and without glycose at approximately 4 × 10 ⁸ cells/ml.
Add chloramphenicol to the culture solution to a concentration of 150 μg/ml, and incubate at 37°C.
After culturing for 15 hours, collect the bacteria by centrifugation. 10 of 50ml
mMTris-HCl (PH8.)-0.015M NaCl-1.5m
Wash the bacterial cells with M sodium citrate and add 3.2ml
Suspend in 25% sucrose-50mM Tris-HCl (PH8.0). Add 1.6ml of 250mM EDTA (PH
8.0), 1ml of 5mg/ml lysozyme, 6ml of 2%
Bridge 58-62.5mMEDTA-50mM Tris-
Add HCl (PH8.0), incubate at 30°C for 15 minutes to gently dissolve the cell wall, and precipitate the bacterial cells by high-speed centrifugation (30,000 rpm, 30 minutes at 4°C) to obtain a supernatant. This is subjected to ethidium bromide-cesium chloride equilibrium density gradient centrifugation (36,000 rpm, 48 hours at 10°C) to purify plasmid DNA. Do this in the same way as (b)
After cutting with Hind, the length of the fragment containing the SAK gene was measured by agarose gel electrophoresis using 1% agarose, and it was found to be 4.9 Kb. (g) Isolation of 4.9 kb fragment 100 μg of SφC DNA was added to 0.2 ml of reaction solution (10 m
M Tris-HCl (PH7.6)-7mM _MgCl2-7
mM β-Mercaptoethanol - 50mM NaCl
-100u Hind) for 20 hours at 37°C, and subjected to agarose gel electrophoresis using 1% agarose. Stain the DNA with ethidium bromide,
Excise only the desired 4.9 kb DNA fragment from the gel. The cut out gel fragments are eluted into a dialysis tube by electrophoresis. The eluted DNA solution was treated with phenol saturated with TE buffer, and the aqueous layer was further treated with ether, followed by ethanol precipitation.
The DNA can be collected and subjected to a recombination reaction with vector DNA. (h) Production, extraction, and purification of SAK The E. coli having SAK-producing ability obtained above was
Broth (polypeptone 1%, yeast extract 0.5%, NaCl 0.5%, glucose 0.1%,
PH7.0) inoculated into 50 ml and cultured overnight at 37°C with shaking. This bacterial solution was inoculated into the same broth 5, and 37
Culture with shaking at ℃ for 12 hours, and collect bacteria by centrifugation.
The cells are suspended in 200 ml of 100 mM Tris-HCl-20% sucrose (PH8.0) and centrifuged again to collect the cells. Add this to 45ml of 100mM Tris-HCl-20%
Suspend in sucrose (PH8.0) and add 5 ml of lysozyme solution (5 mg/ml lysozyme - 20 mM EDTA,
PH8.0) and leave in ice water for 1 hour.
Centrifuge this, collect the supernatant, add ammonium sulfate to 80% saturation, and leave it at 4°C overnight.
Collect the precipitate by centrifugation and dilute with 10mM Tris-HCl.
(PH7.5) Dissolve in 10 ml and dialyze against the same buffer. This was subjected to Sephadex G-75 gel chromatography and 10mM Tris-HCl
(PH7.5), collect the SAK active fraction, and
Adsorb onto a CM-cellulose column equilibrated with mMTris-HCl (PH7.5), and add 0 to 0.5M NaCl.
Elute with a linear concentration gradient of 0.3-0.32M NaCl
Collect the SAK active fraction eluted by . Example 2 This example uses SφC as a DNA donor, Hind and Pst as restriction enzymes for cutting SφC, and vector
This is an example using pBR322 as DNA, EcoR and Pst as restriction enzymes for pBR322 cleavage, and E. coli K12 C600r ^- _K m ^- _K or E. coli K12 WA802r ^- _K m ⁺ _K as the E. coli original strain. . (a) Cleavage of Phage SφC DNA From SφC DNA using the method described in (a) to (f) of Example 1.
Isolate the 4.9Kb fragment when digested with Hind.
This fragment has a single-stranded part consisting of 4 bases each at both ends, so this part is divided into 1u of T4
67m by DNA polymerase and four types of deoxyribonucleoside triphosphates (25μM each)
M Tris-HCl (PH8.0), 6.7mM _MgCl2 , 6.7
The mixture was reacted at 18° C. for 4 hours in a buffer solution (20μ) containing mM β-mercaptoethanol to form double strands. Add 5μ of 1.5M sodium acetate (PH7.0) and 75μ of ethanol to this, and heat to -70℃.
After holding for 10 minutes, centrifuge at 12,000 rpm for 5 minutes,
Wash the precipitate with ethanol. This is 10mM
Tris-HCl (PH7.6) -7mM _MgCl2 -7mMβ
-Mercaptoethanol-Dissolve in a buffer containing 50mM NaCl, add 0.5u of Pst, and react at 37°C for 3 hours to further cut into two fragments. This is subjected to a recombination reaction. (b) Cleavage of plasmid pBR322 1 μg of pBR322 DNA is reacted with 1 u of EcoR and cleaved in the same manner as in Example 1 (b). Since both ends of this cleaved DNA also have single-stranded portions consisting of four bases, they are made into double-strands using T4 DNA polymerase and four types of deoxyribonucleoside triphosphates, as in the case of (a). This is further cleaved with Pst and subjected to a recombination reaction in the same manner as in (a). (c) DNA fragments and vector DNA obtained in recombination reactions (a) and (b)
are mixed and the recombination reaction is carried out in the same manner as in Example 1 (c). However, 1 u of T4 DNA ligase was used. (d) Similar to Example 1 below, a new Escherichia coli [E. coli K12 K600r ^- _K m ^- _K (pSAK-HP2) or E. coli K12 WA802r ^- _K m ⁺ _K
(named pSAK-HP2)], and as a result of plasmid analysis, each E. coli strain was derived from approximately 2.5 Kb SφC.
It had DNA that carries the genetic information for SAK production. Using the obtained new Escherichia coli, it was cultured by a conventional method, and after collection, Talmadge, K. et al.
(1980) Proc.Natl.Acad.Sci.USA 77 , 3369~
The periplasmic fraction was collected by the method described in 3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SAK was subjected to conventional protein purification methods such as salting out, gel filtration,
It was purified using adsorption chromatography. Example 3 This example is an example in which Pφl was used as a DNA donor, pBR322 was used as a vector, Hind was used as a restriction enzyme, and Escherichia coli K12 C600r ^- _K m ^- _K was used as the Escherichia coli original strain. (a) Phage PφlDNA is prepared in the same manner as in Example 1 and cleaved with Hind. Cut the vector DNA in the same way. (b) The recombination reaction, introduction into E. coli, and selection of recombinant E. coli are performed in the same manner as in Example 1. (c) As a result, we were able to introduce the DNA region carrying SAK production genetic information derived from Pφl into E. coli. This novel Escherichia coli bacterium
It was named K12 C600r ^- _K m ^- _K (pSAK601). Using the obtained new Escherichia coli, it was cultured by a conventional method, and after collection, Talmadge, K. et al.
(1980) Proc.Natl.Acad.Sci.USA 77 , 3369~
The periplasmic fraction was collected by the method described in 3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SAK was subjected to conventional protein purification methods such as salting out, gel filtration,
It was purified using adsorption chromatography. [Table] [Table]

Claims (1)

[Claims] 1. Introducing a vector incorporating a DNA fragment carrying the genetic information for staphyrokinase production, which contains the same base sequence as the 1.3 Kb fragment obtained by cleaving SφC, a lysogenic phage of Staphylococcus aureus, with Ava and Acc. A method for producing staphylokinase, which comprises culturing E. coli and collecting staphylokinase accumulated in the cultured cells.