JPH03198775A - Production of staphylokinase - Google Patents

Abstract

PURPOSE:To produce staphylokinase by transducing into Escherichia coli a vector integrated with DNA having staphylokinase-producing genetic information obtained from lysogenic phage of Staphylococcus aureus. CONSTITUTION:Lysogenic phage DNA of Staphylococcus aureus, a DNA donor, is scissored with a restriction enzyme and a DNA fragment having staphylokinase (SAK)-producing genetic information is taken out. Vector DNA is cleft with a restriction enzyme and the cleft part is integrated with the DNA fragment having SAK-producing genetic information. The recombinant DNA is transduced to host Escherichia coli and Escherichia coli having SAK-producing ability is selected and separated. Then the selected and separated Escherichia coli is cultured and SAK accumulated in the culture cells is collected and purified.

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 abbreviated 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) as a result of lysogenization of lysogenic phages that host S. aureus. Conventionally, strains with SAK-producing ability are selected from among S. aureus, or S.
A method has been used in which S. aureus strains that do not produce SAK are lysogenized using a phage that lysogenizes AK, and SAK is collected from the culture solution by culturing these strains.

However, S. aureus is a highly pathogenic bacterium, and its handling requires extremely careful consideration. Therefore, this method is not originally suitable for industrial SAK production methods, 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.

As a result of intensive research into an appropriate method for industrially producing Sl from the standpoint of safety and efficiency, the present inventors found that
It was determined that the gene responsible for AK production information exists on phage DNA, and the lysogenic phage DNA of S. aureus
We succeeded in introducing a fragment of this into E. coli via a vector.

The present invention is based on such achievements, 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 new Escherichia coli used in the method of the present invention are as follows:
This is obtained by the step (2).

(2) The lysogenic phage DNA of S. aureus, which is a DNA donor, is cut with a restriction enzyme.

(2) Extract a DNA fragment containing SAK production genetic information. (This step can be omitted) ■ Vector D
NA is cleaved by restriction enzymes.

■ Add the DNA obtained in ■ or ■ to the cleavage site of the vector DNA.
Incorporate fragments.

■ Introduce this recombinant DNA into host E. coli.

② Escherichia coli that has the ability to produce SAK is selectively isolated by ②.

The lysogenic phage used as the DNA donor mentioned above can be used as long as it is a lysogenic phage isolated from a lysogenic strain of S. aureus that has the ability to produce SAK, and if the lysogenic phage is a phage that has the lysogenic conversion ability. It is possible. Examples are 42D and L42E.

77 (Reference: Mason, R.E., and A11
en, W.E. (1975) Can, J., Micro
biol, 21.1113-1116), Pul.

Pd2. Tu-42D, PI-406C literature:
Kondo, 1. andFujise, M, (
1977) Infect, Immun, 18.266-
272), R519, SmC (Reference: Isamu Kondo et al. (198
0) Tokyo Jikei University School of Medicine Journal 95.1203-1206
)and so on. The means used to prepare lysogenic phage DNA are conventional. That is, a 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,
J.E. and Williams, R.E.O.
(+961) Bull-W, H, 0, 24, 771~7
84). As a result, the phages are released into the culture medium (the bacteria are lysed), and the phages are then subjected to an appropriate method (e.g., cesium chloride equilibrium density gradient centrifugation).
ne, S. (1964) J. Bacteriol.
88.1737-1742), and phage DNA can be further extracted from this 7age by a known method (for example, phenol extraction method, etc.).

Cleavage of lysogenic phage DNA with restriction enzymes is carried out as follows.

By adding appropriate restriction enzymes to the phage DNA and carrying out the reaction under appropriate reaction conditions, the phage DNA is cleaved into various fragments by the added restriction enzymes.

Restriction enzymes that can be used to cleave this phage DNA are:
) capable of cleaving phage DNA, and 2) S
It must not cut the DNA portion that carries genetic information related to AK production. Suitable restriction enzymes include Hindl[, PstI, AccT.

AvaII, EcoRI, HpaI, HpaI [
, )IindII, SstI, C1al.

BstEII, 5stH, XhoI, BclI
, BEI I[, PvuI[, Xorl[.

Examples include Kpn I, Sma I, and Xba I.

Next, DNA fragments containing SAK production information are separated from a group of DNA fragments obtained by cleaving phage DNA with restriction enzymes by various known methods (eg, agarose gel electrophoresis). For this purpose, it is necessary to determine in advance which fragments among the group of phage DNA fragments have 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, vector DNA can be cleaved by adding an appropriate restriction enzyme to the vector DNA and carrying out the reaction under appropriate reaction conditions.

Known vector DNAs can be used. For example, Co1El, pMB9. psclo
l.

p15A and its derivatives (eg pBR322).

pAcYc184, etc.) and Sharon vector derived from λ phage.

Next, the above-mentioned DNA fragment is incorporated into the cleavage site of the vector DNA, but the method itself is a conventional method, and is appropriate depending on the type of phage DNA, vector DNA, and restriction enzyme used. suitable reaction conditions are selected. In addition, 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.

Next, the recombinant DNA is introduced into host E. coli,
As the host E. coli, any E. coli strain lacking the restriction ability among the restriction/modification systems specific to E. coli can be used. In addition, D, which carries SAK production genetic information once modified
Plasmids incorporating NA can also be used for restriction-competent E. coli strains. The host E. coli may be limited depending on the type of vector used. Of the E. coli original strains used, "r" indicates a strain lacking restriction ability, and "mi" indicates a strain lacking modification ability.

The means of introduction is a known method (for example, Cam-ero
Method of n et al.: Reference Cameron, J. R. et al.
al.

(1975) Proc, Natl, Acad, Sci.
U, S, A, 72.3416-3420) can be used.

Even if step ① above is performed, the DNA fragment that carries the SAK production genetic information of the lysogenic phage may not be incorporated, and if step ① is omitted, SAK D not related to productivity
NA fragments 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, literature: K.
ondo, I and Fujise, K, (1
977) In7 fect, Immun, 18.266-272) can be used. 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. After calyx, E. coli with SAK-producing ability is isolated, but SA from E. coli with SAK-producing ability is
Analysis of DNA carrying genetic information for K production is performed 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 phage DNA and vector DNA. . By measuring the molecular weight (that is, the length as DNA) of this cut DNA fragment by a known method (for example, agarose gel electrophoresis), the phage DNA of the DNA fragment carrying the SAK production genetic information is determined.
You can know the position above.

An example of an analytical experiment is shown below.

Experimental example: Using S〆C as phage DNA 1)
When cut with restriction enzyme PstI: 15.8K
b2) When cut with restriction enzyme HindllI
4.9Kb3) Restriction enzyme H4ndl11 and Ava
When cut with I+ 2.0Kb4) Restriction enzyme Avall
SA in the 1.3Kb part when cutting with Acc I
It has been discovered that K-producing genetic information exists.

The SAK souvenir functional E. coli obtained as described above has better bacteriological properties than the original E. coli.
There is nothing different except that it has the ability to produce. Its microbiological properties are shown in the attached table below (examples are given for those obtained in the Examples below).

According to the present invention, SAK accumulated in the cultured cells by culturing the E. coli using the novel E. coli obtained as described above.
An industrial method for producing SAK is provided.

The method for producing SAK according to the present invention involves culturing Escherichia coli having the ability to produce SA obtained as described above by a conventional method,
After harvesting, Talmadge, K. etal (1
980) Proc, Natl, Acad, Sci, U,
Periplasmic fractions were collected by the method described in S.A., 77, 3369-3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SAK was purified using conventional protein purification methods such as salting out, gel filtration, and adsorption chromatography.

By the method of the present invention as described above, it has become possible to produce SAK using E. coli.
aureus, it offers surprising advantages such as safety, growth rate, and large-scale cultivation (large-scale production). Furthermore, in the case of E. coli, since SAK accumulates in the periplasm, it is possible to obtain a concentration more than 100 times higher than in the conventional method using S. aureus and obtaining it from both supernatants, making it possible to disrupt the entire cell. Since there are fewer contaminating proteins compared to the extraction method, this method has the particular advantage that it can be easily separated and purified.

The above phenomenon of accumulation in the periplasm can be seen, for example, when Escherichia coli, Escherichia coli K12 C600r'im, (pSAK 361) obtained in Example 1,
00m12 Noshiichi Pross (Polypeptone 1Og/Q, Ys I Extraf H5g/12, Glucose 1g/
The table below shows an example of the distribution and proportion of SAK activity when cultured at 37°C for 5 hours with 1°NaCQ 5 g/12. In this case, about 60% of SAK is in the periplasm It can be seen that it exists in minutes.

Total activity (,) % Supernatant fraction 950 23 Periplasm fraction 2400 58 Zytoplasm fraction 800 19 Examples of the present invention are listed below.

Example 1 In this example, SφC was used as the DNA donor, pBR322 was used as the vector DNA, and old ndllI was used as the restriction enzyme.
12C60 to Escherichia coli as host E. coli original strain
0r″Kmg or Escherichia coli 12 WA8
This is an example of moxibustion using 021 12 ry and m moxibustion.

a) Preparation and cleavage of 5IC DNA Literature (Blair, J.E. and William)
ms, R, E, O.

(1961) Bull, W. H., 0, 24, 771-7.
Phage SpC was propagated according to the method of 84), and 5M-NaC12100 +
11 (add 2 and further 30% (w/w) polyethylene glycol (average molecular weight 7000-8000) 300 m
4 was added, stirred well, and left at 0°C for several hours. Then centrifuge at 10,000 rpm for 15 minutes to collect the precipitate.
Add this to 20ml (l) of buffer (10mM Tris-HCQ).
, 10mM Mg5O, 5mM CaC (22,0
, 01% gelatin, pH 7.4), and add D to this.
Nase and RNase each at a final concentration of 1ug
/mQ, and reacted at 37°C for 20 minutes. 10. After the reaction is complete. Centrifuge at OOOrpm for 10 minutes,
The supernatant was further added for 30. The precipitate obtained by centrifugation at OOOrpm for 60 minutes was dissolved in 2.5 mQ of the same buffer as above, and about 1.79 cesium chloride was added to it, and 27,00 mQ of cesium chloride was added.
Equilibrium gradient centrifugation for 18 hours at 0 rpm. 10. Collect the phage bands obtained in this way. Dialyze against the same buffer as above 2. 50ttQ of 1.5M NaC (+-0,
15M Sodium Citrate, 5uQ 250mM ED
Add TA (pH 7,0) and 5 μQ of lO% SDS, leave at 37°C for 5 minutes, and then add 0.6++ lQ of 0.15
Add M NaCQ - 15mM sodium citrate solution saturated phenol and mix gently at room temperature for about 1O minutes. Centrifuge this at 3,000 rpm for 10 minutes and collect the upper layer (aqueous layer). This phenol treatment is repeated, followed by three washes with equal volumes of ether, and the resulting aqueous layer is dialyzed three times against 200 mL of 0.15M NaCl2-15mM sodium citrate-1mM EDTA.

To cleave phage DNA, add 3u of HindlI to 111g of DNA, add 10mM Tris-
The reaction is carried out at 37°C for 3 hours in 20 μρ of a buffer of HCl2-7mM MgC (L-7mM β-mercaptoethanol-50mM NaCQ (pH 7,6)), and at the end of the reaction, lμa of 250 mM EDTA (pH 8,0) is added. Add 5μA of 1.5M sodium acetate (pH 7.0) to the DNA solution treated with this restriction enzyme, add 60μA of ethanol, let stand at -70°C for 10 minutes, and centrifuge at 12,000 rpm for 5 minutes. Collect the precipitate and wash again with ethanol.

This was dried under reduced pressure, and 10 μg of 10 mM Tris-
Dissolve in HCQ-1mM EDTA (pH 8-0, TE buffer).

b) Cleavage of plasmid pBR322 For 1 μg of pBR322, 1. Add u of old ndlI+ and incubate at 37° in 20 μρ of the same buffer as above.
React for 3 hours at C, and at the end of the reaction,
Add MEDTA (pH 8,0). Hereinafter, a)
D in the same manner as the treatment of the DNA solution after the completion of the reaction in
Wash the NA solution.

C) Recombination reaction (Ligation) The binding reaction (Ligat 1on) is 50ngpBR32
2DNA (obtained in b) above), 1.5μ
gs$cDNA (obtained in a) above), 30
mM Tris-1tc (2 (pH 7,6), 10m
M MgCQ2. 10mM dithiothreyl, 0
．． 48 hours at 4°C or 20°C in a 20μa reaction containing 1mM ATP and 0.1u T4 ligase.
0.25M EDT of l JIQ
Add A (pH 8,0) to stop the reaction. Then, to inactivate the ligase, heat at 65°C for 5 minutes,
Add 0 μQ of TE buffer and store at -20°C.

d) Introduction of the recombinant plasmid into E. coli.
Q, Yeast extract t・5 g/Q, Na
1 of Cl25 y/12). Inoculated into Qm12 and 5x
After growing to 10'/mρ, collect the cells by centrifugation at 4°C. The bacteria were added to 5 ml of chilled 50 mM CaC.
Suspended in O2, left in an ice water bath for 5 minutes, centrifuged again to collect the bacteria, and further added 0.67 mQ of chilled 50mMCaCl22.
and leave in an ice water bath for 5 minutes. 0.33 m+2 of the recombined plasmid DNA solution (obtained in step C above) is mixed with this E. coli suspension and kept in an ice water bath for 5 minutes. Thereafter, this reaction solution is maintained at 42°C for 2 minutes to stop the introduction of the recombinant plasmid into E. coli. This suspension was diluted 10 times or 100 times as it was, applied to a broth agar medium (agar concentration 1.3%) containing ampicillin (40 μg/mQ), cultured overnight at 37°C, and developed for ampicillin resistance. The resulting E. coli recombinant is isolated.

15 6 e) Selection of Escherichia coli capable of producing SAK Dispense 5 mfl of isolated human plasma into multiple test tubes and incubate at 56°C for 2 hours.
Heat for 0 minutes, and separately dispense 10 m+2 of ordinary agar medium containing dissolved 2% agar into multiple containers and warm them to 56°C. Mix both quickly on a petri dish and leave until solidified. The bacterial suspension of E. coli test bacteria obtained in step d) above was spotted on the plurality of agar plates, and the temperature was kept at 37°C.
Cultivate at E. coli, which has the ability to produce SAK, decomposes fibrin and combs the area around the colony, creating a transparent zone. Each of the new E. coli obtained at this time was transformed into Escherichia coli K12 C600r depending on the host E. coli original strain.
xm-x (psC600rx and Escherichia coli K12 WA802r'iim"x (psAK361)
It was named.

f) Analysis of E. coli plasmids Each E. coli obtained in e) above was treated with ampicillin (40 μg).
/ m12) at 37°C in 400 mQ of glucose-free L-broth.
Grow to mQ and add 1 chloramphenicol to the culture medium.
50Mg/mQ, and further heated at 37°C for 1
After culturing for 5 hours, collect the bacteria by centrifugation. 50m + 2 10m
MTris-HCl2 (pH 8,0)-0,015M
Wash the bacterial cells with NaCQ-1, 5mM sodium citrate, and add 3.2mQ of 25% sucrose-50mM Tris-
Suspend in HCQ (pH 8,0). 1.5 rAI for this
2 of 250mM EDTA (pH 8,0), l+++α
5mg/mQ of lysozyme, 6mQ of 2% Bridge 58
-62.5mM EDTA-50mM Tris-HC (
1 (pH 8,0), incubate for 30°C"clS minutes to gently dissolve the cell wall, and precipitate the bacterial cells by high-speed centrifugation (30.OOOrpm at 4°C, 30 minutes) to obtain the upper groove. This was subjected to ethidium bromide-cesium chloride equilibrium density gradient centrifugation (36
．． 000 rpm for 148 hours) to purify the plasmid DNA. This was cut with HindlI+ in the same manner as in b), and 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) Separation of 4.9Kb fragment 100μg of SdCDNA was added to 0.2mQ of reaction mixture (10
mMTris-HCl2 (pH7,6) -7mM M
gC (h-7mM β-mercaptoethanol-50mM
The fragments are cleaved by reacting in NaCQ-100u (containing H4ndllI) at 37°C for 200 hours, and subjected to agarose gel electrophoresis using 1% agarose. The DNA is stained with ethidium bromide, and only a portion of the desired 4 to 9 Kb DNA fragment is excised from the gel.

The cut out gel fragments are eluted into a dialysis tube by electrophoresis. The eluted DNA solution is treated with phenol saturated with TE buffer, the aqueous layer is further treated with ether, and then the DNA can be collected by ethanol precipitation and subjected to a recombination reaction with vector DNA.

h) Escherichia coli having SAK-producing ability obtained through SAK production, extraction, and purification was added to L-broth (polypeptone 1%, yeast extract 0.5%, N
aC0.0.5%, glucose 0.1%, pH 7.0)
50+n(+) and cultured with shaking at 37°C overnight.

This bacterial solution is inoculated into 5 ρ of the same broth, incubated at 37°C for 12 hours, and collected by centrifugation. This bacterial body is 100
mM Tris-HCQ-20% sucrose (pH 8,0)2
Suspend in 00tQ and centrifuge again to collect bacteria. This is 45
mQ 100mM Tris-)1cQ-20% sucrose (
pH 8,0) and suspended in 5 mQ lysozyme solution (5 m
g/mO lysozyme - 20mM EDTA, pH 8
, 0) and leave in an ice water bath for 1 hour. Centrifuge the mixture, collect the supernatant, add ammonium sulfate to 80% saturation, and leave at 4°C overnight. Collect the precipitate by centrifugation,
Dissolve in 10mM TrisHCO (pH 7,5) at 10mC and dialyze against the same buffer. This was subjected to Sephadex G-75 gel chromatography, and lomM
Tris-IC (eluted at 1° (pi(7,5)),
Collect the active fraction in SA and add lQmMTris-)1c(1
It is adsorbed onto a CM-cellulose column equilibrated with (pH 7,5), eluted with a linear concentration gradient of 0 to 0.5 M NaCQ, and the SAK active fraction eluted with 0.3 to 0.32 M NaCQ is collected.

Example 2 In this example, SIC was used as a DNA donor, old ndll[ and PstI were used as restriction enzymes for cutting SIC, and vector DNA was
This is an example in which pBR322 was used as a restriction enzyme for pBR322 cleavage, EcoRI and Pst I were used as restriction enzymes for cleaving pBR322, and Escherichia 90 12 C600r'11m1 for Escherichia coli or 12 WA802rim was used for Escherichia coli as an original strain of Escherichia coli.

a) Cut t(
The 4.9 Kb fragment upon digestion with [ind] II is isolated.

Since this fragment has a single-stranded portion consisting of 4 bases at each end, this portion was treated with 1 u of 74 DNA polymerase and 4 types of deoxyribonucleoside triphosphates (each with 2 bases).
5 μM) by 67 mM Tris-MCI2 (pH
8,0), 6.7mM 1i1gcI22.6.7mM
A double strand was formed by reacting at 18°C for 4 hours in a buffer containing β-mercaptoethanol (20 μQ). Add 5 μa of 1.5 M sodium acetate (pH 7.0) and 75
After adding μQ of ethanol and keeping at −70° C. for 10 minutes, centrifugation is performed at 12.00 rpm for 5 minutes, and the precipitate is washed with ethanol. This was mixed with 10mM Tris-HCQ (
pH 7,6) -7mM Mg (42-7mM β-mercaptoethanol - 50mM NaCθ) was dissolved in a buffer containing 0.5u of Pstl, and reacted at 37°C for 3 hours to further cleave it into two fragments. is subjected to a recombination reaction.

b) Cleavage of plasmid pBR322 Add 1 u of EcoRI to 1 μ9 of pBR322 DNA, react in the same manner as in b) of Example 1, and cleave. Both ends of this cleaved DNA also have a single-stranded portion consisting of 4 bases, so
As in case a), it is made into double strands using T4 DNA polymerase and four types of deoxyliponucleoside triphosphates. This is further cleaved with PStl and subjected to a recombination reaction in the same manner as in a).

C) Recombination reaction The DNA fragments obtained in a) and b) and vector DNA are mixed, and a recombination reaction is performed in the same manner as in C) of Example 1. However, T4 DNA ligase uses lu.

d) In the same manner as in Example I below, a new Escherichia coli [Escherichia coli K12 C600r'imx (pSAK-HF2) or Escherichia coli K12 C600r'imx (pSAK-HF2) or Escherichia coli K12 C600r'imx (pSAK-HF2)]
As a result of plasmid analysis, each E. coli had approximately 2.5 Kb of DNA carrying SAK-producing genetic information derived from S〆C.

Using the obtained new Escherichia coli, it was cultured by a conventional method, and after collecting the bacteria, Talmadge, K. et al.
0980) Proc, Natl, Acad, Sci,
Periplasmic fractions were collected by the method described in U.S.A., 77, 3369-3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SAK was purified using conventional protein purification methods such as salting out, gel filtration, and adsorption chromatography.

Example 3 This example uses Pul as the DNA donor and p as the vector.
BR322 was used as a restriction enzyme, old n, dI [[ as an Escherichia coli original strain, Escherichia coli K12 C6C600r
This is an example using x.

a) Phage P〆I DNA is prepared in the same manner as in Example 1 and cleaved with Hindll. Vector DNA is also cut in the same manner.

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, the DNA region carrying SAK production genetic information derived from Put could be introduced into E. coli. This new Escherichia coli strain was transformed into Escherichia coli K12 C600.
rxmx (named psC600rx).

Using the obtained new Escherichia coli, it was cultured by a conventional method, and the bacteria were collected.
1980) Proc, Natl, Acad, Sci.
Periplasmic fractions were collected by the method described in U, S, A, 77+ 3369-3373. Most of the SAK activity was recovered from this periplasmic fraction. The obtained crude SA was subjected to salting out, which is a commonly used protein purification method.
The gel was filtered and purified using adsorption chromatography.

3 4 Thu A is 12 CC600rx+ for Escherichia Cori
Represents +.

Kimoto B is Escherichia coli 12 C600rxm
x (represents pSAC600rx.

Kiregi C is Escherichia coli K12 C60C600
rx+) SAK361).

Claims (1)

[Claims] 1) Staphylococcus aureus lysogenic phage D
Staphylokinase, which is characterized by culturing Escherichia coli into which a vector incorporating DNA carrying genetic information for staphylokinase production obtained by cleaving NA is introduced, and collecting staphylokinase accumulated in the cultured cells. manufacturing method.