JPH0679556B2 - Plasmid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid having a thermostable L-alanine dehydrogenase gene.

L-alanine dehydrogenase is used in clinical tests and food analysis.
It is a very important enzyme used for quantification of alanine and enzymatic synthesis of L-alanine. As a microorganism capable of producing this L-alanine dehydratase, Bacillus
Bacteria belonging to the genus Bacillus such as Bacillus sphaericus are known [European Journal.
Of Bio Chemistry (Eur.J.Biochem.) 100,29
~ 39 (1979)] However, L-alanine dehydrogenase obtained from these bacteria usually loses most of its activity within 1 to 3 weeks in an aqueous solution at room temperature. It has a major drawback that it lacks stability.

Therefore, in order to maximize the advantages of the clinical test analysis method using L-alanine dehydrogenase, it is stable to heat,
The appearance of L-alanine dehydrogenase, which does not lose its activity for a long time at room temperature, has been eagerly awaited.

For this reason, Biochem. Biophys. Acta. 615,34-47 (1980) is heat-stable from bacteria belonging to the genus Thermus and has long-term activity at room temperature. It is described that a thermostable L-alanine dehydrogenase which is not lost is obtained.

However, this thermophilic bacterium belonging to the genus Thermus has a low productivity of thermostable L-alanine dehydrogenase, and is not sufficiently satisfactory to efficiently obtain this enzyme.

On the other hand, bacteria belonging to the genus Escherichia originally have no L-alanine dehydrogenase-producing ability at all.

In recent years, genetic recombination technology has made it possible to produce proteins of heterologous organisms in Escherichia coli, and plasmids useful for recombinant DNA genetic engineering and microorganisms transformed with them are well known. For example, in Science 198, 1056 (1978), plasmid p
It is described that an animal protein is produced in Escherichia coli in which a plasmid in which a lactose promoter is connected to BR322 is introduced.

Further, in JP-A-56-5093, a genus of Escherichia transformed by introducing a plasmid having a gene of a bacterium belonging to the genus Thermus (the plasmid pBR322 is used as a vector) is introduced. It has been described that a thermostable enzyme is prepared using a bacterium belonging to it, but nothing is described about a plasmid having a gene for thermostable L-alanine dehydrogenase, and its creation It has not been reported as successful.

Therefore, the present inventors have earnestly studied for a plasmid capable of expressing a thermostable L-alanine dehydrogenase in a room temperature microorganism in order to improve the productivity of the thermostable L-alanine dehydrogenase. It was confirmed that the gene for L-alanine dehydrogenase was isolated from a thermophilic microorganism and that this gene could be introduced into a known plasmid DNA, and that the plasmid thus obtained by introducing the gene has the above-mentioned properties. Found and completed the present invention.

That is, the present invention comprises a thermostable L-alanine dehydrogenase gene derived from a chromosomal DNA of Bacillus stearothermophilus and a vector plasmid, and a plasmid pICR3 having the following physicochemical properties or It is based on the plasmid pICR301.

Molecular weight Plasmid pICR3 is about 7 megadaltons Plasmid pICR301 is about 5 megadaltons The following restriction enzymes are sensitive to the following restriction enzymes.

To obtain the plasmid of the present invention, for example, biochemica
Biochem.Biophys Act
a.) According to the method described in 72,619 to 629 (1963), a chromosomal DNA fragment containing a gene of thermostable L-alanine dehydrogenase is obtained, and the obtained gene and the DNA having a role as a vector are journaled. According to the method described in J. Mol. Biol 96 171-184 (1975), it can be prepared by digestion with a restriction enzyme and then ligation with ligase.

The thermostable L-alanine dehydrogenase gene used in the present invention is Bacillus stearothermophilus (Bacill).
us stearother-mophilus) chromosomal DNA-derived thermostable L-alanine dehydrogenase gene. Specific examples of stearothermophilus include IF012550, ATCC7953, 795.
There are 4,8005,10149,12980, NCA1503 etc.

In addition, examples of DNA having a role as a vector include plasmid DNA, particularly plasmid pBR322.
Is preferred. Moreover, as a restriction enzyme, for example, Sal I
Examples of the ligase include T4 DNA ligase.

Examples of the plasmid of the present invention include plasmids pICR3 and pICR301, which are obtained by introducing the gene for L-alanine dehydrogenase derived from chromosomal DNA of Bacillus stearothermophilus into plasmid pBR322 by the method described above. The plasmids pICR3 and pICR 301 were deposited on February 14, 1984, at the Institute of Microbial Technology, Ministry of International Trade and Industry, and were not accepted. This plasmid pICR 3 and pICR 30
It shows the physicochemical properties of 1.

(1) A thermostable L-alanine dehydrogenase can be expressed in a room temperature microorganism.

(2) It has the following cleavage sensitivities to the following restriction enzymes:

The name of the restriction enzyme is the abbreviation of the restriction enzyme obtained from the following bacterial species.

EcoRI; Escherichia coli HindIII; Haemophilus influenzae Sa1 I; Streptomyces arbus The number of restriction sites digested with restriction enzymes was digested with plasmids pICR3 and pICR301 in the presence of an excess of restriction enzymes, and the digests were digested with agarose gel electrolysis. It is determined by the number of fragments that can be separated by electrophoresis.

(3) Molecular weight The plasmid pICR 3 is about 7 megadalton,
pICR 301 is about 5 megadaltons.

(4) The plasmid pICR 301 is the plasmid plCR 3
The L-alanine dehydrogenase gene of was left as it is, and the other plasmids were partially removed to make the plasmid smaller.
This plasmid was introduced into Escherichia coli C600-
The L-alanine dehydrogenase activity of the pICR 301 strain was determined by the Escherichia coli C600PICR in which the plasmid pICR 3 was introduced.
The value is about 3 times higher than the activity of 3 strains.

The plasmid of the present invention can be introduced into, for example, bacteria that grow at room temperature, and by introducing this plasmid, transformed bacteria can be obtained. Examples of the bacteria that grow at room temperature include Escherichia (Esch.
Bacteria belonging to the genus erichia) are mentioned, and Escherichia Coli is particularly preferable. As a concrete example of the Escherichia coli,
r ^- m ^-, and the like.

In addition, for example, in order to introduce the plasmid of the present invention into the above-mentioned bacteria that grow at room temperature, for example, the journal of
Molecular Biology (J. Mol. Biol.) 53,159-162
According to the method of (1970), it may be carried out by contacting the above-mentioned bacterium treated with calcium chloride at a temperature around 0 ° C. with the plasmid of the present invention.

As an example of the bacteria transformed as described above, Escherichia coli C60 into which the plasmid pICR 301 was introduced was used.
0-pICR 3 strains (Microtechnology Research Institute No. 7447) and C600-p
The ICR 301 strain (Microtechnology Research Institute, No. 7448) is an example. This strain is known as Escherichia coli C600 [Nature 21
See 7,1110-1114 (1986). ] And has the same mycological properties except that it has heat-resistant L-alanine dehydrogenase-producing ability and apicillin resistance. This bacterium is kept safe without converting non-transmissibility to transmissibility and non-pathogenicity to pathogenicity.

The plasmid of the present invention is a useful microorganism as described above,
For example, by transforming bacteria that grow at room temperature, heat-resistant L
-It is very useful because it can endow the ability to produce alanine dehydratase and can easily obtain a large amount of thermostable L-alanine dehydrogenase from transformed bacteria.

Next, the present invention will be specifically described with reference to examples.

The activity of thermostable L-alanine dehydrogenase is shown by the European Journal of Biochemistry [Eu
LJ described in rJBiochem, 100, 29-39 (1979).
A method for measuring alanine dehydrogenase activity, ie pH 11.3
1.25 μmo in 100 μmole of glycine-KCl-KOH buffer
Prepare a mixed solution containing le NAD and 10 μmole L-alanine, and add an appropriate amount of crude enzyme extract to the mixed solution to make the final volume 0.8 ml, and reduce NA at 25 ℃ or 55 ℃.
The increase in D per unit time was measured by measuring the increase in absorbance at 340 nm.

In addition,% in the examples and reference examples indicates% by volume.

Examples 1 and 2 (1) Chromosomal DNA of Bacillus stearothermophilus
Separation of.

From the Bacillus stearothermophilus IF012550 strain, Biochemica et biophysica Actor (Biochem
Chromosomal DNA was isolated according to the method described in Biophys Acta) 72, 619-629 (1963).

First, Bacillus stearothermophilus IFO12550 strain was treated with glycerol medium (polypeptone 10 g /, yeast extract 2.
5g /, Meat Extract 2g /, Glycerol 2g /, Sodium Chloride 5g /, Potassium Phosphate 2g /, Dipotassium Phosphate 2g /, Magnesium Sulfate 0.1g /, Biotin 4
μg /, and adjusted to pH 7.2) 2 liters, shake-cultured at 55 ° C for 12 hours, and then collected by centrifugation.

Next, 12 mg of lysozyme was dissolved in 6 ml of a saline-EDTA solution (containing 0.15M NaCl and 0.1M EDTA and adjusted to pH 8.0), and the collected strain was added to this solution and stirred well. This was heated at 37 ° C for about 10 minutes and immediately frozen when the cells began to lyse.

50 ml of Tris-SDS buffer (10 mg / ml
0.1M Tris buffer adjusted to pH 9.0 containing SDS and 0.1M Nacl. ) Was added and stirred, and the mixture was further heated to 60 ° C. to completely lyse the cells. To this lysate, 56 ml of 80% phenol was added, shaken for about 20 minutes, and extracted with phenol to remove contaminating proteins. 2 to this extracted crude DNA solution
A double volume of cold ethanol was added and the fibrous precipitate was wound up with a glass rod and immersed in 10 ml each of 70, 80 and 90% ethanol for several minutes, and then 20 ml of dilute sarin-citrate (saline). -Citrate) solution (0.015M NaCl, 0,0015M
Na ₃ - Preparation citrate. ), Further concentrated saline
-Citrate solution _{(1.5M NaCl, 0.15M Na 3 -} . Preparation citrate) was added 2 ml, and the crude DNA solution was prepared.

This crude DNA solution was diluted to about 500 μg / ml, ribonuclease [RNase A (manufactured by Sigma)] was added to 50 μg / ml, and the mixture was heated at 37 ° C. for 30 minutes. After cooling, add an equal amount of 80% phenol, perform phenol extraction, and extract
The DNA was recovered by ethanol precipitation, and the diluted sal
Dissolve in 20 ml of ine-citrate solution, and further add the above concentrated saline.
-An extract of chromosomal DNA was prepared by adding 2 ml of citrate solution.

(2) Preparation of vector plasmid pBR322.

Escherichia coli C600 strain into which the plasmid pBR322 (Bethsda Research Laboratories) was introduced was used in 2 L-medium (polypeptone 10 g /, yeast extract 5 g /).
, Glucose 1 g /, sodium chloride 5 g / were added to adjust the pH to 7.2. ), Aerobically cultivated at 37 ℃ until the early logarithmic growth phase, and then 10 ml of chloramphenicol solution (3.6 mg
Prepared with ethanol to make / ml. ) Was added and the mixture was aerobically cultured at 37 ° C for 15 minutes to grow the plasmid pBR322.

Next, the cells collected by centrifugation were treated with 80 ml of TE-sucrose buffer (0.2 g / ml sucrose, containing 20 mM EDTA, pH 8.0.
0.05M Tris buffer prepared in. ), And then 8
ml of lysozyme solution (prepared with the above TE-sucrose buffer so that the concentration becomes 5 mg / ml) was added, and 28 ml of 5 was added.
M NaCl solution and 4 ml of 40 mg / ml SDS solution were added.

The mixture is allowed to react at 37 ° C for 2 hours and then at 0 ° C for about 15 minutes.
After keeping for a while, the crude plasmid DNA was separated by centrifugation.

Next, 1/2 volume of 80% phenol was added and phenol treatment was performed to remove contaminating proteins. The crude plasmid thus extracted was recovered by precipitation with cold isopropanol, and further dissolved in TE buffer (20 mM Tris buffer containing 0.14 M NaCl and 1 mM EDTA, adjusted to pH 7.5). 2m in this mixture
RNase A (g) was added, the mixture was reacted at 37 ° C. for 2 hours, and contaminated RNA was removed by phenol treatment in the same manner as above.

The extracted crude plasmid was recovered by precipitation with 2 volumes of ethanol. This was further dissolved in 10 ml of the above TE buffer, contaminated RNA was further removed by agarose gel filtration, and the obtained crude DNA was recovered again by ethanol precipitation.

This precipitate was dissolved in 23.1 ml of 0.02M Tris buffer (adjusted to pH 8.0), 23.7 g of cesium chloride and 0.6 ml of ethidium bromide solution (prepared to 10 mg / ml) were added, and the time was about 40 hours. By ultracentrifugation, plasmid DNA
Was separated, and then ethidium bromide was removed with normal butanol. 0.01% of this isolated plasmid
M TE buffer (0.0 mM adjusted to pH 7.5 with 0.1 mM EDTA)
1M Tris buffer. ), The purified plasmid pBR322 was obtained.

(3) Creation of plasmid pICR3 (Example 1).

5 μg of chromosomal DNA of Bacillus stearothermophilus obtained by the method (1) and 20 units of restriction enzyme Sal I (Takara Shuzo) were added to 7 mM MgCl ₂ , 150 mM NaCl, 0.2 mM EDT.
A, containing 7 mM 2-mercaptoethanol, 0.1 mg / ml BSA p
Add to 100μ of 10mM Tris buffer prepared to H7.5, and at 37 ℃
After reacting for 2.5 hours to digest the DNA, it was heated at 65 ° C. for 5 minutes to inactivate SalI, and the digested DNA fragment was recovered by precipitation with cold ethanol.

Next, the plasmid pBR3221 μg obtained by the method of (2)
The restriction enzyme SaII3 unit was added to the reaction mixture, and the mixture was reacted in the same buffer as above at 37 ° C. for 10 hours, and the digested plasmid DNA was recovered by the same method as above. The DNA digestion chromosomal and plasmid thus obtained was mixed, using T4DNA ligase (Takara Shuzo Co., _{Ltd.), 6.6mM MgCl 2, 10mM DTT} , at 66mM Tris buffer, prepared pH7.6 containing 66 .mu.m ATP, 13 The plasmid pICR3 was obtained by reacting at 19 ° C for 19 hours and recombining the two digested DNAs.

(4) Creation of plasmid pICR 301 (Example 2).

Plasmid plCR3, 1 μg, created by the method of (3)
The restriction enzyme HIND III3 units in addition to, 7mM MgCl _2, 60
10 mM Tris buffer 10 adjusted to pH 7.5 with mM NaCl
After the reaction was carried out at 37 ° C for 10 hours, the digested DNA was recovered by the same method as in (3) and religated with T4 DNA ligase to obtain plasmid pICR301.

Reference Examples 1 and 2 In order to examine the expression properties of the plasmids plCR3 and pICR 301 obtained in Examples 1 and 2, the following transformation treatment was performed.

(5) Transformation.

First, the host strain Escherichia coli C600r ^- m ^- strain is cultured in 50 ml of the above L-medium, collected by centrifugation, and then 50 ml.
Of it was suspended in 0.1 M MgCl ₂ solution, and more finally by centrifugation was suspended in 0.1MMgCl ₂ solution 2.5 ml.

The thus obtained Eshierichia coli C600r ^- m ^- suspension 0.2ml to (3) of the strain and the mixture was added 0.1ml containing plasmid pICR3 or PICR 301 obtained by the method in (4), 0 ° C.
After treatment for 30 minutes at 42 ° C. for 2 minutes. Next, 3 ml of the above-mentioned L-medium was added thereto, and the mixture was incubated at 37 ° C for 1 hour, and further, L-agar medium containing ampicillin (prepared to 15 µg / ml) (15 g of agar per L-medium was added). The resulting colonies were further cultured on L-agar medium containing tetracycline (prepared to 25 μg / ml.), And the colonies that did not grow were Proc, Natl, Acad.S.
According to the method described in ci.USA, 72, 2274-2278 (1975), it was separated on paper, NBT (nitroblu-tetrazolium) and
By reacting with a reaction solution containing PMS (phenadicimesosulfate), a colony having L-alanine dehydrogenase activity was found and the plasmid pICR was detected.
3 and pICR301-introduced Escherichia coli C60
0-pICR3 or C600-pICR301 was obtained.

Next, the Escherichia coli C600-pICR thus obtained
From 3 or C600-pICR 301 colonies, 100 ml of the above glycerol medium containing ampicillin (prepared to 15 μg / ml) was shake-cultured at 37 ° C. for 16 hours. The cells were collected by centrifugation, washed, and then suspended in 0.01M phosphate buffer containing 5 ml of 0.01% 2-mercaptoethanol adjusted to pH 7.4 and subjected to ultrasonic treatment at 0 ° C for 5 minutes. Crush cells,
A crude enzyme extract was obtained by centrifugation.

The activity of the thermostable L-alanine dehydrogenase of the crude enzyme extract thus obtained was measured.
In Coli C600-pICR3 strain, 0.8 unit / mg protein,
In Escherichia coli C600-pICR301 strain, 2.4 units /
It was mg protein. This was 10 to 30 times higher than the L-alanine dehydrogenase activity of the Bacillus stearothermophilus IFO12550 strain (0.070 units / mg protein), which is a DNA donor.

The crude enzyme extract containing L-alanine dehydrogenase contained 10 mM of 2-mercaptoethanol at pH 7.2 and 0.01%.
When heat-treated in phosphate buffer at 70 ℃ for 60 minutes, 80%
It had the above residual activity.

Then, from this strain, a plasmid was prepared in the same manner as in (2) above.
The pICR3 and pICR301 were separated and the properties of the plasmids pICR3 and pICR301 were examined by horizontal agarose gel electrophoresis.

The buffer used for electrophoresis was 2.5 mM EDTA-Na, 89 mM.
89 mM Tris buffer containing boric acid at pH 8.3 was used, and 7 mg / ml agarose and 0.
The amount of ethidium bromide adjusted to 5 mg / was used, and plasmid pICR 3 and pICR 301, plasmid pBR 322, and HindIII (Takara Shuzo) digested fragments of lambda phage DNA as a molecular weight marker were each added to each of them.
μg was simultaneously electrophoresed on the same agarose gel at a constant voltage of 7 V for 3 to 4 hours per 1 cm in width. Next, the band was judged with an ultraviolet lamp and the sizes of the plasmids pICR 3 and pICR 301 were examined. As a result, the molecular weight of the plasmid pICR 3 was about 7 megadaltons, and that of pICR 301 was about 5 megadaltons. The molecular weight of the plasmid pBR322 is 2.8 megadalton.

In addition, the plasmids pICR3 and pICR301 were reacted under the proper conditions of restriction enzymes to obtain plasmids pICR3 and pICR30.
Digested 1.

The digested sample obtained with each restriction enzyme was subjected to agarose gel electrophoresis in the same manner as above, and the number of cleavage sites by each control enzyme was examined. As a result, plasmids pICR 3 and pICR 3 were obtained.
301 was found to have the following cleavage sensitive sites.

Furthermore, the plasmid pICR3 has a chromosomal DNA fragment containing the L-alanine dehydrogenase gene of Bacillus stearothermophilus IFO12550 strain bound to the plasmid pBR322 DNA at the Sal A cleavage site.
In 301, it is clear that the chromosomal DAN fragment containing the gene for L-alanine dehydrogenase and the plasmid pBR322 DNA are bound at the HindIII and SalI cleavage sites.

 ─────────────────────────────────────────────────── --Continued from the front page Judgment panel for referees Chief referee Hironori Kaneo Referee Masaki Hirota Referee Toshiko Otaka (56) References JP 56-5093 (JP, A) Biochem. Biophys. Acta Vol. 445 No. 3 P. 549 to 557 Eur. J. Biochem. Vol. 77 P.I. 463-470

Claims (1)

[Claims] 1. A Bacillus stearothermophilus (Baci)
llus stearothermophilus) chromosomal DNA-derived thermostable L-alanine dehydrogenase gene and a vector plasmid, and a plasmid pI having the following physicochemical properties.
CR3 or plasmid pICR301. Molecular weight Plasmid pICR3 is about 7 megadaltons Plasmid pICR301 is about 5 megadaltons The following restriction enzymes are sensitive to the following restriction enzymes.