JPH05103668A - Plasmid capable of producing beta-galactosidase and production of beta-galactosidase - Google Patents

Abstract

PURPOSE:To efficiently obtain the subject enzyme useful for genetic manipulation techniques, etc., by transducing a plasmid capable of producing beta-Ralactosidase into Escherichia coil, culturing the resultant transformant in a culture medium containing isopropyl-beta-D-thiogalactopyranoside and collecting the prepared product. CONSTITUTION:A gene, derived from Streptococcus.thermophilus and capable of producing f3-galactosidase is connected to the downstream side of a tac promoter which is a hybrid promoter of a trip promoter and a lacVV5 promoter to prepare a plasmid capable of producing the beta-galactosidase. The resultant plasmid is then transduced into Escherichia coil. Transformation is carried out and the obtained transformant is cultured in a culture medium containing isopropyl-beta-D-thiogalactopyranoside added thereto to accumulate the beta- galactosidase in the cultured microbial cells. After the culture, centrifugation is performed to collect the microbial cells, which are then crushed. The obtained product is collected according to a conventional method to efficiently afford the objective beta-galactosidase.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION This invention is, E. coli (Escherichia c
and a method for efficiently producing β-galactosidase by introduction into E. coli) and a method for producing β-galactosidase in Escherichia coli using the plasmid.

[0002]

2. Description of the Related Art In recent years, genetic engineering techniques centered on recombinant DNA experimental techniques have rapidly developed. This recombinant D
Β-galactosidase genes such as Bacillus stearothermophilus , Thermus quaticus , and Lactobacillus bulgaricus have been cloned using NA experimental technology (Japanese Patent Application Laid-Open No. Sho-U. 61-
No. 81788, JP-A-62-208285, JP-A-2
-5878).

Β-Galactosidase is an enzyme that performs lactose decomposition, galactose transfer reaction and the like, but its activity can be easily measured, and therefore it is one of the most common methods in molecular biology. That is, in conducting a protein or gene fusion assay, the progress of the reaction is judged by using an experiment designed to biosynthesize β-galactosidase. This utilizes the fact that the production of β-galactosidase disappears when an exogenous DNA fragment is inserted into a gene region that contributes to the production of β-galactosidase, and 5-bromo-4-chloro-3-indolyl is added to the medium. -Β-D-galactopyranoside (hereinafter, X-g
(noted as al) is added thereto, those having no exogenous DNA fragment inserted therein will be decomposed to give 5-bromo-4.
-Chloro-3-indigo is formed and the cells are dyed to form blue colonies, whereas in the case of a bacterium having a recombinant having an exogenous DNA fragment inserted, the enzyme cannot be produced and the colony is colorless and easily. It can be selected. For example, Streptococcus thermophilus used as one of the lactic acid bacteria starters of fermented milk
Gene has also been cloned for β-galactosidase in E. coli (R. E. Herman, et al .; Applied Environmental Microbiology, Vol. 52).
45-50 (1986) (RE Herman, et al; Applied and
Environmental Microbiology, 52, 45-50 (1986)):
Simply write "Document 1").

[0004]

However, no attempt has been made so far to attempt large-scale production in a transformant of Escherichia coli using the β-galactosidase-producing gene derived from Streptococcus thermophilus. In Material 1 above, Streptococcus thermophilus
Plasmid pRH116 in which the β-galactosidase producing gene of ATCC 19258 is ligated to plasmid pBR322.
It was disclosed that E. coli JM108 was introduced to express β-gal activity. However, as described in detail in Examples described later, in a plasmid of the same combination prepared by the present inventors, -The galactosidase-producing ability was very low (see Table 1).

The present invention efficiently expresses the β-galactosidase gene derived from Streptococcus thermophilus in the cells of an Escherichia coli transformant strain, and β-galactosidase capable of producing a large amount of β-galactosidase of Streptococcus thermophilus. It is intended to establish an economical production method of β-galactosidase by applying a gene recombination technique for the purpose of obtaining a production plasmid.

[0006]

The β-galactosidase-producing plasmid according to the present invention of claim 1 provides Streptococcus thermophilus ( Streptococcus therm).
ophilus) the β- galactosidase-producing gene from, t
It is linked downstream of the tac promoter, which is a hybrid promoter of the rp promoter and the lacUV5 promoter.

Further, β-according to the invention described in claim 2
The method for producing galactosidase is Escherichia coli into which the β-galactosidase-producing plasmid according to claim 1 has been introduced.
( Escherichia coli ) is cultured in a medium containing isopropyl-β-D-thiogalactopyranoside (hereinafter referred to as IPTG), β-galactosidase is accumulated in the cultured cells, and β-galactosidase is collected. Is the way to do it.

[0008]

In the present invention, the β-galactosidase producing gene derived from Streptococcus thermophilus is transformed into trp.
A β-galactosidase producing plasmid linked to the downstream of the tac promoter, which is a hybrid promoter of the promoter and the lacUV5 promoter, was introduced into Escherichia coli, and the E. coli was cultured in a medium containing IPTG, and β was introduced into the cultured cells.
-Since galactosidase is accumulated and β-galactosidase is collected, β-galactosidase can be efficiently expressed in a large amount in cells of E. coli transformant strain.

The tac promoter is a hybrid promoter composed of the -35 region of the trp promoter, which is known as a promoter having high promoter activity, and the -10 region (Pribnow box) of the lacUV5 promoter,
It is suppressed by the lac repressor and released by IPTG. This tac promoter has a so-called "consensus sequence" with high affinity for RNA polymerase.

The method for obtaining the β-galactosidase gene used in the present invention is described in Document 1 above, but it can be obtained more efficiently by the following method.
That is, Streptococcus thermophilus bacteria are cultured and collected, treated with N-acetylmuramidase SG and lysozyme, and lysed by SDS treatment, lysed after protease treatment, phenol extraction is repeated, and DNA is extracted by ethanol precipitation. After further treatment with ribonuclease A, chromosomal DNA is obtained again by the ethanol precipitation method.

Chromosomal DNA is a vector DNA which is completely or partially digested with a restriction enzyme and then cut with the same restriction enzyme.
To the chromosome D by ligation using T4 ligase
A plasmid incorporating NA is obtained. At this time, when the molecular weight of the target chromosomal DNA fragment is clear, the chromosomal DNA digested with a restriction enzyme is fractionated according to the molecular weight by agarose gel electrophoresis etc., and only the recovered DNA of the target molecular weight is collected. It can also be ligated to a plasmid.

The recombinant plasmid obtained as described above is transformed into Escherichia coli deficient in the β-galactosidase gene. Β of Streptococcus thermophilus
-A transformant of Escherichia coli carrying a plasmid in which a DNA fragment containing the galactosidase gene is incorporated is X-g.
It is obtained by culturing on an agar plate medium containing al and a drug for transformant selection, and then selecting a colony in which X-gal is blue due to the decomposition of β-galactosidase.

Then, the plasmid is separated from the obtained recombinant by the following method. Escherichia coli harboring the plasmid is cultured and collected. A plasmid DNA is obtained from the cells by the alkali method, the clear lysate method, or the like. The obtained plasmid DNA can transform E. coli again. Also, from this plasmid, only the inserted DNA fragment containing the β-galactosidase gene of Streptococcus thermophilus is separated and recovered by restriction enzyme treatment and agarose electrophoresis, and ligated to another vector having a strong promoter, for example. A new plasmid can be prepared with. E. coli can also be transformed with this plasmid.

Escherichia coli harboring the recombinant plasmid obtained as described above is cultivated under the condition of inducing a promoter, and crushed with an ultrasonic crusher to obtain a crude β-galactosidase of Streptococcus thermophilus. The enzyme solution can be efficiently obtained. Β-galactosidase can be purified from the crude enzyme solution by a conventional method.

The β-galactosidase production plasmid according to the present invention is a transformant of E. coli (E. coli K12 JM1).
09 (pTI-ST-β)) has been deposited (Microtechnology Research Institute No. 12447).

[0016]

EXAMPLES The present invention will be further clarified below with reference to Examples. 1. Chromosomes from Streptococcus thermophilus
Preparation and cleavage of DNA Streptococcus thermophilus ATCC 1925
The 8 strains were cultured overnight at 37 ° C. in 250 ml of a modified Rogosa medium (containing 1% lactose) to collect the cells. Bacteria are 50 mM Tris-HCl (pH 8.0), 1 mM
EDTA, 1.2 M sucrose, 10 μg / ml N-acetylmuramidase SG (manufactured by Seikagaku Corporation), 10
Buffer solution containing 0 μg / ml lysozyme 30 m
It was suspended in 1 and treated at 37 ° C. for 30 minutes. Then 25%
SDS (2 ml) was added and treated at 65 ° C. for 30 minutes, and then 20 mM Tris-HCl was added to remove sucrose.
It was dialyzed for 3 hours against 10 mM EDTA (pH 8.0).

Next, proteinase K solution (Merck)
(2 mg / ml) (3 ml) was added, and the mixture was treated at 37 ° C. for 3 hours. After adding an equal amount of phenol and repeating the phenol extraction twice, the aqueous layer was separated, ethanol was added, and the DNA was wound up with a glass rod. 20 mM T of the obtained DNA
It is dissolved in 10 ml of ris-HCl (pH 8.0) and 10 mM EDTA buffer, and a ribonuclease A solution (1 mg
/ Ml) was added and treated at 37 ° C for 2 hours.
After repeating the phenol extraction twice, the aqueous layer was separated, ethanol was added, and the DNA was wound with a glass rod. 80
After washing the DNA with a% ethanol solution, it was dried under vacuum, and 1 ml of 10 mM Tris-HCl (pH
8.0) Dissolved in 1 mM EDTA solution. By this operation, 92 μg of chromosomal DNA was obtained from 250 ml of culture solution.

100 units of restriction enzyme PstI was added to 3 μg of chromosomal DNA and digested at 37 ° C. for 3 hours.
The digested chromosomal DNA is electrophoresed on a 0.7% agarose gel, and the DNA fraction corresponding to the size of 7 Kb in which the β-galactosidase gene of Streptococcus thermophilus is present is eluted from the gel, extracted with phenol, extracted with ether, and ethanol. The DNA was recovered by precipitation.
The recovered DNA was dissolved in water.

2. Cleavage and dephosphorylation of vector DNA
It was added 10 units PstI to pBR322 1 [mu] g is a vector, and 3 hours completely digested with 37 ° C.. The DNA recovered by phenol extraction, ether extraction, ethanol precipitation was dried under vacuum. 100 mM of this
Dissolve in 100 μl of Tris-HCl (pH 8.0) solution, add 5 units of alkaline phosphatase, and 37 ° C.
After reacting for 1 hour, phenol extraction, ether extraction,
The DNA was recovered by ethanol precipitation. The recovered plasmid was dissolved in water.

3. Chromosomal DNA fragment into vector DNA
Insert the above 1. And the chromosomal DNA fragment of, 2. Vector DNA
66 mM Tris-HCl (pH 7.5),
6.6 mM MgCl ₂ , 1 mM DTT, 1 mM A
TP, 5 units of T4-DNA ligase was added, 16
The reaction was conducted at 16 ° C for 16 hours.

[0021] 4. Traits of recombinant plasmids to E. coli
Conversion above 3. Was mixed with competent cells of Escherichia coli JM108 strain and left in ice for 20 minutes.
It was treated for 2 minutes and then returned to ice. Add this to 3 ml of L
B medium (sodium chloride 0.5%, tryptone 1
%, Yeast extract 0.5%), and cultured with shaking at 37 ° C. for 1 hour. Bacteria were collected by centrifugation, and were collected into tetracycline (35 μg / ml) and X-gal (40 μg / m).
A chloramphenicol-resistant transformant was selected by plating on LB agar medium containing 1) and culturing at 37 ° C. for 24 hours.

5. Large intestine producing β-galactosidase
Selection of bacteria 4. From the chloramphenicol-resistant transformant 1500 strain obtained in the above step, X-gal in the agar medium was decomposed in order to produce β-galactosidase, and a strain (3 strains) that became blue was selected. This selected strain is
Plasmid pRH11 in which the β-galactosidase producing gene of Streptococcus thermophilus ATCC 19258 described in Document 1 is ligated to plasmid pBR322.
6 is similar to that of transformant JM108.
(PRH116 ′).

6. The gene that carries the β-galactosidase gene
Purification of rasmid and isolation of this gene 5. Inoculate 100 ml of LB medium supplemented with 35 μg / ml of tetracycline with the strain selected by
After harvesting the culture, the plasmid was purified by the alkaline method. That is, 2 ml of buffer solution A (25 mM Tris-HC
1 (pH 8.0), 10 mM EDTA, 15% sucrose, 2 mg / ml lysozyme) and suspended at 0 ° C for 10
After treatment for 4 minutes, 4 ml of buffer solution B (0.2 M sodium hydroxide, 1% SDS) was added, and the mixture was treated at 0 ° C. for 10 minutes. To this, add 2.5 ml of 3M sodium acetate,
After treating at 0 ° C. for 20 minutes, the precipitate was removed by centrifugation, ribonuclease A was added to the separated supernatant so that the concentration was 50 μg / ml, and the mixture was treated at 37 ° C. for 30 minutes.

After phenol extraction and phenol / chloroform (1: 1) extraction, the aqueous layer was separated. Ethanol was precipitated by adding twice the amount of ethanol to the aqueous layer. Dissolve the precipitate in 0.8 ml of water and add 4M sodium chloride 0.2 ml, 1
1 ml of 3% polyethylene glycol 6000 was added and mixed, and the mixture was left at 0 ° C. for 1 hour. 15000 ×
g A DNA precipitate was collected by centrifugation for 10 minutes, washed with 80% ethanol, and then dried under vacuum. Dissolve the precipitate in water and add 0.5 μg of it to P
Digested with 10 units of stI for 2 hours at 37 ° C.
The gel after agarose gel electrophoresis was immersed in ethidium bromide solution to stain the DNA, and the inserted DNA portion containing the β-galactosidase gene was cut out from the gel under an ultraviolet lamp, and the DNA was eluted, purified by phenol extraction, and purified by ethanol precipitation. ..

7. Β-to the plasmid pKK223-3
Galactosidase gene-introduced plasmid pKK223-3 (Pharmacia) 1 μ
10 units of PstI was added to g and digested at 37 ° C. for 3 hours. The DNA was purified by phenol extraction, ether extraction, ethanol precipitation and dried under vacuum. D
NA in 100 mM Tris-HCl (pH 8.0) solution 1
After dissolving in 00 μl and adding 5 units of alkaline phosphatase and reacting at 37 ° C. for 1 hour, phenol extraction, ether extraction, and ethanol precipitation were performed. The recovered plasmid was dissolved in water. Above 6. Containing the β-galactosidase gene obtained in
The A fragment and the plasmid DNA are mixed, and 66 mM Tri is added.
s-HCl (pH 7.5), 6.6 mM MgCl ₂ , 1 m
MDTT, 1 mM ATP, 5 units of T4 DN
A ligase was added and reacted at 16 ° C. for 16 hours.

8. Recombinant expression plasmid for E. coli
Transformation 7. The reaction solution was added to competent cells of Escherichia coli JM109 strain, left in ice for 20 minutes, treated at 42 ° C. for 2 minutes, and then returned to ice. This is 3 ml of LB
The medium (sodium chloride 0.5%, tryptone 1%, yeast extract 0.5%) was inoculated and shake-cultured at 37 ° C for 1 hour. The bacteria collected by centrifugation were treated with ampicillin (100 μ
g / ml), LB containing X-gal (40 μg / ml)
A blue colony having both ampicillin resistance and β-galactosidase activity was selected by plating on an agar medium and culturing at 37 ° C. overnight.

9. Selection of plasmid pTI-ST-β8 . We randomly selected 6 strains from the blue colonies obtained in
It was named No. 1, 2, 3, 4, 5, 6. Each strain was inoculated into LB medium supplemented with ampicillin (100 μg / ml) and IPTG (0.02 mM), and cultured at 37 ° C. overnight. From the culture solution, dispense 0.1 ml into a test tube and use β-
A method using o-nitrophenyl-β-D-galactoside (ONPG) which is a synthetic substrate for galactosidase activity measurement (Experiments in Molecular Genetics, J.
The color reaction was performed according to H. Miller p403 (1972). N
In the culture solutions of o.4 and No.6, the reaction solution showed a deep yellow color, but the culture solutions of No.1,2,3,5 showed only a pale yellow color. That is, the cultured cells of No. 4 and No. 6 have high β-
Accumulating galactosidase formation, No. 1, 2,
3,5 was shown to be low. No.4 and No.6 are ta
It was expected to be a strain carrying a plasmid in which the β-galactosidase gene was ligated in the forward direction to the c promoter.

In the following method, No. 1, 2, 3, 4, 5,
The plasmids of 6 strains were extracted and their structures were investigated. Each of the 6 strains was inoculated into 3 ml of LB medium containing 100 μg / ml of ampicillin, and cultured at 37 ° C. for one day and overnight.
Buffer A (25 mM Tris-HCl (pH 8.
0), 10 mM EDTA, 15% sucrose, 2 mg /
ml lysozyme) and treated at 0 ° C. for 10 minutes, 0.2 ml of buffer solution B (0.2 M sodium hydroxide, 1% SDS) was added, and the mixture was treated at 0 ° C. for 10 minutes. 0.125 ml of 3M sodium acetate was added to this, and after treating at 0 ° C for 20 minutes, the precipitate was removed by centrifugation, and ribonuclease A was added to the supernatant at 50 µg / ml, and treated at 37 ° C for 30 minutes. did. After phenol extraction and phenol / chloroform (1: 1) extraction, twice the amount of ethanol was added to the separated aqueous layer to carry out ethanol precipitation. Dissolve the precipitate in 0.04 ml of water and add 0.01 ml
4M sodium chloride and 0.05 ml of 13% polyethylene glycol 6000 were added, and the mixture was left at 0 ° C. for 1 hour.
The DNA precipitate was collected by centrifugation, washed with 80% ethanol, and then dried under vacuum.

Each of the plasmid DNAs was dissolved in water,
After digestion with restriction enzymes SalI, PstI, etc., agarose gel electrophoresis was performed to create a restriction enzyme map. N
plasmids extracted from strains o.4 and No.6, namely ta
The plasmid in which the β-galactosidase gene was inserted in the forward direction with respect to the c promoter was designated as pTI-ST-β. The plasmids obtained from Nos. 1, 2, 3 and 5 had the gene insertions in the opposite direction. FIG. 1 is an explanatory view showing the process of preparing the above plasmid pTI-ST-β.

10. Cultivation of recombinant E. coli 9. No. 4 strain obtained in step 1, that is, recombinant JM10
9 (pTI-ST-β) was inoculated into 100 ml of LB medium containing 100 μl / ml of ampicillin and cultured at 37 ° C. for 2 hours, and when the absorbance at 660 nm became about 0.2, the final concentration became 0.02 mM. As described above, IPTG was added, and the culture was further continued under the condition of inducing the tac promoter on the vector. 16 hours and 24 hours after the start of the culture, 10 ml of each culture solution was collected and 6000 × g
The cells were collected by centrifugation for 10 minutes.

As a control, E. coli JM109 (pKK223-3) transformed with the vector alone was treated under the same conditions.
PTG was added and cultured, and the cells were collected at the same time. Also,
The above 5. The transformant JM108 (pRH11 obtained in
6 ′) containing 35 μg / ml tetracycline
Inoculate 100 ml of medium for 16 hours or 24 hours at 37 ℃
The cells were cultivated with shaking at, and the cells were collected by centrifugation in the same manner.

11. Streptococcus thermophile
Scan variant Rogosa medium of culture 100ml (including 1% lactose)
Streptococcus thermophilus ATCC19258 was inoculated into the cells and cultured at 37 ° C. for 16 hours or 24 hours. The culture was centrifuged at 6000 xg for 10 minutes to collect the cells.

12. Β-galactosidase activity in bacterial cells
Measurement of 10. And 11. The microbial cells obtained in 1. were suspended in the same amount of phosphate buffer (20 mM potassium phosphate, pH 6.8) as the original culture solution, and 1 part of this was sonicated to disrupt the microbial cells. .. The precipitate was removed by centrifugation at 100,000 × g for 30 minutes, and β-galactosidase activity was measured using the supernatant. β-galactosidase activity is a synthetic substrate O-nitrophenyl-β-D-galactoside (ON
PG) (Experiments in Molecular G)
enetics), JH Miller p403 (1972)).

On the other hand, the above 10. And 11. Each of the bacterial cells obtained in step 1 was added with the same amount of phosphate buffer (20
The activity was similarly measured for the bacterial cells suspended in mM potassium phosphate, pH 6.8) and treated with the addition of toluene. Table 1 below shows Streptococcus thermophilus and the vector pKK223-3 or plasmid p.
Intracellular β of Escherichia coli JM109 carrying TI-ST-β
-Galactosidase and plasmid pRH1 as a comparison
The activity value of intracellular β-galactosidase of Escherichia coli JM108 having 16 'is shown.

[0035]

[Table 1]

The enzyme activity was determined by detecting the absorbance of nitrophenol molecules produced by the hydrolysis of ONPG at a wavelength of 420 nm, and the activity intensity for liberating 1 nmol of nitrophenol in 1 minute at 37 ° C. was defined as 1 unit. From the results shown in Table 1, it is clear that the activity of β-galactosidase obtained is higher when the recombinant Escherichia coli of the present invention is used (the number of Streptococcus thermophilus and JM108 (pRH116 ′)). 10 times).

13. Purification of β-galactosidase 10. The cells obtained in step 1 were crushed and treated according to a conventional method. β-galactosidase was obtained.

[0038]

As described above, the β-galactosidase-producing plasmid in which the β-galactosidase-producing gene derived from Streptococcus thermophilus is ligated to the downstream of tac promoter which is a hybrid promoter of trp promoter and lacUV5 promoter is introduced into Escherichia coli. Then, the E. coli is cultured in a medium containing IPTG to accumulate β-galactosidase in the cultured cells and collect β-galactosidase.
Β-galactosidase can be efficiently expressed in a large amount in the cells of the Escherichia coli transformant strain, as compared with the case of using the cultured cells of Thermophilus.

[Brief description of drawings]

FIG. 1 is an explanatory view showing a process of constructing a plasmid pTI-ST-β.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:19) (C12N 15/56 C12R 1:46)

Claims (2)

[Claims] 1. Streptococcus thermophilus ( S
A β-galactosidase-producing plasmid comprising a β-galactosidase-producing gene derived from treptococcus thermophilus ) ligated downstream of a tac promoter which is a hybrid promoter of a trp promoter and a lacUV5 promoter. 2. Escherichia col introduced with the β-galactosidase-producing plasmid according to claim 1.
i ) is cultured in a medium containing isopropyl-β-D-thiogalactopyranoside, β-galactosidase is accumulated in the cultured cells, and β-galactosidase is collected.
-A method for producing galactosidase.