JP2007089538A - Method for producing new escherichia coli and tauropine dehydrogenase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new gene recombinant Escherichia coli containing a recombinant vector plasmid gene incorporated with a gene encoding tauropine dehydrogenase, and a method for producing the tauropine dehydrogenase by using the Escherichia coli. <P>SOLUTION: This new Escherichia coli accepted as FERM AP-20609 has the recombinant vector plasmid gene obtained by processing a tauropine dehydrogenase gene derived from Arabella iricolor, inserting the gene into a plasmid without inhibiting the function of tauropine dehydrogenase production ability and contains the tauropine dehydrogenase gene consisting of a specific sequence. By culturing the Escherichia coli, the tauropine dehydrogenase is collected from an obtained cultured material and purified. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel recombinant E. coli containing a recombinant vector plasmid gene into which a gene encoding tauropine dehydrogenase has been incorporated, and a method for producing tauropine dehydrogenase using this E. coli.

Tauropine is an imino acid obtained from marine invertebrates, and is produced in vivo from taurine and pyruvic acid by the action of tauropine dehydrogenase. Taurine is a drug listed in the Japanese Pharmacopoeia.
(1) Blood pressure lowering action, (2) Decrease in total cholesterol level, increase in good cholesterol level,
(3) Prevention and treatment of liver disease, (4) Treatment of heart failure, (5) Effective for bronchial asthma. Examples of foods rich in taurine include oysters, clams, swordfish, octopus, squid, clams and the like.

Tauropine belongs to the opines in which pyruvic acid and other amino acids or taurine are bound in a form sharing an amino group. In addition to tauropine, octopine bound to arginine, thrombin bound to glycine, alanopine bound to alanine, and β-alanopine bound to β-alanine are known as opine. These are speculated to be products of anaerobic respiration of marine invertebrates. Non-patent document 1 describes the entire base sequence of tauropine dehydrogenase of segroisomers.
Marine Biotechnology 6,493-502 (2004)

As described above, taurine is a health functional substance that is abundant in marine invertebrates. However, it is currently measured with an amino acid analyzer for quantification, and there is no simple method. In addition, tauropine dehydrogenase is prepared by extracting and purifying proteins from natural products. However, this method requires a large amount of natural products and tends to lose its activity during extraction and purification. It was difficult to extract in large quantities.
Therefore, the present inventors tried to incorporate a gene encoding tauropine dehydrogenase into an Escherichia coli plasmid. Since pTV118N vector (manufactured by Takara Bio Inc.) was used for integration, it was necessary to slightly modify the gene.

  In addition, when a large amount of foreign protein is produced in a host such as E. coli, the translational product folding by a molecular chaperone does not function normally, so an inactive mass of the foreign protein, a so-called inclusion body is formed. There are many cases. The inclusion body is a change in the three-dimensional structure of the target object, and adversely affects the original activity of the target object. Therefore, a manufacturing method in which such an enclosure is not formed is required.

The composition of the present invention is a novel Escherichia coli received as FERM AP-20609, which is a combination of a tauropine dehydrogenase gene derived from Arabella iricolor and inserted into a plasmid without inhibiting the ability to produce tauropine dehydrogenase. E. coli having a replacement vector plasmid gene, which contains the tauropine dehydrogenase gene described in Sequence Listing 1. Moreover, it is a genetically modified Escherichia coli in which a Tauropine dehydrogenase gene derived from Arabella iricolor is incorporated into pTV118N vector (Takara Bio Inc.) and inserted into Escherichia coli JM109. Tauropine dehydrogenase is collected from the product and purified.

The present invention is a technique capable of producing a large amount of tauropine dehydrogenase, which is an enzyme that synthesizes tauropine from taurine and pyruvate, that is, tauropine dehydrogenase (hereinafter referred to as “Air tadh” ) of a ring-shaped animal, Arabella iricolor . A technique for producing a large amount of Air tadh was completed by incorporating it into pTV118N vector (Takara Bio Inc.) while maintaining the amino acid sequence encoding Air tadh. When this Air tadh is used, taurine or pyruvic acid in a biological component can be easily measured. That is, since only taurine and pyruvic acid specifically become tauropine, it is easy to quantify this. This E. coli is received as FERM AP-20609.

According to the present invention, tauropine dehydrogenase can be produced in large quantities. Taurine is a health functional substance having various biological activities, and taurine can be quantified by a simple method by using tauropine dehydrogenase.
Furthermore, since the amount of tauropine in natural products is very small, its physiological activity has not been sufficiently studied. However, the effectiveness of the present invention may be investigated by the present invention.

  A method for quantifying taurine using tauropine dehydrogenase is as follows. Taurine, pyruvic acid, Air tadh of the present invention, and coenzyme NADH are dissolved in 0.1-0.5 M phosphate buffer. Coenzyme NADH absorbs the ultraviolet region at 340 nm. The coenzyme NADH is oxidized to NAD by the reaction between taurine and pyruvate by Air tadh, and the absorbance at 340 nm decreases. The degree of this decrease can be measured and converted to the content of taurine or pyruvic acid. Since this reaction is characteristic only to taurine and pyruvic acid, it significantly improves the conventional measurement method using an amino acid analyzer that requires cumbersome pretreatment and a long time.

The present invention is prepared by ligating the gene encoding Air tadh of the annelid Arabella iricolor into an expression vector cleaved with an appropriate restriction enzyme.
The actual gene sequence inserted into the recombinant plasmid is as shown in the sequence listing. The first to 44th and 1245th to last in the sequence listing are part of the expression vector of the present invention.
Examples of the recombinant vector plasmid gene in the present invention include plasmids, cosmids, artificial chromosomes or phages. Furthermore, the recombinant vector plasmid gene in the present invention may contain one or more selectable markers capable of selecting E. coli transformed with the gene of the present invention. Examples of such selection markers include constituents such as kanamycin, ampicillin, tetracycline, chloramphenicol and the like.

  In the present invention, the recombinant vector preferably has a promoter or other control sequence for expressing the gene of the present invention linked to the gene of the present invention. Examples of other control sequences include a ribosome binding site for bacterial expression, a transcription termination sequence, an upstream control region, an enhancer, and an operator. Such a promoter or other regulatory sequence is not particularly limited as long as it can express the gene of the present invention, and those known in the art can be used. Specific examples include T7 promoter, Tac promoter and the like. The recombinant vector preferably contains a regulatory sequence capable of regulating protein expression in addition to the above regulatory sequence.

  As a method for preparing a plasmid into which the Air tadh gene has been inserted, a known method can be used. The gene of the present invention can be synthesized by a generally known method. In order to incorporate into a vector, it is preferable to amplify by PCR method using primers with appropriate restriction enzyme cleavage sites located at both ends. The conditions of the PCR reaction can be appropriately determined by those skilled in the art, but amplification is carried out by reacting for 30 cycles with one cycle of denaturation at 98 ° C for 30 seconds, annealing at 57 ° C for 15 seconds, and polymerization at 74 ° C for 30 seconds Gene can be obtained.

In order to insert a DNA fragment containing the gene of the present invention into a recombinant vector, an appropriate restriction enzyme is allowed to act on the above vector. Any restriction enzyme can be used as long as it can be ligated with the inserted DNA fragment. Conditions such as the reaction temperature and reaction time of the digestion reaction with the restriction enzyme can be set according to the selected enzyme. If necessary, these vectors can be purified by a purification means such as a boil method or an alkaline SDS method, and further concentrated by a concentration means such as an ethanol precipitation method or a polyethylene glycol precipitation method.
In the treatment step, the combination of the insert DNA fragment and the restriction enzyme for digesting the vector can be appropriately selected from combinations known per se in this technical field.

Subsequently, the amplified gene of the present invention and the vector can be mixed, and a ligase can be allowed to act thereon to obtain a recombinant vector plasmid gene. Examples of the ligase include T4 DNA ligase and Escherichia coli-derived DNA ligase.
Subsequently, in order to express the gene of the present invention, a competent cell is transformed with the recombinant vector plasmid gene containing the obtained gene. As the competent cell in the present invention, any cell having high transformation efficiency can be used. For example, E. coli JM109 competent cells have high transformation efficiency and can be preferably used. For transformation of competent cells with a recombinant vector plasmid gene containing the gene of the present invention, a commonly used chemical competent cell method, electreporation method or the like can be used.

  E. coli transformed with the gene of the present invention can be easily selected because colonies are formed on LB medium agar plates containing antibiotics such as ampicillin and kanamycin by the marker gene of the vector plasmid gene. However, in order to confirm whether or not the cloned E. coli has been transformed with the gene of the present invention, a part thereof is used to confirm the amplification of the insert by the PCR method or the sequence analysis by the dideoxy method using a sequencer. It is preferable.

  Escherichia coli transformed by the present invention obtained by the above method can be cultured in an appropriate medium to obtain a large amount of recombinant vector plasmid genes. As the medium, a commonly used LB liquid medium, M9 medium, or the like can be used. In addition, the recombinant vector plasmid gene can be recovered using a known method. Among them, a recombinant vector plasmid gene can be obtained under defined conditions using a plasmid recovery kit such as Wizard (manufactured by Promega).

The culture form of Escherichia coli, which is a transformant, may be selected in consideration of the nutritional physiological properties of Escherichia coli. Industrially, it is advantageous to perform aeration and agitation culture using a jar fermenter or the like.
As a nutrient source for the medium, any medium commonly used for culturing Escherichia coli can be widely used. The carbon source is an assimilable carbon compound, and polyols such as glycerin and organic acids such as pyruvic acid, succinic acid or citric acid can be used. Nitrogen compounds that can also be used as a nitrogen source include peptone, meat extract, casein hydrolyzate, soybean koji alkaline extract, ammonia or ammonium salt. In addition, phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc and other salts, specific amino acids, specific vitamins, antifoaming agents and the like can be used as necessary. Moreover, it is preferable to add protein expression inducers, such as isopropyl- (beta) -D-thiogalactopyranoside, to a culture medium as needed.

  Although the culturing time varies depending on the conditions, it is preferable to recover the cells when the cell growth reaches a steady state. Usually, the culture is performed at 37 ° C. for 1.5 to 3.0 hours and at 37 ° C. for about 20 hours with IPTG addition.

  As a method for recovering Air tadh in the culture, the cells are collected from the obtained culture by means such as filtration and centrifugation. Then, the cells can be destroyed and collected by a mechanical method, an enzymatic method using an enzyme such as lysozyme, or a chemical treatment using a surfactant. If necessary, a chelating agent such as EDTA, a surfactant, or the like may be added to solubilize Air tadh and collected as an aqueous solution.

  In order to further purify Air tadh from the crude enzyme solution thus obtained, an ordinary protein purification method can be used. Specifically, ammonium sulfate fractionation method, organic solvent precipitation method, adsorption treatment method using ion exchanger, ion exchange chromatography, gel filtration chromatography, affinity chromatography, electrophoresis method, etc. are used alone or in combination. Can be used.

Preparation of expression vector The Air tadh gene is incorporated into a sequencing vector ZeroBlunt TOPO (trade name, manufactured by Invitrogen).
In order to remove one NcoI cleavage site, a PCR primer described in Sequence Listing 2, Air m1F, and a PCR primer described in Sequence Listing 3, Air m1R were synthesized.
PCR was performed using a plasmid in which the Air tadh gene was incorporated as a template. The above-mentioned primer contains a restriction enzyme Cla 1 cleavage site. By cleaving both ends of the PCR product with Cla 1 and performing self-ligation, the amino acid sequence was not changed and one NcoI cleavage site was removed. .
In this case, the PCR reaction was incubated at 94 ° C. for 2 minutes, then incubated 30 times in a cycle (94 ° C. for 15 seconds, 50 ° C. for 30 seconds, 68 ° C. for 4 minutes 30 seconds), and finally incubated at 40 ° C. did.

In order to incorporate the gene of the present invention having the above sequence, that is, the Air tadh gene, into the recombinant vector, a restriction enzyme, AirNcoI described in Sequence Listing 4 was synthesized and added.
PCR was performed using the mutant Air tadh gene as a template. The AirNcoI primer contains a restriction enzyme NcoI cleavage site, and the template plasmid has an EcoRI cleavage site. By cleaving the PCR product with restriction enzymes NcoI and EcoRI, the mutant Air tadh gene is Cut into two parts. The longer fragment of the two has NcoI cleavage sites at both ends, and the shorter fragment has NcoI on one side and EcoRI cleavage site on the other. First, the shorter fragment was ligated to a recombinant vector (pTV118N, manufactured by Takara Bio Inc.) cleaved with NcoI and EcoRI. After recutting with NcoI, the longer fragment was ligated to complete the expression vector. .
In this case, the PCR reaction was incubated at 94 ° C. for 2 minutes, then incubated 30 times in a cycle (94 ° C. for 15 seconds, 50 ° C. for 30 seconds, 68 ° C. for 1 minute 15 seconds), and finally incubated at 4 ° C. did.

  As a result of analyzing the PCR product and PCR cleaved with NcoI and EcoRI by a commonly known electrophoresis method, the presence of a fragment corresponding to the mutated Air tadh gene and the cleaved mutated Air A fragment corresponding to tadh was confirmed.

Preparation of mutant Escherichia coli The mutant Air tadh gene was subcloned into the pTV118N vector for expression in Escherichia coli.
E. coli JM109 was transformed with the pTV118N vector into which the mutant Air tadh gene obtained above was incorporated. Then, it apply | coated to LB culture medium and incubated overnight at 37 degreeC.
The colonies formed above were arbitrarily picked and cultured overnight in LB liquid medium to recover the plasmid. PCR was performed using the plasmid as a template, and it was confirmed that the target fragment was correctly inserted.

Production of Tauropine Dehydrogenase The recombinant Escherichia coli constructed here was called pTVAirJM109, which was cultured by the following method to obtain a recombinant protein, ie, tauropine dehydrogenase.
Pre-culture is performed in 2 × YT medium (containing 50 μg / ml ampicillin). 50 μl of the preculture was inoculated into 5 ml of 2 × YT (containing ampicillin) medium and cultured at 37 ° C. for 3 hours. To this is added 5 μl of 1M IPTG and induced overnight at 37 ° C. Bacteria were collected by centrifugation (12000 rpm, 4 ° C.). Cells were disrupted in a phosphate buffer solution by ultrasound, and the supernatant was collected by centrifugation (15000 rpm, 4 ° C.). The enzyme of the present invention could be obtained by purifying this.

Quantitative determination of taurine 0.25M sodium phosphate buffer (pH 7.2) 2ml
1.4 mM NADH 0.5 ml
0.08M Pyruvate (pH 7.2) 0.1ml
0.4M Taurine (pH 7.2) 0.1ml
0.3 ml of enzyme (Air tadh)
Using the measurement solution of the above-mentioned formulation, it was incubated at 30 ° C., and the absorbance at 340 nm was measured. Separately, 10 g of raw squid was ground and 40 ml of purified water was added and heated to 90 ° C. or higher for 5 minutes. After cooling, purified water was added to make 50 ml. A measuring solution was prepared by replacing 0.1 ml of the supernatant of this specimen with taurine having the above formulation, and incubated at 30 ° C., and the absorbance at 340 nm was measured. From this absorbance, the taurine content of the sample was 750 mg / 100 g.

The taurine content of the sample was measured by a conventional method. That is, when the polymer compound was previously removed from the ground sample and then measured with an amino acid analyzer using an ion exchange resin column while separating various amino acids, the taurine content was 780 mg / 100 g. From the above results, the taurine dehydrogenase obtained by the present invention can be used to obtain a quick and simple and accurate measurement value of taurine. In addition, if the amount of taurine is kept constant, determination of pyruvic acid is easy.
Furthermore, since tauropine is a rare substance, its pharmacological action has not yet been elucidated. In the future, it is expected that tauropine will be easily produced using tauropine dehydrogenase, and that the useful pharmacological effects of tauropine will be elucidated.

Claims (5)

New E. coli received as FERM AP-20609. Escherichia coli having a recombinant vector plasmid gene inserted into a plasmid without inhibiting the ability to produce tauropine dehydrogenase by processing a tauropine dehydrogenase gene derived from aragro iricolor . E. coli containing the tauropine dehydrogenase gene described in Sequence Listing 1. A genetically engineered Escherichia coli in which a Tauropine dehydrogenase gene derived from Arabella iricolor is inserted into pTV118N vector (Takara Bio) and inserted into Escherichia coli JM109. A method for producing tauropine dehydrogenase, comprising culturing Escherichia coli according to any one of claims 1 to 4 and collecting and purifying tauropine dehydrogenase from the obtained culture.