JP2533671B2 - Method for producing thermophilic bacterium ribonuclease H by recombinant DNA technology - Google Patents

Description

TECHNICAL FIELD The present invention relates to production of thermophile ribonuclease H by a DNA recombination method, and more specifically, expression capable of efficiently expressing thermophile ribonuclease H gene. The present invention relates to a vector, a transformant containing the expression vector, and a method for producing thermophile ribonuclease H using the transformant.

[Problems to be Solved by Prior Art and Invention] Ribonuclease H is RNA of DNA / RNA hybrid.
It has a substrate specificity of specifically cleaving only the chain by the endo action. Based on such substrate specificity, it has extremely high utility value in the field of biochemistry. In particular, in genetic engineering, which has undergone remarkable development in recent years, Escherichia coli-derived enzymes are widely used as enzymes having various actions as described below.

1) Removal of template mRNA during cloning of cDNA.

2) Removal of the poly A region of mRNA.

3) Fragmentation of RNA.

Although the importance of this enzyme is increasing with the development of genetic engineering, the production amount of Escherichia coli ribonuclease H in the bacterial cells is extremely low at a maximum of about 10 μg per culture in the natural state, and the supply amount to the demand is increased. The shortage is obvious. Therefore, an attempt was made to produce a cloned enzyme using Escherichia coli using the DNA recombination method, and the products thus obtained have already been commercialized by various manufacturers [eg, Bethesda Research・ Laboratory (Beth
Esda Research Laboratory), Pharmacia, and Takara Shuzo]. The present inventors have already succeeded in developing a high-producing bacterium of Escherichia coli ribonuclease H (JP-A-01-202286).
issue).

By the way, ribonuclease H is used as the cDNA described above.
When it is used as a reagent for cloning, a highly purified purified sample that does not contain nucleases is required. However, no matter how high the production level of Escherichia coli ribonuclease H is raised, it is extremely difficult to remove contaminants such as nucleases present in an extremely small amount as long as the present enzyme is purified from Escherichia coli. Even if such a highly purified purified sample can be prepared without any contamination, it is only necessary to mix a trace amount of nuclease from the enzyme diluent, the reaction solution, and the air. However, if it becomes possible to prepare an extremely heat-stable ribonuclease H, such contaminating nuclease activity etc. will be removed by heat treatment. Generally, the thermophilic bacterium-derived enzyme has a T ₅₀ value (temperature at which enzyme activity is lost by 50%) of 30 to 40 ° C higher than that of a normal-temperature-derived enzyme, and is inactivated even if treated at 100 ° C for several minutes. It is known not to. Therefore, if thermophilic bacterium ribonuclease H can be produced in Escherichia coli by a gene recombination technique, the nuclease activity contaminated by the heat treatment can be removed, so that the value as a research reagent is expected to be significantly increased. In this case, it would be clear that purification of the enzyme directly from the thermophile is meaningless, since the contaminating nucleases would also be heat stable.

Further, even when the DNA / RNA hybrid has a higher-order structure and thus is less likely to be cleaved by ribonuclease H, it is possible to allow the enzyme to act at a high temperature at which such a structure cannot be obtained. Thermophilic ribonuclease H has the advantage of From the above viewpoints,
Development of a method for preparing cloned thermophile ribonuclease H has been desired. By the way, the structural gene of thermophile ribonuclease H has already been cloned by Mitsubishi Kasei Life Research Institute: Dr. Mitsuyasu Itaya, and its DNA sequence has also been determined (Fig. 1), but since the start codon is GTG, it remains as it is. Therefore, it is predicted that the expression efficiency will be extremely poor even when the expression is performed in E. coli. This is because the initiation codon is generally ATG of Met, and it is known that the efficiency of GTG to encode Met is extremely low. Therefore, in order to efficiently express the cloned thermophile ribonuclease H in E. coli,
It was predicted that modification of the start codon would be necessary.

[Means for Solving the Problem] The present inventors have conducted various studies for constructing an expression vector capable of efficiently expressing the thermophilic ribonuclease H gene for the purpose of expressing thermophilic ribonuclease H in Escherichia coli. As a result, an E. coli host cell transformed with the P _L and P _R promoters of bacteriophage lambda and a thermophilic ribonuclease H structural gene under the control of the promoter efficiently transformed the enzyme into E. coli host cells. The inventors have found that they are produced and have completed the present invention. Here, thermophilic ribonuclease H
In the structural gene of, the start codon is modified from GTG to ATG.

That is, according to the present invention, the structural gene of thermophile ribonuclease H whose start codon is modified is P _{L of} bacteriophage λ.
And under the control of the P _R promoter, there is provided a replication and expression vector capable of expressing in E. coli.

The present invention also relates to a method for producing thermophile ribonuclease H, which comprises transforming an Escherichia coli host with the above expression vector and transforming the resulting transformant into thermophile ribonuclease H.
The present invention provides a method comprising culturing under conditions suitable for gene expression and isolating and purifying ribonuclease H in each culture solution.

The present invention also provides a transformant containing the expression vector of the present invention.

Furthermore, the present invention is a method for isolating and purifying thermophilic bacterium ribonuclease H from a culture broth, wherein after culturing a transformant, the bacterium is collected from the culture broth and sonicated to dissolve the bacterium. Then, the cell lysate thus obtained is subjected to P-11 column chromatography in the presence of 8 M or more of urea.

The expression vector of the present invention, the plasmid pJAL700T, is a structural gene of thermophilic ribonuclease H (hereinafter, simply referred to as ribonuclease H in the present specification) cloned from thermophilic chromosome by Dr. Mitsuyasu Itaya, Mitsubishi Kasei Life Research Institute. Plasmid pRE containing (rnh) (Fig. 1)
It was constructed as follows using T4004-3B6A as a source of rnh (see Figure 2).

In addition, cloning of thermophile ribonuclease H and DN
The A sequence has already been published by Itaya (61st Japan Biochemical Society), and the plasmid pRET4004-3B6A has been published.
Is provided by Itaya.

Construction of plasmid pJAL700T The 700 bp M1uI-HindIII fragment of plasmid pRET4004-3B6A was excised and commercialized plasmid pUC19 with a 29mer synthetic DNA oligomer containing NdeI site (see Fig. 3).
At the EcoRI-HindIII site to obtain plasmid pUC700EH. Here, the 29-mer synthetic DNA oligomer contains the initiation codon of thermophile ribonuclease H. In the synthetic oligomer, the initiation codon is modified from natural GTG to ATG. Saind the HindIII site of plasmid pUC700EH
After conversion to the site, cut out the 650 bp NdeI-Sa1I fragment,
A commercially available expression vector pJLA503 is inserted into the NdeI-Sa1I site to construct the expression vector pJAL700T of the present invention. A restriction map of plasmid pJAL700T is shown in FIG. In plasmid pJAL700T, P _L and P _R promoters of bacteriophage lambda dominates the thermophilic bacteria rnh gene of the 3'-downstream. Furthermore, since this plasmid contains the gene for the heat-sensitive repressor cI ^ts857 , whichever E. coli host is transformed with this plasmid, the thermophilic RN
The expression of aseH is induced by shifting the culture temperature from 30 ° C to 42 ° C.

Expression of ribonuclease H in Escherichia coli Escherichia coli K-12 strain HB was prepared using the above plasmid pJAL700T.
^{101 (F -, hsd.S20 (r} B -, m B -), recA13, ara-14, pro
A2, lacY1, ga1K2, rpsL20 (Sm ^r ), xy1-5, mt1−
1, supE44, λ ^-) to the transformation. In this case, the type of E. coli host transformed as described above does not matter. P _L
Since the promoter does not function in the temperature range where the cI repressor is stable (32 ° C or lower) and functions in the unstable temperature range (40 ° C or higher) for the first time, the trait selected on the basis of ampicillin resistance. Transformant, E. coli HB1
01 / pJAL700T culture temperature in the logarithmic growth phase from 30 ℃ to 40 ℃
When it is raised and cultured, the promoter functions, the expression of ribonuclease H is induced, and the desired enzyme ribonuclease H is mass-produced.

Isolation and purification of ribonuclease H Isolation and purification of ribonuclease H in the method of the present invention are performed in the following order.

1) sonication 2) P-11 column chromatography 3) re-chromatography with P-11 column The ribonuclease H-highly producing bacterium of the present invention obtained as described above is collected and then sonicated. , Solubilizes ribonuclease H in cells. After centrifuging the cell lysate, urea was added to the centrifugation supernatant (crude extract) to a final concentration of 8 to 10 M, and the mixture was subjected to P-11 cellulose column chromatography to obtain a purified sample.

[Operation of the Invention] The transformant containing the vector of the present invention is cultured as described above, and the ribonuclease H isolated and purified from the obtained culture solution according to the method of the present invention is treated at 90 ° C for 10 minutes. However, since 100% of the enzyme activity is retained, it is possible to remove contaminating nuclease activity and the like by heat treatment. Therefore, according to the present invention, an enzyme for genetic engineering, which requires extremely high purity, can be efficiently obtained in high yield. Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Construction of plasmid pJAL700T pRET4004-3B6A was used as the rnh gene source. This plasmid pRET4004-
3B6A (10 μg) was digested with 50 units each of HindIII and M1uI in a 100 μl reaction solution at 37 ° C. for 1 hour. The digest was then subjected to 1.5% agarose gel electrophoresis. r
The 700 bp HindIII-M1uI fragment containing the nh gene was extracted from the agarose gel by electroelution (electroelution) and then purified by DE-52 column chromatography. 700bp HindII recovered by ethanol precipitation
The amount of I-EcoRI fragment was about 1 μg. On the other hand, plasmid vector pUC19 (manufactured by TOYOBO) (2 μg) was added to 50 μl of reaction solution at 37 ° C. with 10 units of HindIII and EcoRI.
The digested product that had been completely digested for 1 hour was subjected to 0.7% agarose gel electrophoresis, and the 2.7 kb EcoRI-HindIII fragment was eluted and purified in the same manner as the 700 bp fragment. The recovery rate was almost 100%.

The 700 bp HindIII-M1uI fragment and the 2.7 kb EcoRI-HindIII fragment thus obtained were each 0.1 μg and 2
9mer Synthetic DNA oligomer (Fig. 3) 0.01 μg was mixed, and 2
5 units of T4 DNA ligase (TOYO
(Manufactured by BO) and reacted at 16 ° C. for 30 minutes to cyclize the plasmid. The circularized plasmid was then used to transform E. coli JM10.
9 was transformed, and the plasmid pUC700EH was obtained from the transformant.

Then, this pUC700EH (2 μg) was completely digested with 10 units of HindIII in 20 μl of the reaction solution at 37 ° C. for 1 hour. After collecting the DNA by ethanol precipitation,
After adding 2 units of DNA polymerase (Klenow) to the reaction solution of 1 and reacting at 37 ° C for 30 minutes, the mixture was treated at 65 ° C for 10 minutes. 20μ of DNA recovered by ethanol precipitation
After adding 1 unit of Escherichia coli alkaline phosphatase to the reaction solution of 1 and treating at 37 ° C. for 30 minutes, the reaction solution was subjected to 0.7% agarose gel electrophoresis. The 3.5 kb DNA fragment was eluted and purified in the same manner as the 700 bp HindIII-M1uI fragment. The recovered amount of DNA was about 1 μg. Obtained DNA 0.1
μg was mixed with 0.01 μg of Sa1I linker (Takara Shuzo) (molar ratio 1:50), and 5 units of T4 DNA in 20 μl of reaction solution.
Ligase was added and the mixture was treated at 16 ° C for 30 minutes. Escherichia coli JM109 was transformed with the plasmid thus cyclized, and a plasmid pUC700ES was obtained from the transformant.

Finally, the expression vector pJAL700T was constructed as follows. The plasmid pUC700ES (10 μg) was completely digested with 50 units of NdeI and 50 units of Sa1I in 100 μl of the reaction solution at 37 ° C. for 1 hour, and then subjected to 1.5% agarose gel electrophoresis. The 650 bp NdeI-Sa1I fragment was cut out and eluted and purified in the same manner as in the case of the 700pb HindIII-M1uI fragment. The amount of DNA recovered by ethanol precipitation was about 1 μg.

On the other hand, plasmid pJLA503 (West Germany: purchased from Medac)
5 μg was added to 100 μl of reaction solution with 50 units of NdeI and 50
After digesting with 1 unit of Sa1I for 1 hour at 37 ℃, 0.7
% Agarose gel electrophoresis. 4.9kb NdeI-Sa1I
The fragment was cut out and eluted and purified in the same manner as in the case of the 700 bp HindIII-M1uI fragment. The amount of DNA recovered by ethanol precipitation was about 1 μg.

650 pb NdeI-Sa1I fragment 0.1 obtained as described above
μg and 0.1 μg of 4.9 NdeI-Sa1I fragment were mixed, and 20 units of reaction solution was added with 5 units of T4 DNA ligase at 16 ℃.
After treatment for 30 minutes, the plasmid was circularized. Escherichia coli HB101 was transformed with the cyclized plasmid, and the plasmid pJAL700T was obtained from the transformant (see FIG. 2).

A detailed restriction enzyme digestion map of the desired plasmid pJAL700T obtained as described above is shown in FIG. The transformant Escherichia coli ( E. coli ) HB101 / pJAL700T has been deposited at the Institute for Microbial Technology, Institute of Industrial Science and Technology (Microbiology Research Institute No. 11416, Deposit date: April 18, 1990). .

Example 2 Cultivation of transformants and preparation of ribonuclease H-containing cells Escherichia coli K-12 strain HB101 (phenotype was the above) transformed with the plasmid pJAL700T described in Example 1 was added to LB medium containing 80 μg / l ampicillin. The culture was performed at 30 ° C with shaking. At the time when the turbidity of the culture broth reached a Klett value of around 100, the culture temperature was raised to 42 ° C., and the mixture was further shaken for 4 hours and then collected. The obtained cells were stored frozen at -20 ° C.

Example 3 Extraction and purification of ribonuclease H from transformants HB101 / pJAL70 collected from 40 ml of the culture broth obtained in Example 2.
About 300 mg of 0T microbial cells was suspended in 5 ml of 10 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, and the microbial cells were disrupted by sonication. Then, it was centrifuged at 15,000 rpm for 30 minutes.
To about 5 ml of the obtained centrifugation supernatant, 3.5 g of urea and 120 μl of 1M acetate buffer (pH 5.5) were added. At this time, the final concentrations of urea and acetate buffer in the centrifugal supernatant are about 9 M and about 20 mM, respectively. By the above operation, the ribonuclease H in the cells was solubilized by almost 80%.

Then, the sample solution was applied to a P-11 column (0.6 ml) equilibrated with a 20 mM acetate buffer (pH 5.5) containing 8 M urea (hereinafter abbreviated as buffer A). After application of the sample, the column was washed with about 2 ml of buffer A and 2 ml of buffer A containing 0.2 M NaCl in this order, and then the enzyme was eluted by increasing the NaCl concentration linearly to 0.7 M. did. P
The recovery rate of ribonuclease H in -11 column chromatography is about 50%, and the purification purity is about 20% to 95%.
Rose to%. Performing so-called re-column chromatography, in which the elution fraction from the P-11 column is diluted 3 times with buffer A and then applied again to the P-11 column (0.5 ml) equilibrated with the same buffer. Ribonuclease H was purified by SDS-PAGE until a single band was shown (Fig. 5). The recovery rate of this enzyme in this re-chromatography with P-11 was about 80%. Therefore, the purification yield of ribonuclease H through the entire purification process is about
40%. The removal of 8M urea from the purified sample was performed by PD-10 (Pharmacia) equilibrated with 10 mM acetate buffer (pH 5.5).
It was carried out by desalting with. Recovery of ribonuclease H in this desalting operation was almost 100%. After all,
The yield of the purified preparation was about 10 mg / l. The specific activity of the thermophilic bacterium ribonuclease H thus obtained against M13.DNA / RNA hybrid hydrolysis was about 10,000 units / mg, which was about 8% of E. coli ribonuclease H, when measured at 37 ° C. It was

Example 4 Thermostability of thermophile ribonuclease H When the thermostability of thermophile ribonuclease H obtained in Example 3 was compared with that of Escherichia coli ribonuclease H,
As shown in FIG. 6, the enzyme activity of Escherichia coli ribonuclease H is almost completely inactivated by treating at 60 ° C. for 10 minutes, whereas the enzyme activity of thermophilic ribonuclease H is at 90 ° C. for 10 minutes. The treatment did not have any effect.

[Effect of the Invention] As described above, ribonuclease H is an enzyme having a high value as a reagent. Up to now, cloned Escherichia coli ribonuclease H is commercially available, but there is a problem that it is not possible to completely remove contaminants such as a trace amount of nuclease activity. The present invention provides a cloned thermophile ribonuclease H capable of removing nuclease activity contaminated by heat treatment.
The present invention provides a method for preparing the above, and promotes the development of research utilizing the enzyme.

[Brief description of drawings]

Figure 1 shows the DNA of the thermophilic ribonuclease H structural gene.
Sequence and a schematic diagram showing the amino acid sequence predicted from the DNA sequence. Fig. 2 is a schematic diagram of the assembly of plasmid pJAL700T. Fig. 3 is a schematic diagram showing the sequence of the synthetic DNA linker (29mer) and restriction enzyme sites. The figure shows the restriction enzyme digestion map of plasmid pJAL700T. Figure 5 is a schematic diagram of the photograph of SDS-PAGE followed by CBB staining. Figure 6 shows the stability against irreversible heat inactivation. It is the graph which compared between.

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Claims (12)

(57) [Claims] 1. A DNA fragment encoding thermophile ribonuclease H, the start codon of which is natural GTG to ATG.
Recombinant expression vector containing the Escherichia coli under control of the gene expression system. 2. A replication origin derived from plasmid pBR322, a gene of heat-sensitive repressor cI ^ts857 , both P _L and P _R promoters of bacteriophage lambda, and a thermophilic bacterium under the control of both P _L and P _R promoters. The expression vector according to claim 1, which contains a structural gene for ribonuclease H and is capable of replication and expression in E. coli. 3. The expression vector according to claim 2, which is the plasmid pJAL700T. 4. The expression vector according to claim 3, which contains an ampicillin resistance gene as a selectable marker. 5. A transformant obtained by transforming an Escherichia coli host with the expression vector according to any one of claims 1 to 4. 6. The transformant according to claim 5, wherein the host is Escherichia coli K-12 strain HB101. 7. The transformant according to claim 6, which is Escherichia coli HB101 / pJAL700T. 8. The transformant according to claim 5 is cultured under conditions suitable for expression of thermophilic ribonuclease H, and thermophilic ribonuclease H is recovered from the obtained E. coli cells. Thermophilic ribonuclease H consisting of
Manufacturing method. 9. The method according to claim 8, which comprises culturing while changing the culture temperature of Escherichia coli HB101 / pJAL700T from 30 ° C. to 42 ° C. in the logarithmic growth phase. 10. After completion of the culture, the transformant cells are sonicated to be disrupted, and the resulting solution is subjected to column chromatography to isolate the desired protein. 9. The method described in 9. 11. The method according to claim 10, which comprises subjecting the desired protein to purification by subjecting it to cation exchange column chromatography in the presence of urea at a concentration of 8 M or more, and then removing the urea by a desalting column. 12. The method according to claim 11, which comprises using phosphocellulose as the cation exchange resin.