JP2535583B2 - Mutant Escherichia coli ribonuclease H - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention provides a mutant Escherichia coli ribonuclease H with improved enzyme activity, a gene encoding the mutant enzyme, and a gene containing the gene, which can be expressed in E. coli. Relates to an expression vector, a transformant containing the expression vector, and a method for producing a mutant Escherichia coli ribonuclease H using the transformant.

[Prior Art and Problems Thereof] Escherichia coli ribonuclease H (hereinafter, simply referred to as ribonuclease H in the present specification) is a hydrolase consisting of 155 amino acids and having a molecular weight of about 17 Kd. It has the substrate specificity that it specifically cleaves by endo action. Based on such substrate specificity, in recent years, in genetic engineering, which has undergone remarkable development, it has been attracting attention as an enzyme having an extremely high utility value as an enzyme 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.

It is believed that the importance of this enzyme will increase with the development of genetic engineering. Since the production amount of this enzyme in E. coli is extremely low, a cloned enzyme by genetic engineering has been produced and already supplied from BRL, Pharmacia, Takara Shuzo and the like. However, these commercially available cloned enzymes also have low productivity and are expensive due to low yield due to loss and inactivation during the isolation and purification steps. Therefore, it is strongly desired that ribonuclease H having an enzymatic activity useful for promoting the above-mentioned various studies be easily available.

Generally, the effective amount of an enzyme is judged based on the level of catalytic activity, regardless of the amount of the enzyme. Therefore, if the specific activity of the enzyme is increased, the enzyme supply amount is substantially increased. By the way, in order to change the functions of proteins such as enzymes and antibodies, a method of inducing mutation in the DNA sequences encoding these proteins to modify the amino acid sequence is often used. As a result of such an operation,
Antibodies with improved substrate specificity and / or antigen binding, enzymes with altered substrate specificity and optimal PH, enzymes with improved thermostability, etc. have been obtained. However, there are almost no examples in which an enzyme having an increased catalytic activity was obtained, and no such report has been made for ribonuclease H before the present invention.

Therefore, if it is possible to obtain a mutant ribonuclease H having an increased specific activity, it means that the supply amount of the enzyme is substantially increased. In addition, since the purified sample of mutant ribonuclease H is less contaminated with contaminating proteins such as nucleases normally associated with natural ribonuclease H, it can be said that a purified sample of higher quality can be prepared as an enzyme reagent. That is, by obtaining an enzyme having a high specific activity, a high quality enzyme preparation can be supplied at a low cost.

[Means for Solving Problems] From the above viewpoints, the present inventors have found that ribonuclease H
For the purpose of enhancing the catalytic activity of the enzyme, site-specific mutation was introduced into the gene of the enzyme to prepare mutants having various amino acid substitutions, and the effect on the enzyme activity was examined. Cys (Cysteine) is Ser (Serine)
The present inventors have found that the mutant ribonuclease H having the amino acid sequence substituted with is about twice as active as the natural ribonuclease H, and completed the present invention.

That is, the present invention provides a mutant ribonuclease H having an amino acid sequence in which the 13th cysteine is replaced with serine.
Is provided.

The present invention also provides Cys of the ribonuclease H structural gene.
TGT, which encodes ¹³ , was transformed into S by site-directed mutagenesis.
has been converted to a TCT encoding er. It provides a mutant ribonuclease H gene.

The present invention also provides an expression vector for expressing mutant ribonuclease H in Escherichia coli, the vector containing the mutant ribonuclease H gene under the control of the tac promoter. Since the expression vector of the present invention contains the origin of replication derived from the plasmid pUC18, it can replicate in E. coli.

The present invention also provides a mutant ribonuclease H-producing transformant transformed with the above expression vector.

Further, the present invention provides a method for producing a mutant ribonuclease H having a high ribonuclease H enzyme activity by culturing the transformant.

The gene encoding the mutant ribonuclease H of the present invention was obtained according to the following steps.

1) M13mp18RF of ribonuclease H structural gene (rnh)
Subcloning into DNA The rnh gene was cloned by the inventors (Kanaya and Crouch,
Journal of Biological Chemistry, 25
8 , 1276-1281 [1983]). This cloned plasmid pSK750 is used as a source of the rnh gene and subcloned into M13mp18RF DNA according to the procedure described in FIG.

First, a 820 bp EcoRI-PstI fragment of plasmid pSK750 was cut out, and the fragment was cut into commercially available plasmid pUC19, EcoRI-PstI.
Insert at the site to obtain plasmid pSK820. This plasmid pSK820 was digested with the restriction enzyme HgiAI and contained the rnh gene.
1bpHgiAI fragment was obtained, the 3'overhang was blunt-ended by treatment with T4 DNA polymerase, and then commercially available plasmid pU
Insert at the SmaI site of C18. The rnh gene whose orientation is opposite to that of the lac promoter of the plasmid pUC18 is selected and named plasmid pKS801, which will be used in the subsequent experiments.

600 bp EcoRI-P containing rnh gene from plasmid pKS801
The stI fragment was cut out and EcoRI-PstI of commercially available M13mp18RFDNA
Insert into the site to create M13mp18 (rnh) RF DNA. M13mp
A schematic diagram of the assembly of 18 (rnh) RF DNA is shown in FIG.

2) Introduction of site-specific mutation into rnh gene Escherichia coli TG-1 is transformed with the above M13mp18 (rnh) RFDNA, and the obtained transformant is cultured. From the supernatant, M13mp18 (rnh) circular single-stranded DNA is prepared according to a conventional method.
25m of this M13mp18 (rnh) circular single-stranded DNA was chemically synthesized.
er (mer) oligonucleotide and mixed with the method of Eckstein et al. (Taylor, JW, Ott, J. and Ec
Using a commercially available kit (Amersham) using ketein, F. (1985) Nucleic Acids Research, 13 , 8674-8785), it operates according to the instruction | indication and introduces a site-specific point mutation. By this operation, Cys ¹³
Mutation rnh in which the codon TGT of is replaced by the codon TCT of Ser
M13mp18 (rnh-13S) RF DNA carrying the gene is obtained (see FIG. 2). Hereinafter, in order to distinguish the mutant ribonuclease H encoded by this mutant rnh gene from the natural ribonuclease H, ribonuclease H (13
S).

3) Construction of ribonuclease H (13S) expression vector The 230 bp EcoRI fragment was deleted from the above plasmid pKS801, and at the same time, the EcoRI site was converted into a SalI site using a SalI linker to obtain a plasmid pKS600. Then, a 600 bp XbaI-SalI fragment containing the rnh gene is excised from the plasmid pKS600. The fragment was used as a plasmid vector for expressing Escherichia coli protease III pDR42S (Patent Application No. 62-218973).
By substituting the 4.3 kb XbaI-SalI fragment containing the ptr gene of p. Then this plasmid pDR600 500b
The pXbaI-SalII fragment was cloned from M13mp18 (rnh-13S) RF DNA
Substitution with the 0 bp XbaI-SalII fragment gives the plasmid pDR601 for expressing the ribonuclease H (13S) of the present invention. A schematic diagram of the construction of plasmid pDR601 is shown in FIG. 3, and its restriction enzyme cleavage map is shown in FIG.

4) Expression of ribonuclease H (13S) in Escherichia coli Escherichia coli K-12 strain JM10 was prepared by using the above plasmid pDR601.
9 is transformed, and the obtained transformant is cultured in a medium containing ampicillin. Isopropyl β-D- during logarithmic growth phase
Ribonuclease H (13S) is expressed by adding thiogalactoside (IPTG) and culturing.

Then, the cells of the ribonuclease H (13S) -producing transformant were harvested, solubilized by lysozyme-EDTA treatment and ultrasonic treatment, and the supernatant obtained by centrifugation was dialyzed, followed by DEAE-cellulose, P -11 Cellulose and Sephacryl S-300 (Super Fine)
Prepare a purified preparation of (13S). The ribonuclease H (13S) thus obtained is purified to the extent that it gives a single band on SDS-PAGE. The degree of contamination with natural ribonuclease H in the above method is considered to be extremely small because the amount of normal ribonuclease H produced by Escherichia coli K-12 strain JM109 is extremely low. The introduction of the desired mutation was confirmed by amino acid sequencing from the amino terminus.

[Operation of the Invention] The mutant ribonuclease H of the present invention is [ ³² P] poly (rA) p
When oly (dT) is used as a substrate, it has about twice the enzymatic activity of natural ribonuclease H. Its enzymatic activity can be used in the field of engineering of various genes described above, just like the natural ribonuclease H. Accordingly, the present invention provides a mutant ribonuclease H having a high ribonuclease H activity, thereby increasing the supply of the enzyme activity.

Hereinafter, the present invention will be described in more detail with reference to examples.

Example 1 Subcloning of the rnh gene into M13mp18RF DNA pSK750 was used as the rnh gene. Plasmid pSK750
(10 μg) in 100 μl reaction solution,
Digested with stI and EcoRI for 1 hour at 37 ° C. The digest was then subjected to 1.5% agarose gel electrophoresis. The 820 bp EcoRI-PstI fragment containing the rnh gene was extracted from agarose gel by electroelution (electroelution) and then purified by DE-52 column chromatography.
The amount of 820 bp EcoRI-PstI fragment recovered by ethanol precipitation was about 1 μg. On the other hand, plasmid vector
pUC19 (TOYOBO) (2 μg) in 50 μl reaction solution
It was completely digested with each unit of PstI and EcoRI at 37 ° C for 1 hour. The digest is run on 0.7% agarose gel. The 2.7 kb EcoRI-PstI fragment was eluted and purified in the same manner as the 820 bp fragment. The recovery rate was almost 100%.

820 bp and 2.7 kb EcoRI-Ps obtained as described above
0.1 µg of each tI fragment was mixed, and 5 units of T4 DNA ligase (manufactured by TOYOBO) were used in 20 µl of the reaction solution to mix with 16 units.
The reaction was carried out at 30 ° C for 30 minutes to circularize the plasmid. E. coli JM109 was then transformed with the circularized plasmid,
Plasmid pSK820 was obtained from the transformant.

Then, 100 μl of this plasmid pSK820 (10 μg)
The reaction solution was digested with 100 units of HgiAI at 37 ° C for 1 hour. DNA was recovered by phenol-chloroform extraction and ethanol precipitation, and then 2.5 units of T4 DNA polymerase was added to 20 μl of the reaction solution, followed by reaction at 37 ° C. for 15 minutes. After stopping the reaction by treating at 65 ° C for 5 minutes, the reaction solution was subjected to 1.5% agarose gel electrophoresis, and the 801 bp HgiAI fragment containing the rnh gene was treated with 820 bp EcoRI.
-Eluted and purified in the same manner as for the PstI fragment. The recovered amount of DNA was 2 μg. On the other hand, plasmid vector pUC18 (T
(Manufactured by OYOBO) (2 μg) was completely digested with 10 units of SmaI at 37 ° C. for 1 hour in 50 μl of reaction solution, and then 1 unit of Escherichia coli alkaline phosphatase was added and further reacted at 37 ° C. for 30 minutes. After stopping the reaction by the phenol-chloroform extraction method, the DNA was recovered by ethanol precipitation. The recovery rate was almost 100%.

The 801 bp HgiAI fragment obtained as described above and SmaI
PUC18 treated with Escherichia coli alkaline phosphatase and 0.1 μg each were mixed in 20 μl reaction solution with 5 units of T4 DNA ligase at 16 ° C.
After reacting for minutes, the plasmid was circularized. Next, Escherichia coli JM109 was transformed with the circularized plasmid, and the plasmid pKS801 was obtained from the transformant.

Then, 100 μl of this plasmid pKS801 (10 μg)
50 units of EcoRI and 50 units of PstI in the reaction solution of
Was completely digested at 37 ° C for 1 hour and the digest was subjected to 1.5% agarose gel electrophoresis. 600bp Ec including rnh gene
The oRI-PstI fragment was eluted and purified as in the case of the 820bp EcoRI-PstI fragment. The recovered amount of DNA was about 1 μg.
On the other hand, 2 μg of M13mp18RFDNA (Takara Shuzo) was added to 20 μl.
10 units of EcoRI and 10 units of PstI in the reaction solution of
It was completely digested at 37 ° C for 1 hour. 0.7% digest
The 7.2 kb EcoRI-PstI fragment was subjected to agarose gel electrophoresis and eluted and purified in the same manner as the 820 bp EcoRI-PstI fragment. D
The amount of NA recovered was about 1.5 μg. The 600 bp and 7.2 kb EcoRI-PstI fragments obtained as described above were used respectively in 0.1
5g T4 D in 20μl reaction solution
RF DNA was circularized by reacting with NA ligase (TOYOBO) at 16 ° C for 30 minutes. Then circularized RF DNA
E. coli TG-1 was transformed with E. coli and the transformant was used to transform M13mp1
8 M13mp18 (rnh) RF DNA with rnh gene inserted in RF DNA
Was obtained (see FIG. 1).

Example 2 Introduction of Site-Directed Mutation in rnh Gene Preparation of single-stranded DNA from the culture supernatant of Escherichia coli TG-1 transformed with M13mp18 (rnh) RF DNA obtained in Example 1 Both phosphorylation and introduction of point mutations into the rnh gene were performed using a kit (Oligonucleotide-Directed In Vitro Mutagenesis System) commercially available from Amersham, exactly following the attached instructions. As the oligonucleotide, chemically synthesized 25-mer 5'-CCGATGGTTCGTC
TCTGGGCAATCC-3 'was used. Cys ¹³ codon TGT is Ser
The RF DNA containing the mutant ribonuclease H gene modified at the codon TCT of was named M13mp18 (rnh-13S) (see FIG. 2) and used in the subsequent experiments.

Example 3 Construction of plasmid pDR601 for expression of ribonuclease H (13S) 1. Construction of plasmid pDR601 for expression of natural ribonuclease H Plasmid pKS801 (see FIG. 1) 2 μ obtained in Example 1
37 g with 20 units of EcoRI in 20 μl reaction solution
Digested completely at 1 ° C for 1 hour. DNA by ethanol precipitation
2 units of DNA in 20 μl reaction solution after
Polymerase (Kreso) was added and reacted at 37 ° C for 30 minutes, and then treated at 65 ° C for 10 minutes. The DNA recovered by ethanol precipitation was added with 1 unit of E. coli alkaline phosphatase in 20 µl of the reaction solution and treated at 37 ° C for 30 minutes, and then the reaction solution was subjected to 0.7% agarose gel electrophoresis. The 3.3 kb DNA fragment was converted to 820 bp Eco described in Example 1.
Elution and purification were carried out in the same manner as for the RI-PstI fragment. The recovered amount of DNA was about 1 μg. 0.1 μg of the obtained DNA was mixed with 0.01 μg of SelI linker (Takara Shuzo) (molar ratio 1:50),
In 20 μl of the reaction solution, 5 units of T4 DNA ligase was added and treated at 16 ° C. for 30 minutes. Escherichia coli JM109 was transformed with the plasmid thus cyclized, and plasmid pKS600 was obtained from the transformant.

Then, the plasmid pKS600 (10 μg) was completely digested with 50 units of XbaI and 50 units of SalI in 100 μl of the reaction solution at 37 ° C. for 1 hour and subjected to 1.5% agarose gel electrophoresis. The 600 pb XbaI-SalI fragment was cut out and eluted and purified in the same manner as in the case of the 820 bp EcoRI-PstI fragment described in Example 1. DN recovered by ethanol precipitation
The amount of A was about 1 μg.

On the other hand, plasmid vector pDR42S (patent application No. 62-218973) for expression of Escherichia coli protease III, 5 μg and 100 μm
50 units XbaI and 50 units SalI in 1 reaction solution
Was digested for 1 hour at 37 ° C. and subjected to 0.7 agarose gel electrophoresis. The 3.0 kb XbaI-SalI fragment was cut out and eluted and purified in the same manner as in the case of the 820 bp EcoRI-PstI fragment described in Example 1. DN recovered by ethanol precipitation
The amount of A was about 1 μg.

600 bp XbaI-SalI fragment 0.1 obtained as described above
μg and 0.1 μg of 3.0 kb XbaI-SalI fragment were mixed,
16 units with 5 units of T4 DNA ligase in 1 l of reaction solution
C. for 30 minutes to circularize the plasmid. Escherichia coli JM109 was transformed with the circularized plasmid, and a plasmid vector pD for expressing native ribonuclease H was obtained from the transformant.
I got R600.

2. Ribonuclease H (13S) expression plasmid pDR601
Construction of plasmid pDR600 obtained in 1 was replaced with the mutant rnh gene as follows. First,
Plasmid pDR600 (2 μg) was added to 10 μl of the reaction solution in 50 μl.
1 unit at 37 ° C using 1 unit of XbaI and 10 units of SatII
It was completely digested for a period of time and subjected to 0.7% agarose gel electrophoresis. The 3.1 kb XbaI-SatII fragment was excised and the 820 bp E
Elution and purification were performed as in the case of the coRI-PstI fragment.
The amount of DNA recovered by ethanol precipitation was about 1.5 μg. On the other hand, M13mp18 (rnh-13S) RF obtained in Example 2
DNA (5 μg) in 100 μl reaction solution, 50 units Xb
It was completely digested with aI and 50 units of SatII for 1 hour at 37 ° C. and the digest was subjected to 1.5% agarose gel electrophoresis. The 500 bp XbaI-SatII fragment was cut out and the above 820 bp Eco
Elution and purification were performed in the same manner as for the RI-PstI fragment. The amount of DNA recovered by ethanol precipitation was about 0.2 μg. 500 bp and 3.1 kb Xb obtained as described above
0.1 μg of each aI-SatII fragment was mixed, and 30 μl was added to 5 μl of T4 DNA ligase in 20 μl of reaction solution at 16 ° C.
It was treated for minutes and the plasmid was circularized. Escherichia coli JM109 was transformed with the cyclized plasmid, and the plasmid pDR601 was obtained from the transformant.

The detailed restriction enzyme digestion map of the ribonuclease H (13S) expression plasmid pDR601 obtained as described above is shown in
As shown in the figure. Transformant Escherichia coli ( E. col
i ) JM109 / pDR601 has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology (Ministry of Microbiology Research Institute No. 9708, deposit date: Showa era)
November 14, 62).

Example 4 Production and purification of ribonuclease H (13S) in Escherichia coli 1. Culture of transformant JM109 / pDR601 Transformant JM109 / pDR601 was shake-cultured at 37 ° C. in LB medium 1 containing 800 μg of ampicillin. When the turbidity of the culture solution grew to a Klett value of around 100, IPTG
Was added to a final concentration of 0.8 mM, and the mixture was further shaken for 5 hours, and then the cells were collected. The cret value at this time is about 12
0, the wet weight of the cells was about 1.5 g.

2. Extraction and purification of ribonuclease H (13S) from bacterial cells After suspending the bacterial cells obtained in 1 above in 5 ml of 10 mM Tris-HCl buffer (pH 8) containing 0.1 M NaCl and 25% sucrose, 1.33 ml of 0.3 M Tris-HCl buffer (pH 8) containing mg lysozyme and 0.1 M EDTA was added. After allowing to stand in ice for 5 minutes, the bacteria were disrupted by ultrasonic treatment. Centrifuge supernatant (crude extract) obtained by centrifugation at 15000 rpm for 30 minutes at 4 ° C.
It was dialyzed against 50 mM Tris-HCl buffer (pH 7.5) containing EDTA, 1 mM β-mercaptoethanol and 10% glycerin. Then, a DE-52 column (~
6 ml) and a P-11 column (-4 ml) in this order.
Under this condition, ribonuclease H (13S) passes through the DE-52 column and is adsorbed on the P-11 column. Ribonuclease H (13S) was eluted from the P-11 column by increasing the NaCl concentration linearly. Finally, the elution fraction from the P-11 column was mixed with 10 mM Tris-hydrochloric acid buffer solution (pH 7. mM) containing 1 mM DTT, 1 mM EDTA, 0.08 M NaCl and 10% glycerin.
Ribonuclease H (13S) was purified by SDS-PAGE to give a single band by loading on a Sephacryl S-300 (Superfine) column (φ1.6 × 190 cm) equilibrated in 5). The recovery rate of ribonuclease H (13S) was about 60% through the above entire purification procedure.
The purification yield is about 3 mg / l culture, which is equivalent to JM109 / pDR
There was little change compared to the purified yield of native ribonuclease H from 600 of 5 mg / l. Since the natural ribonuclease H and ribonuclease H (13S) are not separated by the above-mentioned purification operation, ribonuclease H (13S)
The purified sample is contaminated with natural ribonuclease H derived from JM109 used as a host. However, E. coli
The purified yield of native ribonuclease H from JM109 is
Usually, since it is about 5 μg / l, the mixing ratio is about 0.16%, which is extremely low. Therefore, it can be considered that the properties of this purified preparation reflect the properties of ribonuclease H (13S). To identify the purified sample, the amino acid sequence from the amino terminus was analyzed and the 13th amino acid was the Ser
It was carried out by confirming that it was mutated.

The enzymatic activity of ethanol ribonuclease H (13S) in Example 4 was evaluated as described in the Examples below.

Example Evaluation of Enzyme Activity of Ribonuclease H (13S) The enzyme activity of natural ribonuclease H and that of ribonuclease H (13S) were measured using [ ³² P] poly (rA) poly (dT) as a substrate and compared. Table 1 shows the results. From Table 1, it can be seen that the specific activity of ribonuclease H (13S) is increased about twice as much as that of the natural type.

[Effects of the Invention] The mutant ribonuclease H obtained by the present invention is
Since its enzymatic activity is about twice as high as that of natural ribonuclease H, a smaller amount than the conventional enzyme reagent will show an equivalent catalytic action. Therefore, the present invention substantially increases the supply amount of the enzyme. Further, according to the method of the present invention, the contamination of contaminating proteins with the native ribonuclease H can be reduced, so that a higher quality purified preparation can be obtained.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the outline of subcloning of the rnh gene into M13mpRF DNA. FIG. 2 is a schematic diagram showing a reaction for introducing a site-specific mutation into a natural ribonuclease H gene using a synthetic oligonucleotide (25mer) to obtain a ribonuclease H (13S) gene. In the figure, the point mutation site is indicated by a star, and the amino acid corresponding to the mutation is underlined. Figure 3 is a plasmid
FIG. 7 is a schematic diagram of assembly of pDR601. Figure 4 shows plasmid pDR6
It is a restriction enzyme digestion map of 01.

Claims (8)

(57) [Claims] 1. A mutant Escherichia coli ribonuclease H having an amino acid sequence in which the 13th cysteine in the E. coli ribonuclease H amino acid sequence is replaced with serine. 2. A structural gene encoding Escherichia coli ribonuclease H, wherein the TGT codon of the 13th cysteine is converted to the TCT codon of serine by site-directed mutation. 3. An expression vector capable of replication and expression in Escherichia coli, which contains the mutant Escherichia coli ribonuclease H structural gene according to claim 2 under the control of the tac promoter. 4. The expression vector according to claim 3, which contains an ampicillin resistance gene as a selectable marker. 5. The expression vector according to claim 3 or 4, which is the plasmid pDR601. 6. A transformant transformed with the expression vector according to any one of claims 3-5. 7. The transformant according to claim 6, which is Escherichia coli JM109 / pDR601. 8. A mutant Escherichia coli ribonuclease H comprising culturing the transformant according to claim 6 or 7 under conditions suitable for expression of a ribonuclease H gene and recovering the mutant Escherichia coli ribonuclease H from the resulting culture solution. Manufacturing method.