JPH03180179A - Escherichia coli ribonuclease h having artificial disulfide bond - Google Patents

Abstract

PURPOSE:To provide the subject Escherichia coli polynucleotide He having an amino acid sequence in which asparagine and cysteine at the specific number positions in the amino acid sequence are substituted with cysteine and alanine, respectively, capable of being replicated in Escherichia coli cell and giving a manifestation vector containing a replication source originated from plasmid pUC 18. CONSTITUTION:A reduction type variant ribonuclease H having an amino acid sequence in which asparagine at the 44th position is substituted with cysteine and cysteine at the 63th and 133th positions also with alanine. A transformant transformed with the abovementioned manifestation vector and enabling to produce the variant ribonuclease H is obtained. The culture of the transformant allows to prepare the reduction type variant ribonuclease H. The reduction type variant ribonuclease H is oxidized e.g. with glutathione redox buffer to provide an oxidation type variant ribonuclease H having a S-S bond therein.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention relates to a reduced mutant Escherichia coli ribonuclease H1 gene encoding the mutant enzyme, an expression vector containing the gene and capable of expression in Escherichia coli. A transformant containing a vector, a method for producing reduced mutant Escherichia coli ribonuclease H using the transformant, and a process for treating reduced mutant Escherichia coli ribonuclease H with a glutathione redox buffer to form an S-8 bond in the molecule. The present invention relates to oxidized mutant Escherichia coli ribonuclease H obtained by introducing the present invention.

Escherichia coli ribonuclease H (hereinafter simply referred to as ribonuclease H1 or RNase H) is a hydrolase consisting of 155 amino acids and having a molecular weight of approximately 17 Kd. It has substrate specificity of cleaving. This enzyme has various uses as described below based on its substrate specificity, and is attracting attention as an enzyme with extremely high utility value.

1) Removal of template mRNA during cDNA cloning.

2) Removal of polyA region of mRNA.

3) RNA fragmentation.

The importance of ribonuclease H is expected to increase with the development of genetic engineering, but since the production amount of this enzyme in E. coli is extremely low, attempts have been made to produce this enzyme using recombinant DNA technology. Ribonuclease H produced by recombinant DNA technology has already been supplied by BRL, Pharmacia, Takara Shuzo, and others. These commercially available recombinant ribonucleases H are produced using E. coli as a host.

Generally, when performing structural analysis of RNA, RNA fragments are obtained by partial digestion with a base-specific nuclease.
RNaseH has the specificity of cutting only the RNA of DNA/RNA hybrids with its endo action, and if the reaction conditions can be controlled, such RNaseH can be used.
Fragmentation of A becomes possible. However, it is necessary to control the activation and inactivation of enzymatic action to obtain the desired cleavage. This is because it would be a problem if the reaction progressed while the reactants were being analyzed. DN A/RN A
To stop hybrid cleavage by partial digestion, currently ED
Addition of TA, heat treatment, etc. are performed. However, the heating method unravels the DNA/RNA hybrid, making it difficult to add enzymes to cut it again. In addition, when stopped with EDTA, this enzyme requires M1+ for activity expression, so for reactivation,
Add Mg! ＋Concentration control becomes difficult. Therefore, it is desired to develop a reaction termination system that can replace the EDTA addition or heating method. Furthermore, if this reaction termination system is reversible, expression and termination of the enzyme activity can be regulated simply by changing the environment without re-adding the enzyme. It is expected that this technology will be extremely useful for various purposes.

From the above point of view, the present inventors investigated the regulation of enzyme activity by introducing SS bonds into lysozyme (Matsumura and Himashuse, Science, 243.792-
794, [1989]), S
In order to introduce the -S bond and thereby modulate the enzyme activity, site-directed mutations were introduced into the gene of the enzyme to prepare mutants with amino acid substitutions, and S-8
I tried to introduce a bond. As a result, the 44th A sn
Mutant RNA with an amino acid sequence in which (asparagine) is replaced with Cys (cystine), and Cys (cystine) at positions 63 and 133 are replaced with Ala (alanine).
seH is active in the reduced state and tertiary in the oxidized state.
It was discovered that an S-3 bridge is formed between Cyg at position 44 and Cys at position 44, resulting in loss of activity, and the present invention was completed.

That is, one of the objects of the present invention is to provide a reduced mutant bonuclease H having an amino acid sequence in which the 44th asparagine is replaced with cysteine, and the 63rd and 133rd cysteines are replaced with alanine. be. Furthermore, the present invention provides the A sn'' of the ribonuclease H structural gene.
AAC encoding C75133 was transformed into TGC encoding Cys by site-directed mutation, TGC encoding cys was transformed into GCC encoding Ala, and TGT encoding C75133 was transformed into TGC encoding Cys. The present invention provides a reduced mutant bonuclease H gene that has been converted to OCT encoding.

Furthermore, the present invention provides an expression vector for expressing reduced mutant bonuclease H in E. coli, which contains the mutant bonuclease H gene described above under the control of the tac promoter. . The expression vector of the present invention contains an origin of replication derived from plasmid pUc1B and is therefore replicable in E. coli.

The present invention also provides for
The present invention provides a transformant capable of producing mutant bonuclease H. Furthermore, the present invention provides a method for producing reduced mutant bonuclease H by culturing the transformant.

The present invention also provides an oxidized mutant bonuclease H characterized in that an S-8 bond is introduced into the molecule by oxidizing the reduced mutant bonuclease H using a suitable method, for example, a glutathione redox buffer. The present invention provides a method for producing nuclease H and an oxidized mutant bonuclease H produced by the method.

As mentioned above, the present invention provides acupuncture bonuclease I4 in i.
When oxidized in vitro, an SS bridge is introduced at the Cys''-Cys' position and the enzyme is inactivated, and when reduced, the SS
Taking advantage of the fact that the crosslink is removed and the enzyme activity returns,
This provides a means to control the enzymatic activity of this enzyme. This makes it possible to ideally fragment the RNA of a DNA/RNA hybrid. That is, the mutant bonuclease H of the present invention can activate and deactivate the enzyme action simply by changing the oxidation and reduction states, and is therefore extremely effective against RNA fragmentation due to partial digestion. Useful.

The present invention will be explained in more detail with reference to Examples below.

Example 1 M13mp19RF DN of rnh gene
ps K 750 was used as the rnh gene source for subcloning into A. Plasmid psK750 (10 μg) was digested with 50 units each of Pstl and EcoRI in 100 μQ reaction solution for 1 hour at 37°C. Next, add 1.5% of the digested product.
It was subjected to agarose gel electrophoresis. The 820 bp EcoRI-Pst I fragment containing the rnh gene was extracted from the agarose gel by electroelution and then purified by DE-52 column chromatography. 820 recovered by ethanol precipitation
The amount of bp EcoR1-Pstl fragment was approximately 1 μ9. On the other hand, plasmid vector 9-pUC19 (TOYOB
(2 μg) was completely digested with 10 units each of Pstl and EcoRI in 5 μQ reaction solution at 37° C. for 1 hour.

The digest was subjected to 0.7% agarose gel electrophoresis; 2.
The 7kb EcoRI-Pstl fragment was eluted and purified in the same manner as the 820bp fragment. The recovery rate was almost 100%.

820bp and 2.7kb obtained as above
Mix 0.1 μy of each EcoRI-Pst I fragment and add 5 units of 74 DNA in 20 μQ of reaction solution.
The plasmid was circularized by reacting with ligase (manufactured by TOYOBO) at 16°C for 30 minutes.

E. coli JMI 09 was then transformed with the circularized plasmid, and plasmid pS K 820 was obtained from the transformant.

Next, add conoplasmid psK820 (10 μ9) to 1
Digested with 100 units of HgiAI in 00 μQ reaction solution for 1 hour at 37°C. After phenol-chloroform extraction and ethanol precipitation to recover DNA, 2
2.5 units of 74 DNA in 0 μQ reaction solution
Polymerase was added and reacted at 37°C for 15 minutes. 6
After stopping the reaction by treatment at 5°C for 5 minutes, the reaction solution was subjected to 1°5% agarose gel electrophoresis and rn
The 801 bpHgiΔI fragment containing the h gene was
pEc.

It was eluted and purified in the same manner as for the R1-Pstl fragment.

The amount of DNA recovered was 2 μ2. On the other hand, add 50 μy of plasmid vector 9-pUC18 (manufactured by TOYOBO) (2 μy).
g of the reaction solution at 37° by the IOl unit Smal.
Digest completely for 1 hour at 37°C, then add 1 unit of E. coli alkaline phosphatase and incubate for an additional 30 minutes at 37°C.
I reacted with After stopping the reaction by phenol-chloroform extraction, DNA was recovered by ethanol precipitation. The recovery rate was almost 100%.

The 801 bp Hg1A 1 fragment obtained as described above and pUc18. 0.1 each
5 units of T in 20 μQ reaction solution.
4. The plasmid was circularized by reacting with DNA ligase at 16°C for 300 minutes.

Next, Escherichia coli JM109 was transformed with the circularized plasmid, and plasmid pKS801 was obtained from the transformant.

Next, 10 μg of this plasmid pKs801 was added to
50 units of Ec in 00 μg of reaction solution.

RI and 50 units of PstI for 1 hour at 37°C.
After complete digestion, the digest was subjected to 1.5% agarose gel electrophoresis. 6oobpEcoRI containing rnh gene
-Pst I fragment, 820 bp EcoRI -Ps
It was eluted and purified in the same manner as for the tI fragment. The amount of DNA recovered was approximately lμ9. On the other hand, M13n+p19RFD
2μ2 of NA (manufactured by Takara Shuzo) was mixed with 10 units of EcoRI and 10 units of P+st in a 20μQ reaction solution.
Complete digestion was carried out for 1 hour at 37°C. 0 digested matter
．． 7.2 kb E was subjected to 7% agarose gel electrophoresis.
The coRI-Pst I fragment was converted into a 82Obp EcoRI fragment.
- It was eluted and purified in the same manner as the Pst I fragment. The amount of DNA recovered was approximately 1.5 μ9. 600bp and 7.2kb EcoRI Pstl obtained as above
Mix 0.1 tt9 of each fragment, add 5 units of 74 DNA ligase (TOYO
(manufactured by BO) at 16°C for 300 minutes, and RF
DNA was circularized.

Next, E. coli TG-1 was transformed with the circularized RF DNA, and M13+++p19RF was extracted from the transformant.
M13mp19 (r
nh) RF DNA was obtained (see Figure 1).

Example 2 Construction of pSS13 RNaseH has two free cys at positions 13.63 and 13333, but the S-
In order to prevent other Cys from interfering with the cross-linking of 8 bonds, all Cys were first converted to Ala, and then the amino acid at the desired position was converted to Cys. As a method, Ml 3
M13mpl is obtained by converting mpl 9 (rnh) RF DNA from three Cys to Ala by point mutation.
9 (rnh -13,63,L 33A) was produced, and using this RF DNA, the 13th Ala was converted to Cys.

The 44th Asn was converted to Cys. Ml 3a+pl 9(rnh-44C,63,13
A DNA fragment containing the mutant RNaseH gene was excised from 3A) and incorporated into the expression plasmid pDR600 (Zenhō et al., JP-A-1-202284) to generate the mutant R
NaseH expression plasmid pss13 was obtained. Actually, M l 3 mp 19 (rnh
) Preparation of single-stranded DNA from the culture supernatant of E. coli TG-1 transformed with RF DNA, phosphorylation of oligonucleotides, and introduction of point mutations into the rnh gene.
All experiments were carried out using a commercially available kit (Oligonucleotide Buelefted In Vitro Mutagenesis System) from Amerjam and following the attached instructions exactly. Each primer is shown in FIG.

RF DNA M1 containing a mutant ribonuclease H gene prepared by changing the 63rd Cys codon TGC to Ala GCC using Brimer No. 4
3mpl 9 (rnh-63A) was created. next,
This Ml 3apl 9 (rnh-63A) and ply 7
-No.

1 and 5 to create the 13th Cys codon TG
T to GCC of Ala, and codon T of Cys at position 133
Mutant RNA prepared by modifying GT to Ala GCT
RF DNA containing seH gene, M13mp1
9 (rnh-13,63,133A) was obtained.

After creating Cys-free RF DNA in this way, we used Brimer No. 2 and 3 to introduce Cys into the desired positions 13 and 44, and converted the 13th Ala codon GCC into Cys TGT. RF DNA M containing a mutant RNaseH gene prepared by modifying the 44th ASN codon AAC to Cys TGC.
13mp19 (rnh-44C, 63°133A) was created. M13n+p19(r
nh-44C,63,133A) and pDR600 were digested with XbaI and 5stII, respectively, at 37°C for about 1 hour. M 13apl 9(rnh-44C,6
The digested product of 3,133A) was subjected to 1.5% agarose gel electrophoresis, and a 500 bp Xbal-SstII fragment was excised and extracted. Similarly, the PDR600 digest was subjected to 0.7% agarose gel electrophoresis, and the 3.1 kb XbaI-3stn fragment was excised and extracted. 500bp X obtained in this way
bal-8stII fragment 0.025 μ9;
3. lkb Xbal-5stII fragment 0
．． 05μ9 and DNA ligation kit (
The plasmid was circularized using Takara Shuzo (Takara Shuzo) according to the attached instructions. With this circularized plasmid, E. coli J.
MI 09 was transformed, and plasmid p was obtained from the transformant.
ss13 was obtained (see Figure 3).

The transformed bacterium E. coli J M 109/pS S 13 obtained as described above has been deposited at the National Institute of Microbial Technology, Agency of Industrial Science and Technology (
Microtechnology Research Institute No. 11140, Entrusted date: December 5, 1989
Day).

Example 3 Production and purification of RNase H (S S 13) in Escherichia coli 1, transformant JM109/pSS, culture of 13 Transformant JM109/pss13 was cultured with shaking in LB medium le containing 80μ9 ampicillin at 37°C. did. When the turbidity of the culture solution reached a Klett value of around 100, IPTG was added to give a final concentration of 1111 M, and after continued shaking for an additional 4 hours, the bacteria were collected. At this time, the Clett value was approximately 200, and the wet weight of the bacterial cells was approximately 1.59.

2. Extraction and purification of glue bonuclease H (S813) from bacterial cells After suspending the obtained bacterial cells in 30 volumes of 105 M Tris-HCl buffer (TEX pH 7,5) containing 0.1 mM EDTA, the cells were sonicated in water. The bacterial cells were disrupted. l 5.
The centrifugation supernatant (crude extract) obtained by centrifugation at OOOrpm for 30 minutes at 4°C was dialyzed against TE (pH 7,5) 512 at 4°C. It was passed in this order through DE-52 column (41112) and P-11 column (21) equilibrated with the same buffer.
(SS13) passes through the DE-52 column and P-1
Adsorb to one column. TE (pH 7,5) 4x (7, then TE (pH 7,5) with O, LM NaCl2
After flowing 41112, mutant ribonuclease H (SS13) was eluted from the P-11 column by linearly increasing the NaCQ concentration to 0.5M. After concentrating the P-11 elution fraction containing mutant ribonuclease H (SS13) to approximately 2ytQ, it was further enriched with 0.1M NaC.
10mM sodium acetate buffer (pH 5,5
Mutant ribonuclease H (SS13) was purified by applying it to a Sephacryl S-300 (Superfine) column (φ1.8 x 90 cm) equilibrated with ). The purified sample gave a single band on 15% 5DS-PAGE and also showed a single peak on reversed phase HPLC. The purification yield was 819/l culture fluid. This corresponds to approximately 80% of the amount of protein present in the bacterial cell crude extract fraction. The purified sample was identified by digestion with Achromobacter protease ■. The peptide fragments obtained were mapped using reverse phase PLC to confirm the elution position of each fragment peak, and the peptide containing the amino acid at position 13.44 was fractionated and subjected to amino acid sequence analysis.

The obtained purified sample O, IM containing l○m NaCl2
20 in M sodium acetate buffer (pH 5,5)
The CD spectrum from 0 to 260 nm was identical to that of the native type, and no detectable changes in secondary structure were observed in the CD spectrum.

Example 4 Introduction of SS bond The purified sample obtained in Example 3 did not have a disulfide bond in its original state, so it was added to a glutathione redox buffer (ERINK, Peters, Kim, .
Science, 243, 538-541 (1989))
A disulfide bond was introduced. The redox buffer was 1mM oxidized glutathione, 1mM reduced glutathione, 0. IMT ris (pH 9, 0), ln+
Contains M EDTA, 0.2M KCQ, where RNa
seH (S S 13) was added at 34 μM and incubated at 30 °C for 4
Let stand for ~5 hours. After that, desalt with PD-10, change the buffer to O, 10mMAB (pH 5) containing 1M NaCQ.
, 5). When peptide mapping was performed on the enzyme subjected to such treatment in the same manner as described above, the peaks containing Cys13 and Cys4 disappeared;
A new peak was detected instead. This peak was fractionated and amino acid sequence analysis was performed.
Each fragment containing Cy s
The respective peaks containing 3 and Cys44 reappeared. In this way RNa5eH (S S 13)
It was confirmed that the SS bond was introduced. Hereinafter, experiments were conducted by naming the purified enzyme without SS cross-linking as reduced 5813, and the enzyme into which SS cross-linking was introduced by redox buffer treatment as oxidized 5S13. The enzyme activity of the reduced form and oxidized form 5813 shown in Example 4 was evaluated by performing the same as in the following experimental example.

1[f+] Comparison of the enzyme activities of reduced and oxidized 5S13 was performed using ['H]-M13DNA-RNA as a substrate, and the enzyme activity to release 1 nmol of CMP in 15 minutes at 37°C was expressed as 1 unit (U). ) was defined.

The amount of protein was determined by measuring the absorbance of mutant ribonuclease H and natural ribonuclease H, which have the same extinction coefficient. Furthermore, as shown in Table 1, the concentration of DTT in the solution for measuring enzyme activity was varied and measurements were carried out with each solution.

In this way, reduced 5S13 consistently has a 30-5
It has 0% activity. On the other hand, oxidized 5S1
In the absence of DTT, 3 is almost inactivated at 0.3% of the native type, and as the concentration of DTT is increased, 1
The activity returned to 0.7% at 0mM DTT and 30% at 100++M. This reaches 55% of the reduced type. DTT
When added at the dilution stage, approximately 76% of the activity of the reduced form is recovered. It was thus revealed that 5313 is inactivated in the oxidized form and activated in the reduced form. In other words, the expression and termination of enzyme activity of this enzyme can be controlled simply by adjusting the oxidation/reduction state.

Table 1 Activity was measured using ['H]-M13DNA-RNA as a substrate. Specific activity is the enzyme amount (unit) divided by the protein amount (19), which is 0.91%.
Calculations were made assuming that the ultraviolet absorption of the aqueous solution at 280 nm was 2.0.

[Brief explanation of drawings]

FIG. 1 is a schematic diagram showing the outline of subcloning of the rnh gene into Ml 3mpRF DNA. FIG. 2 is a schematic diagram showing the sequence of oligonucleotides used for site mutagenesis. FIG. 3 is a schematic diagram of the assembly of plasmid pSS13. 1↑

Claims (1)

[Scope of Claims] 1. A reduced mutant E. coli ribonuclease H having an amino acid sequence in which asparagine at position 44 is replaced with cysteine, and cysteine at positions 63 and 133 are replaced with alanine. . 2. A mutant E. coli ribonuclease H structural gene encoding the reduced mutant E. coli ribonuclease H according to claim 1. 3. In the structural gene encoding E. coli ribonuclease H, the AAC codon encoding asparagine at position 44 of the corresponding amino acid sequence is a TGC codon encoding cysteine, and the TGC codon encoding cysteine at position 63 is GCC encoding alanine
TG that encodes the 133rd cysteine in the codon
3. The mutant E. coli ribonuclease H structural gene according to claim 2, wherein the T codon is converted to a GCT codon encoding alanine by site-directed mutagenesis. 4. An expression vector capable of replicating and expressing in E. coli, which contains the mutant E. coli ribonuclease H structural gene according to claim 2 or 3 under the control of the tac promoter. 5. The expression vector according to claim 4, which contains an ampicillin resistance gene as a selection marker. 6. The expression vector according to claim 5, which is plasmid pSS13. 7. A transformant transformed with the expression vector according to any one of claims 4 to 6. 8. The transformant according to claim 7, which is E. coli JM109/pSS13. 9. A reduced mutant E. coli comprising culturing the transformant according to claim 7 or 8 under conditions suitable for expression of the ribonuclease H gene, and recovering reduced mutant E. coli ribonuclease H from the resulting culture solution. Method for producing ribonuclease H. 10. In the amino acid sequence of E. coli ribonuclease H, the 44th asparagine is cysteine, and the 63rd asparagine is cysteine.
An oxidized mutant E. coli ribonuclease H having an amino acid sequence in which cysteine No. 1 and No. 133 cysteine are substituted with alanine. 11. The method for producing oxidized mutant E. coli ribonuclease H according to claim 10, which comprises treating the reduced mutant E. coli ribonuclease H according to claim 1 with a glutathione redox buffer.