JPH0538286A - Variant type escherichia coli ribonuclease h - Google Patents

Abstract

PURPOSE:To obtain a new readily handleable variant type Escherichia coli ribonuclease H having a high denaturation temperature. CONSTITUTION:A variant type Escherichia coli ribonuclease H having an amino acid sequence in which lysine at the 91-position, aspartic acid at the 94-position and lysine at the 95-position are respectively replaced with arginine, glutamic acid and glycine. The aforementioned ribonuclease H is obtained by generic manipulation for respective replacing Lys at the 91-position, Asp at the 94-position and Lys at the 95-position with Arg, Glu and Gly and the general technique is according to a manual [Molecular Cloning (1982)]; A laboratory Manual and Specification of Reagents by Maniatis et al.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mutant Escherichia coli ribonuclease H having improved stability, a gene encoding the mutant E. coli ribonuclease H, and an expression vector containing the gene and expressible in E. coli. , A transformant containing the expression vector, and a method for producing a mutant Escherichia coli ribonuclease H using the transformant.

[0002]

PRIOR ART AND PROBLEM TO BE SOLVED BY THE INVENTION Natural type ribonuclease H of Escherichia coli (in the present specification, simply referred to as ribonuclease H or RNase H means natural type E. coli ribonuclease H) The molecular weight of about 155 amino acids is about 17
It is a hydrolase of Kd and has a substrate specificity of specifically cleaving only an RNA chain of a DNA / RNA hybrid by an endo action. 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 cloning of cDNA. 2) Removal of poly A region of mRNA. 3) Fragmentation of RNA.

Although the importance of ribonuclease H is expected to increase with the development of genetic engineering, the production of this enzyme in Escherichia coli is extremely low. It has been attempted and has already been recombined from BRL, Pharmacia and Takara Shuzo etc.
Ribonuclease H produced by NA technology is supplied. These commercially available recombinant ribonuclease H
Is produced by using E. coli as a host (Kanaya et al., Journal of Biological Chemistry, 264 , 11546-11549 (1989)).

If the stability of highly useful ribonuclease H, for example, the resistance to denaturing agents and heat can be increased, it can be used under conditions that conventional recombinant ribonuclease H could not.

To date, the present inventors have used recombinant DNA technology to treat natural ribonuclease H against denaturants.
Although a mutant ribonuclease H having a higher resistance than that of the natural type enzyme was produced, its degree of stabilization was not sufficiently satisfactory, and the enzyme activity thereof did not exceed that of the natural enzyme (Japanese Patent Application No. 01-284454). ). In addition, a method for producing thermophilic bacterium ribonuclease H having extremely higher stability than Escherichia coli ribonuclease H by Escherichia coli has been reported (Kanaya et al., 2nd Annual Meeting of the Protein Engineering Society of Japan Program / Summary (1990), Page 69; Japanese Patent Application No. 02-1110
65), the enzyme activity of thermophile ribonuclease H produced using E. coli was lower than that of E. coli ribonuclease H. In the latter case, the purification method requires solubilization with urea, and the production method thereof is more complicated than that of Escherichia coli ribonuclease H. Therefore, if it is possible to produce a mutant ribonuclease H that is more excellent in enzyme activity and stability and that is easier to manufacture and purify than the thermophilic bacterium ribonuclease H, it is considered to have higher utility value in industry. ..

[0006]

From the above viewpoints, the present inventors refer to the amino acid sequence of thermophile ribonuclease H for the purpose of enhancing the stability of Escherichia coli ribonuclease H while retaining the enzymatic activity thereof. As a result of trying various modifications, by introducing a site-specific mutation into the gene of the enzyme, the 91st Lys (lysine), the 94th Asp (aspartic acid), the 95th Lys (lysine) is Arg (arginine), G respectively
It was found that the mutants substituted with lu (glutamic acid) and Gly (glycine) are more stable to heat than the natural ribonuclease H, and their enzymatic activity is almost the same as that of the natural ribonuclease H. It was confirmed that the present invention was completed.

That is, the present invention relates to the 91st Lys, 94th Asp and 9th of natural E. coli ribonuclease H.
Mutant Escherichia coli ribonuclease H consisting of an amino acid sequence in which the fifth Lys is replaced with Arg, Glu, and Gly, respectively
Is provided.

The present invention also provides a codon encoding Lys at position 91 of natural E. coli ribonuclease H,
The 94th Asp in the codon that encodes Arg for AAA
A codon that encodes Glu, a codon that encodes Glu, and a codon that encodes the 95th Lys, A
The present invention provides a mutant ribonuclease H structural gene in which AA is converted to a codon encoding Gly.
The present invention also provides an expression vector for expressing the mutant ribonuclease H 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 high stability by culturing the transformant. Gene manipulation methods using E. coli are available (Maniatis
Et al., Molecular Cloning (1982): A Laboratoy M.
anual, Cold Spring Harbor Laboratoy). The above-mentioned mutant ribonuclease H structural gene, expression vector, and transformant can be prepared according to the general method described in this document, for example, as described in Examples below. It can be manufactured according to the law. The expression vector of the present invention is not particularly limited as long as it functions in E. coli, but contains the above mutant ribonuclease H gene under the control of the P _L and P _R promoters of bacteriophage λ. Is preferred.

[0010]

INDUSTRIAL APPLICABILITY The mutant Escherichia coli ribonuclease H having improved stability according to the present invention does not denature even at a high temperature which would denature with conventional ribonuclease H (see Example 3). It can be handled under such conditions. Furthermore, the mutant Escherichia coli ribonuclease H of the present invention is expected to have improved durability and the like, is less likely to be inactivated than the conventional ribonuclease H, and can be stably stored, thus facilitating handling. Will be understood. Further, the mutant Escherichia coli ribonuclease H of the present invention has an enzymatic activity equivalent to that of natural ribonuclease H, and by using this, the same amount of enzymatic activity as that of natural ribonuclease H can be obtained.

The present invention will be described in more detail below with reference to examples. The general method of genetic manipulation described below is
Manual (Maniatis et al., Molecular Cloning (19
82): A Laboratoy Manual, Cold Sping Harbor
Laboratoy) and reagent specifications were followed.

Example 1 Construction of plasmid pJL7 An outline of construction of plasmid pJL7 is shown in FIG. First,
Expression plasmid of natural ribonuclease H in E. coli
A point mutation was introduced into the rnh gene by the PCR method using the synthetic oligonucleotide primer with the rnh gene of pAK600 (see JP-A-03-065188) as a template.

A chemically synthesized oligonucleotide was used as the synthetic oligonucleotide. The oligonucleotides used are shown in FIG. As shown in FIG. 1, first, pA
5'-primer (1) containing a restriction enzyme site of SphI and 3'-primer for mutagenesis using K600 as a template DNA
A DNA fragment of about 300 bp was prepared by (4), and a DNA fragment of about 240 bp was prepared by the 5'-primer (3) for mutagenesis and the 3'-primer (2) containing SalI site.
Amplified by R reaction, Lys ⁹¹ was changed to Arg ⁹¹ and Asp ⁹⁴ was changed to Glu.
^{At 94} , mutant rnh gene fragment 2 in which Lys ⁹⁵ was converted to Gly ⁹⁵
Got seeds. The PCR reaction is a commercially available Gene Amp Kit (Takar
Follow the instructions in a). After the above rnh gene fragment was purified by agarose gel electrophoresis, both were mixed,
By adding 5'-primer (1) and 3'-primer (2) and performing PCR again, a mutant rnh gene fragment having SphI and SalI restriction enzyme sites at both ends was obtained. The SphI and SalI sites at both ends of the obtained mutant rnh fragment of about 500 bp were cleaved with restriction enzymes SphI and SalI, and the digest was subjected to 1.5% agarose gel electrophoresis.
A mutant rnhSphI-SalI gene fragment of about 500 bp was cut out, extracted and purified.

Next, the mutant gene fragment thus obtained was subcloned into an expression vector in Escherichia coli to prepare an expression vector. Plasmid pJLA50
4 (Purchased from Medac, Germany) was digested with SphI and SalI, and a fragment of about 4.9 kb was purified by 0.7% agarose electrophoresis.
It was circularized by ligation with the hI-SalI fragment. Ligation is a commercially available ligation kit
(Takara) was used according to the attached instructions. With the thus obtained plasmid, Escherichia coli HB1
The strain 01 was transformed, and a plasmid vector pJL7 for expressing mutant ribonuclease H was obtained from the transformant.

The transformant Escherichia coli HB101 / pJL7 obtained as described above has been deposited at the Institute of Microbial Technology, Institute of Industrial Science and Technology (Ministry of Industrial Research, No. 12321, Deposited) Date: June 21, 1991).

Example 2 Mutant ribonuclease H (L
Production and purification of 7) in E. coli 100 μg / l of E. coli transformant HB101 / pJL7
In 500 ml of LB medium containing ampicillin, cultured at 30 ° C. with shaking. When the turbidity of the culture broth reached a Klett value of about 80, the culture temperature was raised to 42 ° C., shaking was continued for another 3.5 hours, and then the cells were centrifuged to collect the cells. The clet value at this time was about 270.

The resulting cells were treated with 10 mM containing 1 mM EDTA.
After suspending in 15 ml of mM Tris-HCl buffer (TE) (pH 7.5), the cells were crushed by sonication in ice. Fifteen,
The centrifugation supernatant (crude extract) obtained by centrifugation at 000 rpm for 30 minutes at 4 ° C. was dialyzed against 5 liters of TE (pH 7.5) at 4 ° C. overnight. The crude extract after dialysis was equilibrated with the same buffer solution DE-
52 columns (5 ml) and P-11 columns (2 ml) were passed in this order. Under these conditions, mutant ribonuclease H
(L7) passes through the DE-52 column and is adsorbed on the P-11 column. After flowing 4 ml of TE (pH 7.5), NaCl
P- by increasing the concentration linearly to 0.5M
Mutant ribonuclease H (L7) was eluted from the 11th column. P-1 containing mutant ribonuclease H (L7)
The 1 elution fractions were combined and used as a purified standard. 15 for purified sample
% SDS-PAGE gave a single band, reverse phase HPLC
However, it showed a single peak. The purification yield was 52.4 mg / l culture. The purified sample was identified by mapping the peptide fragment obtained by digestion with Achromobacter protease I by reverse-phase HPLC to confirm the elution position of each fragment peak, and the peptide containing the amino acids at positions 91, 94, and 95. Was collected and then analyzed by amino acid sequence analysis.

2 of the purified preparation obtained in 10 mM sodium acetate buffer (pH 5.5) containing 0.1 M NaCl.
The CD spectrum at 00 to 250 nm was the same as that of the native type, and no change in the secondary structure which could be detected by the CD spectrum was observed. When the enzyme activity was measured by the following method and the specific activity was measured, the mutant enzyme obtained had a specific activity of 18 U / mg, which was about 9% that of the natural enzyme.
It retained 0% enzyme activity. The comparison results obtained are shown in Table 1 below.

The activity was defined as 1 unit (U) of the enzyme activity using [ ³ H] -M13 DNA / RNA as a substrate to release 1 μmol of an acid-soluble substance at 37 ° C. for 1 minute.
The protein amount was determined by measuring the absorbance at 280 nm using ε ₂₈₀ = 1576 M ⁻¹ · cm ⁻¹ under the assumption that the mutant ribonuclease H and the natural ribonuclease H have the same absorption coefficient.

[Table 1] Comparison of enzyme activities of native and mutant ribonuclease HEnzyme name Specific activity (U / mg)  Natural Ribonuclease H 20 Mutant Ribonuclease H (L7) 18

Example 3 Mutant Ribonuclease H (L
7) Stability evaluation of natural ribonuclease H and mutant ribonuclease H
The thermal stability of (L7) was measured and compared. The measurement is
The rate of denaturation at pH 5.5 was determined by measuring the heat denaturation curve with the CD value at 220 nm. The heat denaturation experiment was performed using a cell with an optical path length of 2 mm and 1 M guanidine hydrochloride and 1 m.
Performed in 20 mM sodium acetate (pH 5.5) with M dithiothreitol (DTT). The denaturation curve obtained by the measurement is shown in FIG. Mutant ribonuclease H (L
The heat denaturation curve of 7) was shifted to a higher temperature side than the natural type heat denaturation curve, and the thermal stability was obviously increased. Comparing the temperatures when 50% of the whole was denatured, the mutant ribonuclease H (L7) was more stable at 5.6 ° C than the natural type. The results obtained are shown in Table 2 below.

Table 2 Comparison of thermostability of native and mutant ribonuclease HFermentation temperature at 50% denaturation (℃)  Natural Ribonuclease H 51.8 Mutant Ribonuclease (L7) 57.4

From the above results, the 91st Lys, 94th Asp and 9th of the native Escherichia coli ribonuclease H were confirmed.
It was confirmed that the stability against heat was improved by converting the fifth Lys to Arg, Glu, and Gly, respectively.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the construction of plasmid pJL7.

FIG. 2 is a schematic diagram showing a synthetic oligonucleotide for introducing a site-specific mutation, in which the underline indicates the restriction enzyme site, and the ● mark indicates the base (A) corresponding to the mutant amino acid.
rg ⁹¹ , Glu ⁹⁴ , and Gly ⁹⁵ ).

FIG. 3 is a graph showing heat denaturation curves of natural ribonuclease H and mutant ribonuclease H (L7).

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Claims (7)

[Claims] 1. A mutant form of natural E. coli ribonuclease H having an amino acid sequence in which the 91st lysine, the 94th aspartic acid, and the 95th lysine are substituted with arginine, glutamic acid, and glycine, respectively. E. coli ribonuclease H. 2. A mutant E. coli ribonuclease H structural gene encoding the mutant E. coli ribonuclease H according to claim 1. 3. A recombinant plasmid containing the mutant Escherichia coli ribonuclease H structural gene according to claim 2 under the control of an Escherichia coli gene expression system. 4. The recombinant plasmid according to claim 3, which is pJL7. 5. An Escherichia coli strain transformed with the recombinant plasmid according to claim 3. 6. The Escherichia coli strain according to claim 5, which is HB101 / pJL7. 7. A method for producing a mutant Escherichia coli ribonuclease H, which comprises culturing the Escherichia coli strain according to claim 5 or 6, and recovering the mutant Escherichia coli ribonuclease H from the obtained culture solution.