JPH08205862A - Escherichia coli ribonuclease hi stabilized by filling intramolecular void - Google Patents

Abstract

PURPOSE: To easily obtain the subject RNase HI with its purification facilitated, improved in resistance to heat or modifiers, by substituting valine, leucine or isoleucine for the 52nd alanine on natural-type Escherichia coli ribonuclease HI to effect filling the intramolecular voids of this enzyme. CONSTITUTION: This variant Escherichia coli ribonuclease HI is obtained by substituting Val, Leu or Lle for the 52nd Ala on natural-type Escherichia coli ribonuclease HI to effect virtually filling the intramolecular voids existing in the hydrophilic core enclosed by the αI-helix, αIII-helix, αIV-helix, βD-chain and βE-chain of the RNase HI with the side chains of the Val, Leu or Ile. This variant RNase HI is obtained by culturing bacteria transformed with a plasmid containing under the control of Escherichia coli gene manifestation system a variant Escherichia coli RNase HI structural gene coding this variant Escherichia coli RNase HI [e.g. coli k12/HB101/pJAL521 (FERM P-14674)].

Description

Detailed Description of the Invention

[0001]

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

The ribonuclease HI targeted by the present invention is a substance conventionally recognized as ribonuclease H. Recently, the existence of a substance that is different from the conventional ribonuclease H but has a similar enzymatic activity has been found [Itaya, Proceeding of National Academy of Sciences, USA, 8
7 , 8587-8591 (1990)]. Therefore, in order to distinguish between the conventional ribonuclease H and its newly found substance, the conventional ribonuclease H was named ribonuclease HI and the newly found substance was named ribonuclease HII. As such, the term ribonuclease HI as used herein is the term used to refer to conventional ribonuclease H.

A natural ribonuclease HI of Escherichia coli (in the present specification, simply referred to as ribonuclease HI or RNase HI means natural E. coli ribonuclease HI) has a molecular weight of about 155 amino acids of about 18 Kd. A hydrolase of D,
It has a substrate specificity of specifically cleaving only the RNA chain of the NA / RNA hybrid by the endo action. This enzyme has various uses as described below based on its substrate specificity, and is attracting attention as an enzyme having a very high utility value. 1) Removal of template mRNA during cloning of cDNA. 2) Removal of poly A region of mRNA. 3) RNA editing. The importance of ribonuclease HI having such utility value is expected to increase with the development of genetic engineering.

[0004]

2. Description of the Related Art If the stability of ribonuclease HI having a high utility value as described above, for example, the resistance to denaturing agents and heat can be increased, it can be used under conditions that conventional recombinant ribonuclease HI could not use. It will be possible. To date, several mutant ribonucleases HI having certain mutations, which have higher stability than the natural ribonuclease HI, have been developed (Japanese Patent Laid-open No. HEI 0)
3-147785, JP-A-05-038286, JP-A-05-003787, JP-A-05-064585, and JP-A-05-076359). Most recently, the combination of these mutations was shown to further enhance the stability of ribonuclease HI [Kimura et al., Journal of Biological Chemistry, 267 , 2;
1535-21542 (1992); JP-A-05-076.
358, Japanese Patent Application No. 5-314750], the development of another mutation that enhances the stability of ribonuclease HI is important in the sense of broadening the range of means for combining.
And this also increases the possibility of obtaining a ribonuclease HI with higher stability.

A ribonuclease H derived from a thermophile having a much higher stability than that of Escherichia coli ribonuclease HI is known, and a method for recombinantly producing this thermophile ribonuclease H in E. coli has been reported ( Kanaya et al., Journal of Biological Chemistry, 267 , 10184-10192 (1992); JP-A-04-008294). However, the enzyme activity of thermophile ribonuclease H produced using E. coli at 37 ° C was considerably lower than that of E. coli ribonuclease HI (about 5%). Further, in the case of thermophilic ribonuclease H produced from Escherichia coli, the operation of solubilizing it with urea is necessary for its purification, and its production method is more complicated than that of Escherichia coli ribonuclease HI. There was also. Therefore, more enzyme activity,
It is considered that if a mutant ribonuclease HI, which is an enzyme having excellent stability and is easier to produce and purify than thermophilic bacterium ribonuclease H, can be constructed, it will have higher industrial utility value.

[0006]

The E. coli ribonuclease HI enzyme is an αI-helix at the molecular level.
Although it has a large intramolecular cavity (cavity) in the hydrophobic core portion surrounded by αIII-helix, αIV-helix, βD-chain and βE-chain (Katayanagi et al., Nature)
Vol. 347, p. 306-309 (1990)), the present inventors have identified the 74th V facing this intramolecular cavity.
It has been found that the stability of this enzyme is increased by substituting Le (leucine) for al (valine) (Japanese Patent Application No. 04-329470). Then, the three-dimensional structure of this mutant with increased stability was analyzed using X-rays, and the reason why the stability of this enzyme was increased by substituting Leu at the 74th Val was It was clarified that the intramolecular voids in the hydrophobic core were partially filled (Ishikawa et al., Biochemistry, 3
2 , 6171-6178 (1993)). However, the filling of the intramolecular void is not easy because there are many combinations of the amino acid site in the hydrophobic core to be selected and the number of substituted amino acids to be selected, and that the enzymatic activity of this enzyme should be retained. Must also be considered.

[0007]

Therefore, from the above viewpoints, the present inventors tried various modifications for the purpose of enhancing the stability of Escherichia coli ribonuclease HI enzyme while retaining its enzymatic activity. As a result, the 52nd Ala (alanine, A), which is another amino acid that constitutes the intramolecular void, is a bulky amino acid such as Val (valine,
V), Leu (leucine, L), or Ile (isoleucine, I) was found to be more stable to heat than the natural ribonuclease HI, and its enzymatic activity was found to be higher. It was confirmed that it was not damaged. Furthermore, by replacing Val at position 74 of this mutant with Leu, ribonuclease HI more stable than the single mutant at position 52 or 74 could be obtained. That is, the present invention relates to the 52nd E. coli ribonuclease HI enzyme.
A method for stabilizing the enzyme by filling the intramolecular void by substitution of the other amino acid at position Ala with another amino acid, and simultaneous substitution with the other amino acid at position Ala at position 52 and another amino acid at Val at position 74, The present invention provides a stabilized mutant ribonuclease HI.

Further, the present invention provides a structural gene encoding the above mutant ribonuclease HI, an expression vector containing the structural gene, a transformant introduced with the expression vector, and culturing the transformant. The present invention provides a method for producing a mutant ribonuclease HI having high stability. Gene manipulation methods using Escherichia coli have been published (Maniatis et al., Molecular Cloning (1982):
A Laboratory Manual, Cold Spring Harbor Lab
The mutated ribonuclease HI structural gene, expression vector, and transformant described above are prepared in accordance with the general method described in this document, for example, by a conventional method as shown in Examples below. be able to. The expression vector of the present invention is not particularly limited as long as it functions in Escherichia coli, but it is not limited to P _L and P _R of bacteriophage λ.
The above mutant ribonuclease H under the control of a promoter
Those containing the I gene are preferred.

[0009]

INDUSTRIAL APPLICABILITY The mutant Escherichia coli ribonuclease HI having improved stability according to the present invention does not denature even at a high temperature which would denature with conventional ribonuclease HI (see Test Example 1). It can be handled under such conditions. Furthermore, since the mutant Escherichia coli ribonuclease HI of the present invention is expected to have improved durability and the like, it is less likely to be inactivated than the conventional ribonuclease HI, can be stably stored, and is therefore easy to handle. Be understood.

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 Laboratory Manual, Cold Sping Harbo
r Laboratory) and reagent specifications.

Example 1 Preparation of mutant ribonuclease HI (A52I) Construction of plasmid pJAL52I The outline of construction of plasmid pJAL52I is shown in FIG.
First, a point mutation was introduced into the rnh gene by PCR using the rnh gene of the expression plasmid pJAL600 of E. coli expressing the natural ribonuclease HI as a template and the PCR method. The starting plasmid pJAL600 was prepared by using plasmid pJAL135C (Japanese Patent Laid-Open No. 05-0.
This was carried out by replacing the mutant rnh gene part in (03786) with the natural rnh gene part (derived from the plasmid pDR600 described in JP-A 01-202286).

As the primer, the chemically synthesized oligonucleotide shown in FIG. 2 was used. As shown in FIGS. 1 and 2, first, pJAL600 was used as a template DNA and Bg
5'-primer (1) containing the restriction enzyme site of lII and SstI
Approximately 150 bp D by 3'-primer (2) containing site
The NA fragment was treated with the 5'-primer (3) for mutagenesis and the 3'-primer (4) containing a SalI site to give about 350 b.
Each DNA fragment of p was amplified by PCR reaction to obtain two gene fragments. The two gene fragments are partially annealed between the former 3'end and the latter 5'end. Then, using the partially annealed fragment as the template DNA, 5'-primer (1) and 3'-primer
According to (4), a DNA fragment of about 500 bp was amplified by PCR reaction. The PCR reaction is a commercially available Gene Amp Kit (Takar
Follow the instructions in a). The obtained gene fragment of about 500 bp was digested with restriction enzymes BglII and SalI, and the obtained digestion product was subjected to 1.5% agarose gel electrophoresis to obtain a DNA fragment of about 500 bp.

Next, the mutant gene fragment thus obtained was subcloned into an expression vector in Escherichia coli to prepare an expression vector. Plasmid pJAL60
0 was digested with BglII and SalI, a fragment of about 4.9 kb was purified by 0.7% agarose electrophoresis, and then ligated with the above-mentioned mutant rnh gene fragment of about 500 bp for cyclization. Ligation was carried out using a commercially available ligation kit (Takara) according to the attached instructions. Escherichia coli HB101 strain was transformed with the thus obtained plasmid, and a plasmid vector pJAL for expressing mutant ribonuclease HI was obtained from the transformant.
I got 52I. It was confirmed by determining the entire DNA sequence of the mutated gene that the mutated gene had no mutation introduced at sites other than the intended site. The transformant E. coli K12 / H obtained as described above
B101 / pJAL52I has been deposited at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (FERM P-14).
674, contract date: November 29, 1994).

B. Mutant ribonuclease HI (A52
Production and purification of I) in E. coli 100 ml of E. coli transformant HB101 / pJAL52I
30 ml in 200 ml LB medium containing g / l ampicillin
The cells were cultured with shaking at ℃. The turbidity of the culture solution is about 10 in terms of Klett value.
At the time of growing to 0, the culture temperature was raised to 42 ° C., shaking was continued for another 3.5 hours, and then the cells were collected by centrifugation. The clet value at this time was about 250.

The obtained 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 disrupted 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 L of TE (pH 7.5) at 4 ° C. overnight. The crude extract after dialysis was P- equilibrated with the same buffer.
Pass through 11 columns (2 ml). Under this condition, the mutant ribonuclease HI (A52I) is adsorbed on the P-11 column. After flowing 4 ml of TE (pH 7.5), the NaCl concentration was adjusted to 0.
Mutant ribonuclease HI (A52I) was eluted from the P-11 column by linearly increasing to 5M. P-containing mutant ribonuclease HI (A52I)
The 11 elution fractions were combined and used as a purified standard. 1 for purified standard
5% SDS-PAGE gave a single band, reverse phase HPL
C also showed a single peak. The purification yield was 52 mg / l culture. To identify the purified sample, the peptide fragment obtained by digestion with lysyl endopeptidase was mapped by reverse phase HPLC to confirm the elution position of each fragment peak, and the peptide containing the amino acid at the 52nd position was isolated and the amino acid sequence was determined. It was done by analysis.

2 of the obtained purified standard 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 natural type, and no change in the secondary structure which could be detected by the CD spectrum was observed.

Example 2 Mutant Ribonuclease HI (A52L and A52
Preparation of V) Substantially following the procedure described in Example 1, mutant ribonucleases HI, A52L and A in which Ala at position 52 of natural ribonuclease HI was replaced with Leu or Val.
52V was prepared respectively. The starting plasmids and primers used are as follows. It should be noted that for the primer, only the bases corresponding to the mutant amino acids in FIG. 2 are shown.

[Table 1] Starting plasmid Primer A52L pJAL600 CTG A52V pJAL600 GTT

Example 3 Mutant Ribonuclease HI (A52I / V74L, A
52L / V74L, A52V / V74L) . Plasmid pJAL52I74L, pJAL52L7
Construction of 4L, pJAL52V74L Plasmid pJAL52I74L, pJAL52L74
An outline of the preparation of L and pJAL52V74L is shown in FIG.

First, pJAL52I, pJAL52L, p
Using the rnh gene of JAL52V as a template and the primers (1) and (5) shown in FIG. 2, the Afl II restriction enzyme sites were introduced at positions 66 to 68 of the rnh gene by the PCR method. The PCR reaction was performed according to the instructions of a commercially available GeneAmp Kit (Takara). After digesting the amplified gene fragment of about 190 bp with restriction enzymes Bgl II and Afl II,
The resulting digest was subjected to 1.5% agarose gel electrophoresis to obtain a DNA fragment of about 190 bp.

Next, a plasmid pJAL74L (Japanese Patent Laid-Open No. 06-169767) having a mutant rnh gene in which Val at position 74 was replaced by Leu was digested with restriction enzymes BglII and AflII.
The fragment of about 5.2 kb was purified by 0.7% agarose gel electrophoresis, and ligated with the above-mentioned mutant rnh gene fragment of about 190 bp for cyclization. Ligation was performed using a commercially available ligation kit (Takara) according to the attached instructions. Escherichia coli HB101 strain was transformed with the thus obtained plasmid, and the transformant was used to express the mutant ribonuclease HI expression plasmid vectors pJAL52I74L and pJAL5.
2L74L and pJAL52V74L were obtained. In the mutated gene, the fact that the mutation has not been introduced at sites other than the target site is
It was confirmed by determining the total DNA sequence of the mutant gene.

B. Mutant ribonuclease HI (A52
I / V74L, A52L / V74L, A52V / V74
Production and purification of L) in E. coli Substantially following the procedure described in Example 1, Ala at position 52 of the native ribonuclease HI was Ile, Leu or Val.
To the mutated ribonuclease HI, in which Val at position 74 was replaced with Leu, A52I / V74L, A52L / V74
L and A52V / V74L were prepared respectively.

Test Example 1 Increased Stability of Mutant Ribonuclease HI The thermal stability of the natural ribonuclease HI and the mutant ribonuclease HI prepared in Examples 1 to 3 was measured and compared. The measurement was carried out using the rate of denaturation at pH 3.0 of 22.
This was done by measuring the heat denaturation curve with the CD value at 0 nm. The results obtained are shown in Table 2 below.

[Table 2] Fermentase name Tm (° C) ΔTm (° C) WT RNaseHI 56.4-A52I 62.6 6.2 A52L 60.7 4.3 A52V 61.9 5.5 V74L 60.1 3.7 A52I / V74L 60.3 3.9 A52L / V74L 61.3 4.9 A52V / V74L 64.4 8.0 Mutant ribonuclease HI The curve was shifted to the higher temperature side than the natural type heat denaturation curve, and the thermal stability was obviously increased. From the above results, it was confirmed that the stability against heat was improved by introducing an amino acid mutation that fills the intramolecular cavity of the natural Escherichia coli ribonuclease HI. Also, in the single mutants at positions 52 and 74,
The most stabilized A52I and A5 that combines V74L
2I / V74L was equal to or lower than the stabilization by each mutation, and their effects were canceled out. However, A52V
In the case of / V74L, it showed an additive effect, and was the most stabilized among the double substitution products. Further, when comparing A52I and A52V / V74L in which the volume change of the intramolecular void is the same, it is found that the latter is more stable. From this, it can be said that as a method of filling the stabilizing intramolecular void, it is more effective to combine amino acid substitutions with smaller changes in capacity than from only one side.

Reference Example Measurement of Enzyme Activity Mutant Ribonuclease H Prepared in Examples 1-2
The enzyme activity of I was measured by the following method, the specific activity was measured, and the percentage of the native enzyme was calculated. The comparison results obtained are shown in Table 3 below. The activity is [ ³ H]-
Using M13 DNA / RNA as a substrate, the enzyme activity of releasing 1 μmol of an acid-soluble substance in 1 minute at 30 ° C. was defined as 1 unit (U). The amount of protein is based on the assumption that the mutant ribonuclease HI and the natural ribonuclease HI have the same extinction coefficient.

[Equation 1] Was measured by measuring the absorbance at 280 nm. here,

[Equation 2] Means the absorbance at 280 nm of an aqueous solution having a protein concentration of 1 mg / ml.

[Table 3] Comparison of enzymatic activities of native and mutant ribonucleases HI Enzyme specific activity (U / mg) vs. natural type% WT RNase HI 9.45-A52I 3.61 38% A52L 0.28 3% A52V 5.67 60% V74L 9.45 100% A52I / V74L 0.36 3.8% A52L / V74L 0.23 2.4% A52V / V74L 1.51 16%

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the construction of plasmid pJAL52I.

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.
bases corresponding to la ⁵² ).

FIG. 3 is a schematic diagram showing the construction of plasmid pJAL52I74L.

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Claims (9)

[Claims] 1. A native E. coli ribonuclease HI-5
The second alanine is replaced by valine, leucine, or isoleucine, whereby the αI-helix, αIII-helix, αIV-of the ribonuclease HI is replaced.
A variant in which an intramolecular cavity existing in a hydrophobic core surrounded by a helix, a βD-chain and a βE-chain is substantially filled with a side chain of the substituted amino acid residue, thereby enhancing stability. E. coli ribonuclease HI. 2. A natural E. coli ribonuclease HI
The mutant Escherichia coli ribonuclease HI according to claim 1, wherein the 74th valine is substituted with leucine. 3. The mutant Escherichia coli ribonuclease HI according to claim 2, wherein the 52nd alanine is substituted with valine. 4. A mutant Escherichia coli ribonuclease HI structural gene encoding the mutant E. coli ribonuclease HI according to any one of claims 1 to 3. 5. A recombinant plasmid containing the mutant Escherichia coli ribonuclease HI structural gene according to claim 4 under the control of a gene expression system of Escherichia coli. 6. pJAL52I or pJAL52I74
The recombinant plasmid according to claim 5, which is L. 7. An E. coli strain transformed with the recombinant plasmid according to claim 5 or 6. 8. Escherichia coli K12 / HB101 / pJAL5
2I or E. coli K12 / HB101 / pJAL52I
The E. coli strain according to claim 7, which is 74 L. 9. The mutant Escherichia coli ribonuclease HI is recovered from the culture medium obtained by culturing the Escherichia coli strain according to claim 7 or 8.
The method for producing the mutant Escherichia coli ribonuclease HI according to 1.