JP2747128B2 - Mutant Escherichia coli ribonuclease H with improved stability - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

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

[0002]

BACKGROUND OF THE INVENTION Natural ribonuclease H of Escherichia coli (in the present specification, ribonuclease H or RNaseH simply means natural Escherichia coli ribonuclease H). Is a molecular weight of about 17 consisting of 155 amino acids.
A Kd hydrolase having the substrate specificity of specifically cleaving only the RNA strand of a DNA / RNA hybrid by an endo effect. This enzyme has various uses as described below based on its substrate specificity and is attracting attention as an extremely valuable enzyme. 1) Removal of template mRNA during cloning of cDNA. 2) Removal of the poly A region of the mRNA. 3) RNA fragmentation.

[0003] Although the importance of ribonuclease H is likely to increase with the development of genetic engineering, the production of this enzyme by recombinant DNA technology is extremely low due to its extremely low production in E. coli. Attempts have already been made from BRL, Pharmacia and Takara Shuzo, etc.
Ribonuclease H produced by NA technology is supplied. These commercially available recombinant ribonucleases H
Is produced using Escherichia coli as a host (Kanaya et al., Journal of Biological Chemistry, 264 , 11546-11549 (1989)).

[0004] If the stability of ribonuclease H, which has a high utility value, can be improved, for example, the resistance to denaturing agents and heat, it can be used under conditions that cannot be used with conventional recombinant ribonuclease H.

To date, we have used recombinant DNA technology to modify native ribonuclease H against denaturing agents.
Although mutant ribonuclease H having higher resistance was produced, the degree of stabilization was not sufficiently satisfactory, and the enzyme activity was not superior to that of the natural enzyme (Japanese Patent Application No. 01-284454). ). In addition, a method for producing thermophilic ribonuclease H having extremely higher stability than Escherichia coli ribonuclease H using Escherichia coli has been reported (Kanaya et al., The 2nd Annual Meeting of the Protein Engineering Society of Japan (1990), 69 pages; Japanese Patent Application No. 02-1110
65), the enzyme activity of thermophilic ribonuclease H produced using Escherichia coli was lower than that of Escherichia coli ribonuclease H. In the latter case, purification requires an operation of solubilization using urea, and has a disadvantage that the production method is more complicated than that of Escherichia coli ribonuclease H. Therefore, it would be more useful in industry if mutant ribonuclease H, which is an enzyme having better enzyme activity and stability and easier to produce and purify than thermophilic ribonuclease H, could be produced. .

[0006]

Means for Solving the Problems From the above viewpoints, the present inventors have tried various modifications for the purpose of enhancing the stability of Escherichia coli ribonuclease H while maintaining the enzyme activity. A mutant in which the 62nd His (histidine) of the enzyme has been replaced with Pro (proline) by introducing a site-specific mutation into the gene has better stability to heat and denaturing agents than natural ribonuclease H. The present inventors have also found that the enzyme activity is higher than that of the conventional stabilized mutant ribonuclease, and is substantially equivalent to that of the natural ribonuclease H. Thus, the present invention has been completed.

That is, the present invention provides a mutant E. coli ribonuclease H comprising an amino acid sequence in which His at position 62 of natural E. coli ribonuclease H has been substituted with Pro.

[0008] The present invention also provides a codon encoding the 62nd His of natural E. coli ribonuclease H,
The present invention provides a mutant ribonuclease H structural gene in which CAT has been converted to a codon encoding Pro. The present invention also provides an expression vector for expressing the above mutant ribonuclease H in Escherichia 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 using Escherichia coli is well-known (Maniatis
Et al., Molecular Cloning (1982): A Laboratoy M.
anual, Cold Spring Harbor Laboratoy), and the above-mentioned mutant ribonuclease H structural gene, expression vector, and transformant are routinely used in accordance with the general procedures described in this publication, for example, as described in Examples below. It can be manufactured according to the method. Incidentally, the expression vectors of the present invention is not particularly limited as long as it functions in E. coli, those containing the mutant ribonuclease H gene under the control of the P _L and P _R promoters of bacteriophage λ Is preferred.

[0010]

EFFECT OF THE INVENTION The mutant Escherichia coli ribonuclease H having improved stability according to the present invention does not denature even at a higher concentration or at a higher temperature than the concentration of a denaturing agent which is inactivated by conventional ribonuclease H. (See Example 3), so that handling under such conditions is possible.
Furthermore, the mutant E. coli ribonuclease H of the present invention
It is expected that durability and the like are also improved, and it is easy to handle because it is less inactivated than conventional ribonuclease H and can be stored stably. Also, the mutant Escherichia coli ribonuclease H of the present invention is used.
Has the same enzymatic activity as natural ribonuclease H,
An equivalent enzyme activity can be obtained with a smaller amount than the conventional stabilized mutant ribonuclease H.

Hereinafter, the present invention will be described in more detail 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.

Example 1 Construction of Plasmid pJH62P The construction of plasmid pJH62P is schematically shown in FIG. First, the expression plasmid pAK600 (see Japanese Patent Application Laid-Open No. 03-065188) for expression of natural ribonuclease H in E. coli was used.
Point mutations were introduced into the rnh gene by PCR using the nh gene as a template and synthetic oligonucleotide primers.

As the synthetic oligonucleotide, a chemically synthesized oligonucleotide was used. The oligonucleotides used are shown in FIG. As shown in FIG. 1, first, pA
5′-primer (1) containing Kph as template DNA and restriction site of SphI and 3′-primer for mutagenesis
A DNA fragment of about 200 bp was obtained by (4), and a DNA fragment of about 340 bp was obtained by 5′-primer (3) for mutagenesis and 3′-primer (2) containing a SalI site.
Mutation rn amplified by R reaction and converted from His ⁶² to Pro ⁶²
Two types of h gene fragments were obtained. The PCR reaction was performed using a commercially available Gene Am
Performed according to the instructions of p Kit (Takara). After purifying the rnh gene fragment by agarose gel electrophoresis, the two were mixed, and 5′-primer (1) and 3′-primer
By adding (2) and performing a PCR reaction again, Sph
Mutant rnh gene fragments having both I and SalI restriction enzyme sites were obtained. The SphI and SalI sites at both ends of the obtained about 500 bp mutant rnh fragment were restricted with the restriction enzymes SphI and SphI.
After digestion with alI, the digest was subjected to 1.5% agarose gel electrophoresis, and a mutant rnhSphI-SalI gene fragment of about 500 bp was cut out and 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
No. 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 cyclized by ligation with the hI-SalI fragment. Ligation is a commercially available ligation kit
(Takara) according to the attached instructions exactly. Escherichia coli HB1
01 strain was transformed, and a plasmid vector pJH62P for expressing mutant ribonuclease H was obtained from the transformant.

The transformant E. coli HB101 / pJH62P obtained as described above has been deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology. Date: June 21, 1991
Day).

Example 2 Mutant ribonuclease H (H
Production and purification of E. coli 62P) 100 μg of E. coli transformant HB101 / pJH62P
The cells were shake-cultured at 30 ° C. in 2 L of LB medium containing 1 / L of ampicillin. When the turbidity of the culture had grown to a Kret value of about 80, the culture temperature was raised to 42 ° C., shaking was further continued for 3.5 hours, and then the cells were collected by centrifugation. The Kret value at this time was about 180.

The obtained cells were cultured in 10 cells containing 1 mM EDTA.
After suspending in 40 ml of mM Tris-HCl buffer (TE) (pH 7.5), the cells were disrupted by sonication in ice. Fifteen,
The centrifuged supernatant (crude extract) obtained by centrifugation at 000 rpm for 30 minutes at 4 ° C. was dialyzed overnight at 4 ° C. against 5 l of TE (pH 7.5). DE-equilibrated crude extract after dialysis with the same buffer
52 columns (10 ml) and P-11 column (4 ml) were passed in this order. Under these conditions, the mutant ribonuclease H
(H62P) passed through the DE-52 column,
Adsorb to column. 10 ml of TE (pH 7.5) and then 0.1 ml.
After flowing 10 ml of TE (pH 7.5) containing 1M NaCl,
By increasing the NaCl concentration linearly to 0.5 M, the mutant ribonuclease H (H6
2P) was eluted. Mutant ribonuclease H (H62
Fractions eluting with P-11 containing P) were combined and used as a purified sample. The purified sample gave a single band on 15% SDS-PAGE, and also showed a single peak on reverse phase HPLC. Purification yield was 21.7 mg / l culture. Identification of the purified sample was performed by mapping peptide fragments obtained by digestion with Achromobacter protease I by reverse phase HPLC to confirm the elution position of each fragment peak,
The peptide containing the amino acid at position 62 was fractionated and analyzed by amino acid sequence analysis.

The CD spectrum of the obtained purified sample in a 10 mM sodium acetate buffer (pH 5.5) containing 0.1 M NaCl at 200 to 250 nm is the same as that of the natural form, and can be detected by the CD spectrum. No change in secondary structure was observed. When the enzyme activity was measured by the following method and the specific activity was measured, the obtained mutant enzyme had a specific activity of 21 U / mg, and retained about 110% of the enzyme activity of the natural enzyme. Was. The obtained comparison results are shown in Table 1 below.

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

[Table 1] Comparison of enzymatic activities of native and mutant ribonuclease HEnzyme name Specific activity (U / mg)  Natural ribonuclease H 20 Mutant ribonuclease H (H62P) 21

Example 3 Mutant ribonuclease H (H
Evaluation of stability of 62P) The stability of natural ribonuclease H and variant ribonuclease H (H62P) to denaturing agents was measured and compared. 20 mM containing urea of 0 to 8 M as a denaturant
Using a sodium acetate buffer (pH 5.0), the denaturation ratio at 25 ° C. was measured by the CD value at 220 nm.
The measurement was performed using a cell having a 2 mm optical path length, and J-6 manufactured by JASCO Corporation
00. The denaturation curve obtained by the measurement is shown in FIG.
Shown in Both enzymes were almost completely denatured with 7M urea.
The denaturation curve of the mutant ribonuclease H (H62P) was shifted to a higher urea concentration than the native denaturation curve, and the stability to the denaturant was clearly increased.
When comparing the urea concentration when 50% of the whole was denatured, the mutant ribonuclease H (H62P) was about 0.47 M larger than the natural type. The results obtained are shown in Table 2 below. The denaturation with urea was performed in a 20 mM sodium acetate buffer (pH 5.5) at 25 ° C.

TABLE 2 Hydrochloric acid of natural and mutant ribonuclease H
Comparison of stability to guanidineEnzyme name Urea concentration required for 50% denaturation (M)  Natural ribonuclease H 4.99 Mutant ribonuclease (H62P) 5.46

The thermostabilities of natural ribonuclease H and mutant ribonuclease H (H62P) were also measured and compared. The measurement was performed by measuring the rate of denaturation at
The measurement was performed by measuring a heat denaturation curve with a CD value in nm. The heat denaturation experiment was performed using a cell having an optical path length of 2 mm, using 1 M guanidine hydrochloride and 1 mM dithiothreitol (DTT).
In 20 mM sodium acetate, pH 5.5. FIG. 4 shows the denaturation curve obtained by the measurement. The heat denaturation curve of the mutant ribonuclease H (H62P) was shifted to a higher temperature than the heat denaturation curve of the natural type, and the heat stability was clearly increased. Comparing the temperature at which 50% of the whole is denatured, the mutant ribonuclease H (H62
P) was stabilized at 4.1 ° C. more than the natural form. The results obtained are shown in Table 3 below.

TABLE 3 Comparison of thermostability of native and mutant ribonuclease HEnzyme name Temperature for 50% denaturation (℃)  Natural ribonuclease H 51.8 Mutant ribonuclease (H62P) 55.9

From the above results, it was confirmed that by converting the 62nd His of natural Escherichia coli ribonuclease H to Pro, the stability to denaturing agents and heat was improved.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the construction of plasmid pJH62P.

FIG. 2 is a schematic diagram showing a synthetic oligonucleotide for introducing a site-specific mutation. In the figure, an underline indicates a restriction enzyme site, and a closed circle indicates a base (P) corresponding to the mutation site.
(base corresponding to ro ⁶² ).

FIG. 3 is a graph showing urea denaturation curves of natural ribonuclease H and mutant ribonuclease H (H62P).

FIG. 4 is a graph showing heat denaturation curves of natural ribonuclease H and mutant ribonuclease H (H62P).

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/22 C12R 1:19)

Claims (7)

(57) [Claims] 1. A mutant Escherichia coli ribonuclease H having an amino acid sequence in which histidine at position 62 in the amino acid sequence of natural Escherichia coli ribonuclease H has been substituted with proline. 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 pJH62P. 5. An Escherichia coli strain transformed with the recombinant plasmid according to claim 3 or 4. 6. The E. coli strain according to claim 5, which is HB101 / pJH62P. 7. A method for producing a mutant Escherichia coli ribonuclease H, which comprises culturing the Escherichia coli strain according to claim 5 and recovering the mutant Escherichia coli ribonuclease H from the obtained culture solution.