JP2641263B2 - Heat-resistant RNase T1 - Google Patents

Description

The present invention relates to a novel thermostable RNase T1, more particularly,
With the tyrosine at position 24 from the amino terminus of natural RNase T1
A heat-resistant RNase T1 in which the asparagine at position 84 is substituted with cysteine to improve its heat resistance, the heat-resistant RNase
The present invention relates to a DNA sequence encoding ase T1, a recombinant DNA expression vector containing the DNA sequence, a host cell transformed with the vector, and a method for producing the heat-resistant RNase T1.

Technical background and prior art Ribonuclease T1 (EC3.1.27.3, hereinafter referred to as RNase T1) is an Aspergillus oryzae (Aspegillus oryzae).
rgillus oryzae) is an extracellular ribonuclease that has extremely strict substrate specificity and has only the phosphodiester bond of the guanosine 3'-phosphate moiety in single-stranded ribonucleic acid and ribonucleotides. Disconnect selectively.
Due to this very tight guanine specificity, RNase T1
It plays a very important role in RNA structural analysis.

The primary structure of this RNase T1 has already been determined, and this enzyme is a polypeptide as shown in FIG. 1 consisting of 104 amino acid residues and having two disulfide bonds in the molecule. Are known (References 1 and 2). The three-dimensional structure of this RNase T1 has also been clarified by X-ray crystal structure analysis (References 3, 4, and 5).

OBJECT OF THE INVENTION The present inventors have proposed this RNase T useful for RNA structural analysis.
As a result of conducting various studies to obtain a more useful enzyme by modifying 1, the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1 were replaced with cysteine, respectively. It has been found that the present invention can be improved, and the present invention has been completed. That is, the present invention provides a heat-resistant RNase T1 in which the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1 are each substituted with cysteine. Further, the present invention provides a DNA sequence encoding the heat-resistant RNase T1,
It is intended to provide a recombinant DNA expression vector containing the DNA sequence, a host cell transformed with the vector, and a method for producing the heat-resistant RNase T1.

Configuration and Effect of the Invention The heat-resistant RNase T1 of the present invention can be produced as follows.

First, it contains a DNA sequence encoding a heat-resistant RN ase T1, and 5 to 'Bg1 II site and 3' side side Bg1
A converter gene having a II site is prepared. On the 5 'side of this fragment is a codon beginning with a T to create a Bg1 II site, for example a TTC corresponding to Phe in this case, and a codon ATG corresponding to Met which is later required for cleavage from the fusion protein.
Was placed before the DNA sequence encoding the thermostable RNase T1. On the 3 'side, a codon corresponding to Met-G1n was added after the structural gene of the heat-resistant RNase T1, and a Bg1 II site was further provided. The entire DNA sequence of this fragment and the amino acid sequence encoded by the DNA sequence are shown in the following Sequence Formula 1. When natural RNase T1 is desired, the codon TGT corresponding to the 24th amino acid residue and its complementary sequence ACA are respectively set to TAC and GTA, and
The codon TGT corresponding to the fourth amino acid residue and its complementary sequence ACA may be substituted with AAC and GTT, respectively (this changes the 24th and 84th amino acids and cysteine to tyrosine and asparagine, respectively). .

This DNA fragment can be prepared, for example, as follows. That is, the entire DNA sequence is divided into several oligonucleotide units, and each oligonucleotide is synthesized by a known method such as the phosphotriester method or the phosphite method (References 6 and 7). Next, the obtained oligonucleotide was ligated as shown in FIG. 2 (References 6 and 10), and 5 ′
A desired DNA fragment having a Ba1 II site on both the 3 ′ and 3 ′ sides can be obtained.

Next, a recombinant DNA expression vector incorporating the above fragment is constructed as shown in FIG. That is,
Plasmid pIGF8 (Reference 11) was converted to Bg1 II according to the conditions of Reference 16.
Digestion with restriction enzymes, the resulting linear vector,
The Bg1 II fragment is ligated to obtain plasmid pT1S. The plasmid pIGF8 used here is Escherichia coli ( Escheri
chia coli, hereinafter referred to as E. coli) trp promoter gene, referred to as human growth hormone (hereinafter hGH) portion of the gene, referred to as insulin-like growth factor (hereinafter IGF-I) containing a gene and ampicillin resistance gene (Ap ^r) doing. The IGF-I gene is 2 / from the 5 'side of the hGH gene.
Connected via the Bg1 II site at position 3 (third
The amino acid sequence encoded on the upstream side of the Bg1 II site of this hGH gene is referred to as the hGH AB portion. Therefore, the resulting plasmid pT1S contains two heat-resistant RNase T1s.
It expresses a sandwich fusion protein sandwiched between the AB portion of hGH and IGF-I via two methionines (see FIG. 4). Since the RNase T1 molecule does not contain methionine, this fusion protein can be subsequently separated from the fusion protein by the action of cyanogen bromide, which cleaves specifically at the C-terminal end of methionine.
Incidentally, the reason a plasmid pIGH8 the construction of the plasmid pT1S is, E. Direct expression unsuccessful the RN-ase T1 gene by E. coli trp promoter, whereas stable synthetic gene of hGH is under the control of the E. coli trp promoter And that it is expressed efficiently.

Next, the plasmid pT1S was transformed into ampicillin-sensitive E. coli, for example, E. coli HB101 (F ⁻ , hsdS20 [r
B ^-, mB ^-], recA13, ara-14, proA2 , lacY1, galK2, rpsL20
(Sm ^r ), xy1-5, mt1-1, supE44, λ ⁻ ), and transformants are selected on a medium containing ampicillin. This transformant is grown in an appropriate growth medium containing ampicillin, plasmid pT1S is taken out, and it is confirmed that the plasmid has the correct nucleotide sequence by the dideoxy method (Reference 14).
Confirm with. Plasmid pT confirmed in this way
A transformant containing 1S, that is, Escherichia coli HB101 / pT1S has been deposited with the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology (Deposit date: August 2, 1988, Deposit No. 10167 of Microtechnological Laboratory).

The transformant is grown and the fusion protein is expressed by adding 3-indoleacrylic acid when the appropriate OD ₆₆₀ value is reached.

 The outline of purification of the thermostable RNase T1 is described below.

First, the cells are disrupted by a freeze-thaw method. The precipitate is separated from the soluble fraction by centrifugation. The fusion protein recovered in the precipitate fraction is dissolved in a buffer containing 7M urea and separated by anion exchange column chromatography (Whatman DE52) to recover the fusion protein part. After removing urea by dialysis, it is cleaved at methionine by bromide ion treatment, and heat-resistant RNase T1 is cut out from the fusion protein. After dialysis, anion exchange column chromatography (Pharmacia, Q
(Sepharose) and recover the heat-resistant RNase T1. The collected fractions are further purified by HPLC (Pharmacia, Mono Q).

Enzyme activity was measured using the substrate ³² pGpC, whose 5 ′ hydroxyl group was labeled with [γ ³² -P] ATP and polynucleotide kinase.
Heat-resistant RNase T1 and react at various temperatures, measure the initial production of ³² pGp by homochromatography (Reference 12), plot this against time, and determine the initial velocity from the slope In this way, an enzyme activity is measured.

The measurement results are shown in Table 2 below, and it was found that the activity of the heat-resistant RNase T1 of the present invention was at least 10 times higher at a high temperature of 55 ° C. and 60 ° C. than that of the natural RNase T1.

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

The polynucleotide kinase, DNA ligase and Bg1 II restriction enzyme used in the examples were obtained from Takara Shuzo. D
Nase I was obtained from Boehringer Mannheim. Lysozyme was obtained from Sigma. The substrate pGpC is
Dinucleotide Gp according to the method of Otsuka et al.
After synthesizing C, the 5 ′ hydroxyl group was phosphorylated with polynucleotide kinase to prepare (Reference 9).

Example 1 Synthesis of oligodeoxynucleotides A. Synthesis of oligodeoxynucleotides other than MU5, ML6, MU17, ML17, U21C and L21C by solid-phase method A-1. Synthesis of nucleotide resin As shown in the following flowchart 1, nucleosides Resin 10 was synthesized.

1) Synthesis of 3 '-[5'-O-dimethoxytrityl- (deoxyribonucleoside)] monosuccinate (7) 3'-(5'-O-dimethoxytritylthymidinyl)
An example of the synthesis of monosuccinate will be described.

After dimethoxytritylthymidine 2.72 g (5 mmol) was azeotroped with pyridine, it was dissolved in methylene chloride (20 ml), and 750 mg (7.5 mmol) of succinic anhydride and 916 mg (7.5 mmol) of 4-dimethylaminopyridine (DMAP) were added. And stirred for 2 hours. After confirming the disappearance of the raw materials by TLC (CHCl ₃ : MeOH = 20: 1), and washing with 0.5 M KH ₂ PO ₄ (pH 5) (20 ml × 2 times)
After washing with water (20 ml × 2), the solvent was distilled off. Purification was performed using a C18 silanized silica gel column. Yield 3.14g (4.8
8 mmol), yield 97.5%.

2) Synthesis of pentachlorophenyl 3 '-[5'-O-dimethoxytrityl- (deoxyribonucleoside)] succinate An example of the synthesis of pentachlorophenyl 3'-(5'-O-dimethoxytritylthymidinyl) succinate is shown.

Dimethoxytritylthymidinyl monosuccinate 3.14
g (4.9 mmol) was dissolved in 34.1 ml of N, N-dimethylformamide, and 1.43 g (5.4 mmol) of pentachlorophenol and N, N
1.51 g (7.3 mmol) of N'-dicyclohexylcarbodiimide was added and the mixture was stirred at room temperature overnight. TLC (CHCl ₃ : MeOH = 1
After confirming the completion of the reaction in 0: 1), the precipitate was separated by filtration and the solvent was distilled off. 15 ml of benzene was added to the residue, and the precipitated insoluble matter was separated by filtration, and benzene was distilled off. This residue was dissolved in 15 ml of chloroform, and dropped into n-pentane to form a powder. Yield 3.89 g (4.35 mmol), yield 89.2%.

3) Synthesis of nucleoside resin ( 10 ) An example of synthesis of a thymidine-polystyrene resin (NT) will be described.

2.5 g (0.5 mmol) of aminomethylated polystyrene ( 9 , 1% divinylbenzene, NH ₂ : 0.20 mmol / g) was suspended in 15 ml of N, N-dimethylformamide, and pentachlorophenyl 3 ′-(5′-O 1.34 g (1.5 mmol) of dimethoxytritylthymidinyl) succinate and 0.2 of triethyleneamine
3 ml (1.15 mmol) was added and shaken overnight. The reaction solution was filtered, and the resin was washed with N, N-dimethylformamide (20 ml × 2 times).
And pyridine (20 ml x 2), and then washed with 0.1M
4.5 ml of a pyridine solution of dimethylaminopyridine and 0.5 ml of acetic anhydride were added and shaken for 10 minutes. The reaction solution was filtered, and the resin was treated with pyridine (20 ml × 3 times) and dichloromethane (20 ml ×
3 times) and ether (20 ml × 3 times) and then dried.

Quantification of nucleosides is determined by thymidine-polystyrene resin
Methylene chloride 2% benzenesulfonic acid to 10 mg - methanol (7: 3v / v) was added a solution 2ml shaken for 1 minute, after distilling off the solvent taking 1/100 of the reaction solution, HClO the residue ₄ −EtOH
(3: 2 v / v) was performed by adding 3 ml and measuring the absorbance at λ = 500 nm. As described above, the nucleoside in which the base is thymine has been described. In the case of a nucleoside resin having another base, the nucleoside resin was prepared in the same manner.

A-2. Synthesis of oligodeoxynucleoside The procedure shown in Table 1 below was repeated for 20 to 30 mg (3 to 5 μmol) of the nucleoside resin obtained in A-1. In steps 1 to 5, methylene chloride-methanol (7:
The dimethoxytrityl group, which is a protecting group for the 5 'hydroxyl group, is removed with 2% benzenesulfonic acid in 3v / v). Then, in steps 6 to 9, a dimer block or monomer synthesized by a conventional method is added to the resin by 3%. Condensed using ~ 4 equivalents. The condensing agent mesitylenesulfonylnitrotriazolide is used in an amount of 15 to 20 equivalents to the resin, and the condensation reaction is carried out at 40 ° C for 20 minutes, 30 minutes.
C. for 30 minutes and room temperature (20-25.degree. C.) for 40 minutes. After the condensation, the unreacted 5'-hydroxyl group in the steps 10 to 12 of the above-mentioned flowchart is replaced with 4-dimethylaminopyridine catalyst.
Capping was performed with acetic anhydride. This series of operations was performed until the target chain length was reached. (See Compound No. 11-13 of the flow chart 1).

A-3. Cleavage of oligodeoxynucleotide from the resin, deprotection, purification and identification After washing the nucleotide resin reaching the target chain length with dioxane, 0.5 M 1,1,3,3-tetramethylguanidium-s
1 ml of a solution of yn-pyridine-2-aldoximate in dioxane-water (9: 1) was added, and the mixture was shaken at 30 ° C. overnight. After filtering the solution, the resin was washed with 50% aqueous pyridine. The filtrate and washings were distilled off, and 0.5 ml of pyridine and aqueous ammonia (28
(% NH ₄ OH), and the tube was sealed. After heating at 55 ° C. for 5 hours, the solvent was distilled off and the residue was purified by silica gel column chromatography.

After evaporating the solvent from the fraction containing the target substance, 1 ml of 80% acetic acid was added. After 20 minutes, the solvent was distilled off and azeotroped with water. Dissolve the residue in 3 ml of water, and add ethyl acetate (3
After extracting and removing tritanol with (ml × 3 times), the aqueous layer was distilled off. The obtained completely deprotected gene fragment was analyzed and fractionated by C18 HPLC. This was further analyzed by ion-exchange HPLC, and when an impurity was observed, it was separated and purified to a single peak by HPLC. The purified gene fragment is subjected to two-dimensional homochromatography (Mobility Shift Ana
lysis) and 5'-end analysis confirmed that the base sequence was correct.

B. oligodeoxynucleotide MU5, ML6, MU17, ML17,
Synthesis of U21C and L21C Oligonucleotides were synthesized using a DNA synthesizer (Model 380A) manufactured by Applied Biosystems. Raw materials and reagents were purchased from Applied Biosystems.

Deprotection was carried out in a DNA synthesizer for protecting groups other than dimethoxytrityl group, which is a protecting group for the 5 'hydroxyl group. Thereafter, purification and identification of the oligonucleotide were carried out in the same manner as in A-3.

Example 2 Preparation of DNA Fragment Encoding Heat-Stable RNase T1 A. 5 'Phosphorylation 0.1 OD of each oligonucleotide other than U1 and L21C (≒ 750p
Mol, 5 μg) with 50 mM Tris-HCl (pH 8.0), 10 mM M
_{gCl 2, 10mM β-ME,} 1mM spermine buffer, 10 [mu] l by adding polynucleotide kinase 10μ moles of ATP and 6U
And reacted at 37 ° C for 1 hour to phosphorylate the 5 'hydroxyl group. (Because the 5' hydroxyl group of U1 and L21C is a restriction enzyme site, phosphorylation first and then the binding reaction leads to a dimer. Therefore, phosphorylation was performed after the binding reaction was performed). After the reaction, the enzyme was inactivated by heating at 90 ° C. for 3 minutes.

B. Preparation of Segments The U1, L21C and 5 'phosphorylated oligonucleotides were collected and mixed in one tube for each component of each segment (I-IVC) shown in FIG. 2 and mixed with 66 mM Tris.
HCl (pH 7.6), 6.6 mM MgCl ₂ , 0.5 mM ATP buffer 150 μl
The mixture was heat-treated at 90 ° C. for 3 minutes and immediately immersed in ice for rapid cooling. Ten minutes later, the mixture was heated again to 75 ° C., and then allowed to cool to room temperature (20 ° C. to 25 ° C.) over 1 to 3 hours to perform annealing. After that, after keeping at 15 ° C. for 10 minutes, β-
Add ME to 10 mM, add 30 U of T4 DNA ligase,
The reaction was carried out at 15 ° C. for 12 to 15 hours (Segment III was annealed in two to increase the yield of the reaction, allowed to undergo a binding reaction for 2 to 3 hours, and then mixed into one). After the reaction, the enzyme was inactivated by heating at 65 ° C. for 5 minutes, and then the DNA was recovered by ethanol precipitation. 1
0% PAGE (polyacrylamide electrophoresis, size (0.1 ×
20 × 20 cm), CV175V, 3-4 hours), recover DNA by electroelution from the band portion of the segment of the desired chain length, perform phenol treatment and ethanol precipitation, and perform 15-25 μg (225-375p) of each segment. Mol).

C. Preparation of Transformer Gene Using 3.3 μg (50 pmol) of each segment, 15 U of T4 DNA ligase was added in the above-mentioned buffer solution to perform a binding reaction.
At this time, the reaction was performed at 20 ° C. for 12 to 15 hours without annealing. After the reaction, the mixture was treated at 65 ° C. for 5 minutes, precipitated with ethanol, and subjected to 5% PAGE (size 0.1 × 20 × 20 cm). RN
The band corresponding to the chain length of the ase T1 gene was collected by electroelution, and subjected to phenol treatment and ethanol precipitation. Further, both 5'-ends of the fragment were phosphorylated using 6 U of polynucleotide kinase under the conditions described above to obtain about 1 μg (3.8 pmol) of the fragment containing the DNA sequence encoding the heat-resistant RNase T1. I got

Example 3 Construction of Plasmid pT1S A recombinant DNA expression vector containing a DNA sequence encoding a heat-resistant RNase T1 and a plasmid pT1S were prepared as follows according to the method of Nishikawa et al. See figure).

Buffer [20 mM Tris-HCl (pH 7.6), 7 mM MgCl ₂ , 10 m
M β-ME, 175 mM NaCl]
(5 μg) was digested with 12 units of Bg1 II restriction enzyme at 37 ° C. for 1.5 hours. Furthermore, the calf small intestine was treated with alkaline phosphatase (0.04 unit) to remove the phosphate group at the 5 'end. The enzyme was removed by phenol extraction of this solution followed by ether washing. Then linear pIGF8
Was precipitated with ethanol and dissolved in 25 μl of 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA solution. This linear pIGF8
(0.67 pmol) and the transformant gene (2
55 pmol) was ligated with T4 DNA ligase (1.2 units) overnight at 20 ° C. (total volume 20 μl). After phenol extraction of this solution, the DNA was precipitated with ethanol, and the resulting pellet was used for E. coli transformation.

Example 4 Transformation of E. coli HB101 100 μl of competent cells prepared in advance by the method of Morrison et al. (Reference 15) and 10 μl of the vector DNA solution prepared in Example 3 were mixed, allowed to stand for 1 hour, and then left at 42 ° C.
For 75 seconds and left at room temperature for 5 hours. LB medium
900 µl was added, and the cells were cultured at 37 ° C for 1 hour. This was collected, suspended in 100 μl of LB medium, and agar medium [1.5% (w / v)
Agar, LB, 40 μg / ml ampicillin] and cultured overnight at 37 ° C. The transformants were then grown on M9-casamino acid medium containing 20 μg / ml ampicillin. The cells were collected and lysed, and the plasmid was recovered. The base sequence of this plasmid was examined by the dideoxy method (Reference 14).
After confirming that the transformant gene was correctly inserted, this plasmid was designated as pT1S.

Example 5 Expression of fusion protein and preparation of crude enzyme A. Expression of fusion protein E. coli HB101 / pT1S prepared in Example 4 was inoculated in 50 ml of LB medium containing 40 μl / ml ampicillin, and incubated at 37 ° C. for about 8 hours. The cells were cultured with shaking (pre-pre-culture).

M9 medium containing 0.2% (weight / volume) casamino acid and 40 μg / ml ampicillin [5.5 g / l Na ₂ HPO ₄ , 3 g / l K
H ₂ PO ₄ , 5 g / l NaCl, 1 g / l NH ₄ Cl, 0.4% (w / v) dextrose, 1 mM MgSO ₄ , and 0.1 mM CaCl ₂ ] 20
To 0 ml, 5 ml of the pre-preculture was added, and the mixture was cultured with shaking at 37 ° C. for about 15 hours (pre-culture). Next, 40 ml of this preculture solution was added to the above M9 medium 1 containing casamino acid and ampicillin, followed by shaking culture at 37 ° C. After 1 to 1.5 hours, OD ₆₆₀ = 0.01
IAA (3-indoleacrylic acid) when it reaches ~ 0.02
Was added to a final concentration of 40 μg / ml to induce expression of a fusion protein of AB portion of hGH and heat-resistant RNase T1 and IGF-I, followed by further culturing for 7 hours.

B. Purification of heat-resistant RNase T1 After collecting the cells, the cells were washed with physiological saline, and then added to a solution consisting of 50 mM Tris-HCl (pH 8.0), 30 mM NaCl, and 1 mM EDTA. And suspended. Lysozyme was added thereto at a concentration of 1 mg / ml, and left at 0 ° C. for 1 hour. Subsequently, the freeze-thaw operation was repeated 5 times, and 1/50 (vol / vol) of the total volume of 150 mM Tris-HCl (pH 7.5), 2
80 mM MgCl ₂ , 4 mM CaCl ₂ solution was added, and 1 mg / ml DNase I was added.
100 μl of the solution was added and left at 0 ° C. for 60 minutes. This is 35,
The precipitate was collected by centrifugation at 000 × g for 40 minutes. This precipitate was dissolved in 80 ml of a solution containing 7 M urea, 20 mM Tris-HCl (pH 7.5), and 10 mM mercaptoethanol, applied to DE52 column chromatography (φ3 cm × 6 cm) equilibrated with the same solution, and then subjected to 0 to 0.3. Elution was performed with an M salt concentration gradient.
It was confirmed by SDS-PAGE (Reference 17) that the target fusion protein was eluted in the range of 0.1 M to 0.2 M NaCl. This fraction was collected, dialyzed, desalted, and freeze-dried. Total protein was 247 mg. This was dissolved in 82 ml of 70% formic acid, 656 mg of cyanogen bromide was added, and after dissolution, the mixture was allowed to stand at 4 ° C. for 20 hours. After the reaction, the solution was dialyzed against 50 to 10 mM NH ₄ HCO ₃ to remove formic acid and cyanogen bromide, and then concentrated under reduced pressure.
This was subsequently separated by Q Sepharose column chromatography (φ φ) equilibrated with 20 mM Tris-HCl (pH 7.5) and 0.15 M NaCl.
2.4cm x 13.5cm) and 0.15M to 0.45M NaCl
Elution was performed with a concentration gradient. The desired heat-resistant RNase T1 is 0.28M
Eluted from 0.31M NaCl to SDS-PAG
E accepted. This fraction was collected, dialyzed against 10 mM NH ₄ HCO ₃ , concentrated under reduced pressure, separated and purified by MonoQ column chromatography (φ5 × 50 mm), and single band (purity 90% or more) by SDS-PAGE. RNase T1 was obtained.

C. Measurement of RNase T1 activity The concentration of substrate ³² pGpC whose 5 ′ hydroxyl group was labeled with [γ- ³² P] ATP and polynucleotide kinase was 150 μM,
The concentration of Nase T1 was 0.5 ng / μl (45.1 nM) and the concentrations of Tris-HCl (pH 7.5) and EDTA were 50 mM and
A total of 20 μl of 1 mM solution was added at 37 ° C, 45 ° C, 55 ° C,
The reaction was carried out at 70 ° C and 70 ° C, respectively. 0, 2, from the start of the reaction
After 5, 10, and 15 minutes, aliquot 3 μl each, and add 1N HCl
0.6 μl was added to stop the reaction. This was spotted on a DEAE-cellulose plate, and homochromatography (Reference 12) was performed using Homomix VI. The X-ray film was exposed to light, and each spot on the plate was cut out into a vial based on the light, and the radioactivity was measured by a liquid scintillation counter. ^The amount of ³² pGp produced was plotted against time, and the initial velocity was determined from the slope. The results obtained are shown in Table 2 together with the results obtained by similarly testing the natural type RNase T1.

References 1. Takahashi (K. Takahashi), Journal of Biological Chemistry (J. Biol. Chem.), 240 , 211
7 (1965) 2. Takahashi (K. Takahashi), Journal of Biochemistry (J. Biochem.), 98 , 815 (1985) 3. Heinemann et al. (U. Heinemann et al.), Nature, 299 , 27 (1982) 4. Sugio et al. (S. Sugio et al.), FEBS Letters (FEBS L
ett.), 181 , 129 (1985) 5. Sugio et al. (S. Sugio et al.), Journal of Biochemistry (J. Biochem.), 103 , 354 (1988) 6. Ikehara et al. et al.), Proceedings of National Academy of Sun-Eng., USA (Proc. Natl. Acad. Sci. USA), 81 , 5956 (19
84) 7. Baron et al. (ADBarone et al.), Nucleic Acids Res., 12 , 4051
(1984) 8. E. Ohtsuka et al., Tetrahedron, 40 , 47 (1984) 9. CCRichardson, Brothings of National Academy of Sanen Seeds USA (Proc. Nat1. Acad. Sci. USA), 54 , 158 (1
965) 10. Ikehara et al. Proceedings of National Academy of Sun Encies (Proc. Nat1. Acad. Sci.), 83 , 4695 (1986) 11. Nishikawa et al. (S. Nishikawa et al.)
Engineering (Protein Engineering), 1 , 487 (1
987) 12. J. et al. (E. Jay et al.), Nucleic Acids Res., 1 , 337 (1974) 13. Keiichi Kusama, Tracer Experimental Method (above) (Biochemical Laboratory Course 6) ), Edited by The Biochemical Society of Japan, Tokyo Kagaku Doujin, 201 (1977) 14. Sanger et al. (F. Sanger et al.), Proceedings of National Academy of Sun Encies (Proc. Nat1. Acad. Sci.) .), 74 , 5463 (1977) 15. Morrison et al. (PAMorrison et al.), Methods Enzymol., 68 , 326
(1979) 16. Nishikawa et al. (S. Nishikawa et al.), European Journal of Biochemistry (Eur. J. Bioc)
173 , 389 (1988) 17. UK Laemmli, Nature, 22
7 , 680 (1970)

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the primary structure of native RNase T1, and FIG. 2 is a process diagram for preparing a Bg1 II fragment containing a DNA sequence encoding heat-resistant RNase T1,
FIG. 3 is a process diagram of construction of a recombinant DNA expression vector and a plasmid pT1S, and FIG. 4 is a schematic diagram showing a fusion portion of a sandwich-like fusion protein containing a heat-resistant RNase T1.

Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical indication (C12N 15/09 ZNA C12R 1:69)

Claims (8)

(57) [Claims] 1. A heat-resistant RNase T1 in which the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1 are each substituted with cysteine. 2. A DNA sequence encoding a heat-resistant RNase T1 in which tyrosine at position 24 and asparagine at position 84 from the amino terminus of natural RNase T1 are each substituted with cysteine. 3. The DNA according to claim 2, which is represented by the sequence formula 1.
Array. 4. A recombinant DNA expression vector comprising a DNA sequence encoding a heat-resistant RNase T1 in which cysteine has been substituted for the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1. 5. The vector according to claim 4, which is a plasmid pT1S. 6. A recombinant DNA expression vector containing a DNA sequence encoding a heat-resistant RNase T1 in which the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1 are each substituted with cysteine. Converted microorganism. 7. The microorganism according to claim 6, which is E. coli HB101 / pT1S. 8. A method for producing a heat-resistant RNase T1 in which the tyrosine at position 24 and the asparagine at position 84 from the amino terminus of natural RNase T1 are each substituted with cysteine, (a) the heat-resistant RNase T1 (B) preparing a recombinant DNA expression vector containing the DNA sequence; (c) transforming a host cell with the recombinant DNA expression vector to prepare a heat-resistant RNase A method comprising: expressing T1 as a fusion protein; and (d) cleaving the fusion protein to obtain a heat-resistant RNase T1.