JPH06343468A - Enzyme composition - Google Patents

Abstract

PURPOSE:To obtain a restriction enzyme composition containing a stabilizing agent useful for the long-term stabilization of restriction enzyme. CONSTITUTION:The enzyme composition is a stabilized restriction enzyme composition containing a calcium salt. The restriction enzyme is e.g. SfiI and SdiI and the calcium salt is e.g. calcium chloride, calcium nitrate and calcium acetate.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to stabilized restriction enzyme compositions.

[0002]

2. Description of the Related Art A restriction enzyme is an endonuclease capable of recognizing and cleaving a specific base sequence in DNA and is an essential and useful enzyme in the field of genetic engineering. However, many restriction enzymes are unstable, and means for stabilizing them have been studied. For example, the restriction enzyme SfiI is a restriction enzyme produced by Streptomyces fimbriatus, which is a double-stranded DNA 5'-GGCCNNN.
Narrow NGGCC-3 '(G is guanosine, C is cytidine, N is an arbitrary nucleoside) A small number of 8 bases that recognizes the base sequence (represented by SEQ ID NO: 1 in the sequence listing) and cleaves at the position of the arrow It is one of the restriction enzymes for recognition [Nucleic Acids Research (Nucleic Acids Rese
arch), vol. 12, p. 4507-4516 (198)
4)], for its manufacturing method, Methods in Enzymology, Volume 155,
15 to 21 (1987). This S
fiI is a very unstable enzyme, regardless of its concentration,
During the purification operation, dilution, storage at -20 ° C, etc., the activity is considerably reduced. Therefore, it is very difficult to adjust the concentration of SfiI and maintain a constant quality for a long time. JP-A-4-239
No. 87 describes that a stabilized SfiI composition can be obtained by using magnesium chloride as a stabilizer for SfiI.

[0003]

[Problems to be Solved by the Invention] However, Sfi
Even if magnesium chloride is added to the stabilizer of I, deactivation still progresses over a long period of time. Therefore, it has been desired to develop a strong stabilizer for restriction enzymes. An object of the present invention is to provide a restriction enzyme composition containing a stabilizer useful for long-term stabilization of restriction enzymes.

[0004]

SUMMARY OF THE INVENTION In general terms, the present invention relates to a composition of stabilized restriction enzymes, characterized in that it contains a calcium salt.

As a result of intensive studies, the inventors of the present invention found that a long-term stabilized composition of a restriction enzyme can be obtained by using a calcium salt as a stabilizer, and completed the present invention.

The present invention will be described in detail below. The restriction enzymes used in the present invention include all restriction enzymes that are stabilized with calcium salts, and the above-mentioned SfiI is typical. In addition, calcium salts have an effect on relatively unstable restriction enzymes such as ClaI and EcoO65I.

As a restriction enzyme having the same recognition sequence and cleavage site as SfiI, a restriction enzyme produced by Streptomyces diastaticus can be mentioned. The restriction enzyme is a novel enzyme found by the present inventors, and the present inventors named it SdiI.
It has a slightly different substrate specificity from SdiI and SfiI. That is, SfiI cleaves double-stranded DNA methylated with HaeIII methylase [Nucleic Acids Research, Vol. 19, Supplement, 2045-20].
71 (1991)]. In other words, the following formula (Formula 2):

[0008]

[Chemical 2]

(Wherein G is guanosine, C is cytidine,
^5m C also recognizes a base sequence represented by 5-methylcytidine and N represents an arbitrary nucleoside (represented by SEQ ID NO: 2 in the sequence listing), and cleaves at the position of the arrow. On the other hand, SdiI has a remarkably low activity of cleaving the double-stranded DNA.

Other physicochemical properties of SdiI are as follows. (1) Optimum reaction temperature about 50 ° C (2) Optimum salt concentration 50 to 120 mM in the case of sodium chloride and potassium chloride (3) Optimum pH around 8.0

The restriction enzyme used in the present invention may be collected from a culture of a restriction enzyme-producing bacterium, or may be produced by a transformant in which the gene for the restriction enzyme is cloned and introduced into another organism. . The enzyme composition of the present invention may be in the form of a solution or a freeze-dried product.

The calcium salt used in the present invention may be any water-soluble one, and examples thereof include calcium chloride, calcium acetate and calcium nitrate. The concentration of calcium salt may be appropriately selected depending on the type of salt and the like.

The enzyme composition of the present invention may contain a magnesium salt such as magnesium chloride as a stabilizer. A synergistic effect of stabilization can be expected by adding magnesium salt. Glycerol may be contained in the enzyme composition of the present invention. As for the concentration of glycerol,
It may be in a range that does not freeze at −20 ° C. during storage, and is usually 40 to 60% (v / v), preferably 50%. The enzyme composition of the present invention may contain a buffer. An ordinary buffer is used as the buffer, and the pH of the enzyme composition is
Is preferably 7 to 8. For example, Tris-hydrochloric acid buffer solution, Tris-phosphate buffer solution and the like. If necessary, the enzyme composition of the present invention may contain other components. Other components include sodium chloride, potassium chloride, ethylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), bovine serum albumin (BSA), Triton X-100 and the like.

As a reference example, one example of restriction enzyme will be shown below. Reference Example 1 A 30-liter jar fermenter was charged with 20 liters of the medium shown in Table 1 below, and the medium was sterilized by a conventional method.

[0015]

Table 1 Glycerol 17.5 g Yeast extract 3.5 g Polypeptone 3.5 g Meat extract 3.5 g Sodium chloride 2.0 g Dipotassium hydrogen phosphate 1.0 g Magnesium sulfate 0.5 g Deionized water 1 liter (pH 7. 2-7.4)

200 ml of a Streptomyces diasterticus seed culture solution, which had been shake-cultured at 30 ° C. for 48 hours in the same medium as in Table 1, was transplanted to the jar fermenter,
The culture was performed at an aeration rate of 2/3 vvm, a stirring rotation speed of 250 rpm, and a culture temperature of 30 ° C. for 24 hours. After completion of the culturing, the cells were collected using a cooling centrifuge, and approximately 20
220 g of wet cells were obtained. Next, 200 g of wet bacterial cells was added to 400 ml of buffer A [10 mM Tris-HCl pH.
7.5, 10 mM 2-mercaptoethanol, 20 m
M CaCl ₂ , 5% (v / v) glycerol] and ultrasonically disrupted. 11 ml of 8% was added to the resulting extract.
(V / v) Add polyethyleneimine solution and add 1 at 4 ° C
After being left for an hour, centrifugation was performed at 10,000 × g for 10 minutes, and the supernatant was collected. The obtained supernatant is treated with 0.3M NaCl
Dialysis was carried out against Buffer A containing 1 overnight. Next, the dialyzed solution was adsorbed on a column of 50 ml of heparin-Sepharose (Pharmacia), which had been equilibrated with the buffer solution A containing 0.3 M NaCl in advance, to obtain 0.3 M NaCl.
After washing with a buffer solution containing the above, the elution was performed with a buffer solution having a linear concentration gradient of 0.3 to 1.0 M NaCl. The obtained active fractions were collected, dialyzed against buffer A for 4 hours, and the dialyzed solution was adsorbed on a 10 ml column of aminohexyl-sepharose (Pharmacia) previously equilibrated with buffer A. After washing the column with buffer A, 0-0.8M
Elution was performed with buffer A having a linear concentration gradient of NaCl. Next, the obtained active fractions were combined to obtain 0.15M N
The solution A containing aCl was dialyzed for 4 hours, and the dialysis solution was preliminarily adjusted to a solution A containing 0.15M NaCl.
It was adsorbed onto a column of 5 ml of heparin-sepharose equilibrated with. After thoroughly washing the column with buffer A containing 0.15 M NaCl, 0.15-1.0 M NaCl was added.
Elution with buffer A having a linear concentration gradient of 1
An enzyme preparation of diI was obtained. This enzyme preparation was not contaminated with non-specific DNA degrading enzyme and phosphatase. According to the method described above, 20,000 units of activity were obtained from 200 g of wet cells.

The activity is measured by a buffer solution for activity measurement (10 m
M Tris-hydrochloric acid pH 7.5, 10 mM MgC
l ₂ , 50 mM NaCl, 1 mM DTT) 50 μl
The enzyme reaction was carried out at 50 ° C. using adenovirus DNA as a substrate, and after the reaction, agarose gel electrophoresis was performed to detect the degree of decomposition of the substrate. 1μ per hour at 50 ° C
One unit was defined as the enzyme activity that completely cleaved g substrate.

[0018]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example 1 An enzyme preparation of SfiI was obtained by the method described in the method of enzymology described above. Next, 20 mM Tris-
An enzyme composition comprising hydrochloric acid pH 7.5, 10 mM 2-mercaptoethanol, 0.025% (v / v) Triton X-100, and 2 units / μl SfiI was prepared,
The substances shown in Table 2 below were added to the enzyme composition, and the activity after storage at 5 ° C. for 3 weeks was compared.

[0020]

[Table 2] Table 2 Sample No. Additive Additive final concentration 1 None −2 EDTA 1 mM 3 CaCl ₂ 10 mM 4 Ca (NO ₃ ) ₂ 10 mM 5 Ca (CH ₃ COO) ₂ 10 mM 6 MgCl ₂ 10 mM 7 MgSO ₄ 10 mM 8 Mg (CH ₃ COO) ₂ 10 mM 9 Mg (NO ₃ ) ₂ 10 mM 10 MnCl ₂ 10 mM 11 CoCl ₂ 10 mM 12 ZnCl ₂ 10 mM 13 CuCl ₂ 10 mM 14 NiCl ₂ 10 mM

A comparison of the activity is made with the Pm of adenovirus DNA.
The aCI digested fragment (7563 bp) was ligated to Hin of pUC18.
Substrate DNA inserted into the cII site and cut at one site with XbaI
And the enzyme composition in 5 μl of the above-mentioned buffer for activity measurement.
The reaction was carried out at 0 ° C. for 1 hour, and the degree of cleavage of the substrate DNA was compared by agarose gel electrophoresis.

As a result, CaCl ₂ , Ca (N
In the samples (Sample Nos. 3, 4, and 5) to which O ₃ ) ₂ and Ca (CH ₃ COO) ₂ were added, the enzyme stably retained the activity and the substrate DNA was completely cleaved. On the other hand, EDTA
(Sample No. 2), various magnesium salts (Sample Nos. 6, 7, 8, 9), other metal salts (Sample No. 10,
The enzyme was inactivated and the substrate DNA was not cleaved in the sample added with 11, 12, 13, 14) and the sample not added (Sample No. 1).

Example 2 50 mM Tris-hydrochloric acid pH 7.5, 10 mM 2-
A final concentration of MgCl ₂ or CaCl ₂ of 10 was added to an enzyme composition consisting of mercaptoethanol and 2 units / μl of SdiI.
The enzyme activity was compared in the same manner as in Example 1 after the addition of mM so that the mixture was allowed to stand at 5 ° C. for 4 days. As a result, CaC
The sample to which l ₂ was added stably retained the activity and the substrate D
NA was completely cut. On the other hand, in the sample to which MgCl ₂ was added, the activity was decreased and the cleavage of the substrate DNA was incomplete. In the sample without addition, the enzyme is inactivated and the substrate DN
A was not cut.

Example 3 A stabilized enzyme composition having the composition shown in Table 3 below was prepared.

[0025]

Table 3 30 Units / μl SfiI 10 mM Tris-hydrochloric acid pH 7.5 1 mM DTT 0.1 mM EDTA 200 mM KCl 10 mM CaCl ₂ 0.15% (v / v) Triton X-100 50% (v / v) Glycerol The enzyme composition was stable at -20 ° C for at least 3 months.

[0026]

INDUSTRIAL APPLICABILITY The present invention provides a restriction enzyme composition containing a stabilizer useful for long-term stabilization of a restriction enzyme useful as a reagent for genetic engineering.

[0027]

[Sequence list]

SEQ ID NO: 1 Sequence length: 13 Sequence type: Nucleic acid Number of strands: Double-stranded Sequence features: N at the 5th to 9th positions are arbitrary nucleosides. Sequence: GGCCNNNNNG GCC 13

SEQ ID NO: 2 Sequence length: 13 Sequence type: Nucleic acid Number of strands: Double stranded Sequence characteristics: C at the 3rd position is m5c. The 5th to 9th Ns are arbitrary nucleosides. The 12th C is m5c. Sequence: GGCCNNNNNG GCC 13

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Fusao Kimizuka 3-4-1 Seta, Otsu City, Shiga Prefecture Central Research Institute, Minami Shuzo Co., Ltd. (72) Inventor Kazuo Nakajima 3-4-1 Seta, Otsu City, Shiga Prefecture No. Sake Brewing Co., Ltd. Central Research Institute

Claims (2)

[Claims] 1. A stabilized enzyme composition comprising a restriction enzyme, which contains a calcium salt. 2. The following formula (Formula 1) wherein the restriction enzyme is double-stranded DNA: (Wherein G represents guanosine, C represents cytidine, and N represents any nucleoside), which is a restriction enzyme capable of specifically recognizing the base sequence and having the ability to specifically cleave at the site indicated by the arrow. The enzyme composition according to claim 1.