JPS6317436B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an extremely simple method for purifying deoxyribonuclease from a mammalian pancreatic extract to a high degree of purity.

In general, deoxyribonuclease is a useful substance that is expected to be used not only in academic research related to nucleic acids but also as a reagent and in medicine. It is difficult to remove various contaminant proteins, especially trypsin, which is a proteolytic enzyme contained in large amounts. If trypsin is present in the mixture, attempts to purify deoxyribonuclease from pancreatic extracts using common purification methods such as ion-exchange chromatography will fail.
easily degraded by deoxyribonuclease,
As a result, the yield was significantly reduced.

In recent years, affinity chromatography has been developed as a means of enzyme purification, and there are examples of attempts to apply affinity chromatography to purify deoxyribonuclease from the pancreas. Namely, JCSchabort, Journal of
In the method described in Chromatography., Vol. 73, 253-256 (1972), deoxyribonucleic acid-immobilized agarose (hereinafter referred to as
Deoxyribonuclease is adsorbed using DNA-Sepharose, and then
This is a method of elution with 0.35M NaCl. Although this method has the advantage of specifically adsorbing and eluating deoxyribonuclease,
Since the pH for buffering Sepharose is approximately 7.5, which is the optimum pH for deoxyribonuclease,
This purification method had drawbacks such as difficulty in reusing DNA-Sepharose because the DNA of DNA-Sepharose was degraded as a substrate, and it was impossible to commercialize the purification method as it was.

Therefore, the present inventors conducted various studies in order to eliminate the above drawbacks while taking advantage of the advantages of the purification method by affinity chromatography.
When deoxyribonuclease was adsorbed onto DNA-Sepharose at pH 5.5, it was surprisingly found that the adsorption capacity was much higher in this pH range than at pH 7.5, and elution at ionic strengths below 0.3 was inhibited. DNA-Sepharose can be used as a purification method because it is not degraded and does not degrade using DNA as a substrate.
The present invention has been completed because it has been found that it can be used repeatedly.

That is, the present invention provides a solution containing a mammalian pancreas extract;
DNA-Sepharose under acidic conditions, preferably PH
4 to PH5 to detect the deoxyribonuclease in the mammalian pancreatic extract-containing solution.
After adsorbing to DNA-Sepharose, 0.3-1.0M
This is a method for purifying deoxyribonuclease, which is characterized by eluting the deoxyribonuclease from the DNA-Sepharose with potassium chloride.
Since the DNA-Sepharose is brought into contact with the mammalian pancreatic extract-containing solution at pH 5, the DNA-Sepharose after adsorption and elution of the deoxyribonuclease contained therein can be used repeatedly with only simple washing.

This DNA-Sepharose specifically adsorbs deoxyribonuclease.
Other enzymes contained in the pancreas, such as trypsin,
Since ribonuclease etc. are hardly adsorbed,
Only deoxyribonuclease can be easily isolated from pancreatic fluid.

In the present invention, DNA-Sepharose used
The adjustment was performed using the method of Arndt-jovin et al. (Donna J.
Arndtjovin et al European Journal of
Biochemistry 54 , 411 (1975)). Also, DNA-Sepharose is used after being buffered at pH 4 to 5. In this case, the buffer may be an acetate buffer, a citrate buffer, etc., but the action of deoxyribonuclease is suppressed as much as possible. It is preferable to use a citrate buffer, which is convenient for this purpose. Furthermore, any salt solution can be used as long as it has an ionic strength of 0.3 or more as the salt solution used to elute the deoxyribonuclease adsorbed on DNA-Sepharose.

Next, examples of the present invention will be described, and the enzyme activity measurement method used in the test examples and examples is as follows.

1 Deoxyribonuclease activity U.Lindberg's method [Biochimica et Biophysica Acta, Vol. 82,
237 (1964)], the deoxyribonucleic acid (DNA) decomposition activity was measured by the increase in absorbance at 260 nm.

2 Trypsin activity BCWHummel's method [Canadian Journal of Biochemistry and Biophysics]
P-toluenesulfonyl-L-arginine methyl ester (P-toluenesulfonyl-L-arginine methyl ester)
Degradation activity of L-arginine methlester)
Measured by increase in absorbance at 247 nm.

3 Ribonuclease activity MRMcDonald et al. method [Metbod in
Enzymology Vol. 2, 427-436 (1955)], the ribonucleic acid (RNA) separation activity was measured by the increase in absorbance at 260 nm.

Test example 1 100mM Britton- containing 0.2% neopeptone
Add 1 g of DNA-Sepharose (wet weight) buffered to pH 3-10 with 9 ml of Robinson's broad-spectrum buffer.
Add 0.5 ml of deoxyribonuclease (manufactured by Sigma) solution (1 mg/ml) at room temperature, and after stirring for 5 minutes, remove the deoxyribonuclease activity of the supernatant and the deoxyribonuclease activity adsorbed on DNA-Sepharose (however, 1M KCl will reduce the adsorption). The activity recovered in the extract after being eluted from the body was measured, and the results shown in Figure 1 were obtained.

As is clear from Figure 1, below PH5.5
The adsorption power of DNA-Sepharose and deoxyribonuclease is large, but at pH 6 or above,
It can be seen that because DNA is degraded by deoxyribonuclease, the adsorption power between the two is apparently reduced.

Example 1 1 kg of raw pig pancreas was minced, suspended in 1 water, left overnight at 4°C, adjusted to pH 4.0 with 6N-acetic acid,
The precipitate was removed by filtration to obtain 600 ml of an extract.
Meanwhile, 100 ml of pre-equilibrated with 50 mM citrate buffer (PH4.0) containing 0.2% neopeptone.
After passing 200 ml of the above extract through a DNA-Sepharose column to adsorb deoxyribonuclease, the same buffer solution is passed through the column to remove proteins other than deoxyribonuclease, such as trypsin and ribonuclease contained in the extract. After washing, the deoxyribonuclease adsorbed on the DNA-Sepharose column is eluted using 0.3M and 1.0M potassium chloride.
The eluate contained 3 million units of deoxyribonuclease, and the specific activity increased approximately 110 times. The recovery rate of this product from the porcine pancreas extract was 94%, and it contained almost no trypsin or ribonuclease activity.

The elution pattern here is shown in FIG.

Example 2 200g of defatted pork pancreas powder was suspended in 2 water,
After adjusting the pH to 4.0 with 6N-acetic acid and extracting at 4°C for 3 hours,
The extraction residue was removed by filtration to obtain extract 2. Meanwhile, pre-contains 0.2% neopeptone
600 ml of the above extract was passed through a 100 ml DNA-Sepharose column equilibrated in advance with 50 mM citrate buffer (PH4) to adsorb deoxyribonuclease, and then the same buffer was passed through it to remove the DNase contained in the extract. After washing out trypsin, which is a protein other than deoxyribonuclease,
Deoxyribonuclease is eluted from the adsorbent with 1.0 M potassium chloride solution. In the elution section, 94% of the deoxyribonuclease in the extract was recovered, with a totality of 3.5 million units.
Furthermore, the DNA-Sepharose column was washed with 50mM citrate buffer (PH4.0) containing 0.2% neopeptone, and then the extract from the defatted pig pancreas powder was
Pour 600 ml in the same way as before to adsorb deoxyribonuclease, then pour the same buffer solution,
After washing out contaminant proteins such as trypsin,
Deoxyribonuclease is eluted from the column with 1.0M potassium chloride solution. Deoxyribonuclease was recovered in this eluate with a yield of 95%, and the totality was 3.6 million units.

This allows repeated columns to be used.

The elution pattern here is shown in FIG.

[Brief explanation of the drawing]

Figure 1 shows the results at each PH in Test Example 1.
FIG. 2 is a diagram showing the elution pattern in Example 1, and FIG. 3 is a diagram showing the elution pattern in Example 2. FIG. A...Supernatant liquid, B...Eluate with 1M KCl, C
...Trypsin, D...RNase, E...Deoxyribonuclease, F...Porcine pancreas extract.

Claims (1)

[Claims] 1. A solution containing a mammalian pancreatic extract and deoxyribonucleic acid-immobilized agarose are heated under acidic conditions, preferably about
1. A method for purifying deoxyribonuclease, which comprises adsorbing deoxyribonuclease by contacting under conditions of pH 4 to 5, and then eluting deoxyribonuclease by increasing ionic strength. 2. The method according to claim 1, wherein the adsorption of deoxyribonuclease is carried out in the presence of a citrate buffer.