JPH038196B2 - - Google Patents

Description

[Detailed Description of the Invention] [Background of the Invention] Technical Field The present invention relates to protein arginine residue deiminase (hereinafter referred to as PADIase = peptidylarginine deiminase).
Concerning the purification method of deiminase). In other words, the present invention relates to a method for producing purified PADIase. PADIase is a soluble enzyme that has been widely confirmed to exist in various animal tissues, such as the brain and skeletal muscle, and it specifically acts on arginine residues in peptide chains, converting them into citrulline through a deimination reaction. It is a useful protein-modifying enzyme that has the activity of converting into residues. Regarding the enzymatic properties of PADIase, the one derived from rabbit skeletal muscle was reported by the present inventor [J.Biochem. 94, 1945 (1983)], and the one derived from bovine brain was reported by Kubirus ( Joseph
(Biochimica et Biophysica acta) by Kubilus et al.
Acta) 745 (1983), 285~] Each is reported in detail. Prior Art Several proposals have already been made regarding methods for purifying PADIase. In the above report, Kubirus et al. reported that PADIase derived from bovine brain was subjected to five-step column chromatography (DEAE-cellulose,
DEAE-cephadex, hydroxyapatite, arginine-agarose-lysine-agarose) is used to purify the product until a single band is obtained on SDS-PAGE, but the yield is only 4% at most, and the enzyme specific activity [protein The concentration of enzyme activity per unit weight (the same applies hereinafter) was 1100 times.
Furthermore, Gubils et al.
A method for purifying PADIase by three-step column chromatography (DEAE-cellulose, SP-cephadex, arginine agarose) was also reported [The Journal of Investigative Dermatology].
Investigative Dermatology) 85:232-234
(1985)]. The yield of this method was 26% and the enrichment of enzyme specific activity was 472 times. On the other hand, the present inventors previously reported that
A purification method for PADIase that basically consists of six steps was reported [J.Biochem. 94, 1945-1953 (1983)], but the yield of this method was 16%. , the enrichment degree of enzyme specific activity was 879 times. In addition, this purification method employs affinity chromatography using soybean trypsin inhibitor as a ligand, and in this step, PADIase bound to the ligand is eluted with an aqueous sodium hydroxide solution. , by this affinity chromatography
The yield of PADIase was 49%, and the concentration of specific enzyme activity was 3.8 times. As mentioned above, various methods have been proposed as PADIase purification methods, but all of them require complicated operations and have low yields, and so far no satisfactory purification method has been proposed. [Summary of the Invention] Summary The present inventors have conducted various studies aimed at solving the above problems and providing a relatively simple and high-yield method for purifying PADIase. The present invention was completed by discovering that it is effective to adsorb and elute peptides or proteins under specific conditions. That is, the method for purifying PADIase according to the present invention is as follows:
The protein arginine residue deiminase in the solution is adsorbed in the presence of calcium ions to an adsorbent made of trypsin inhibitor supported on an insoluble carrier, and after washing this adsorbent with a washing solution containing calcium ions, calcium This method is characterized in that protein arginine residue deiminase is eluted from this adsorbent using an aqueous solution of a chelating agent that has the ability to form a chelate with ions. Effects of the Invention According to the purification method of the present invention, PADIase in a solution can be concentrated in a yield of 60% or more and the enzyme specific activity can be concentrated by 20 times or more in a single step treatment. moreover,
As one of the preferred embodiments of the present invention, if the purification method of the present invention is used in combination with ion exchange chromatography, PADIase can be purified from animal tissue extracts very efficiently ( Yield: 50% or more, Enzyme specific activity enrichment rate: 1790 times, the obtained PADIase was subjected to SDS-PAGE
(see Example 2). The fact that the present invention was able to obtain high yields and high enrichment rates in one step was unexpected in view of conventional purification methods that required multiple steps. The uniqueness of the elution with the calcium chelating agent of the present invention is unexpected when compared with the yield and concentration, especially the latter, when sodium hydroxide is used as the eluent in a unit purification step consisting of affinity chromatography. You could say that it happened. [Detailed Description of the Invention] Adsorbent Generally speaking, the peptide or protein used as the adsorption group of the adsorbent in the present invention has an arginine residue protruding from its molecular surface. Examples of such peptides or proteins include trypsin inhibitors having an arginine residue as an active site residue; a specific example is soybean trypsin inhibitor (STI).
Inhibitor), onion trypsin inhibitor, bovine pancreatic secretory trypsin inhibitor, and polypeptides showing homology thereto. In the present invention, these peptides or proteins are supported on an insoluble carrier, that is, immobilized, and used. As the immobilization method, any suitable known method can be used. Specifically, insoluble carriers include agarose, dextran, cellulose, polyacrylamide, etc.; immobilization involves covalent bonding using amino groups, carboxyl groups, etc. as functional groups, and physical containment of the insoluble carrier in a matrix. etc. The adsorbent made of an insoluble carrier on which peptides or proteins are immobilized (supported) in this manner is usually prepared in the form of granules that are easy to handle during purification operations. PADIase The PADIase to be purified in the present invention is not particularly limited, and any substance that can adsorb to the peptide or protein used as an adsorption group in the present invention can be used. Specific examples of such PADIases include enzymes (derived from rabbit skeletal muscle, bovine brain, bovine epithelium, etc.) reported in the above-mentioned literature. In the present invention, "PADIase" refers to an enzyme having the activity of acting on an arginine residue in a protein or peptide molecule and converting it into a silthrin residue. Purification of PADIase In the present invention, an aqueous PADIase solution is the target of purification, but when purifying PADIase in animal tissue, PADIase is extracted in advance by subjecting the tissue to a treatment such as homogenizing it in an aqueous solution. After that, the extract may be purified according to the present invention.
Furthermore, when purifying PADIase to a high purity, it is effective to purify PADIase in a solution in advance by anion exchange chromatography (prepurification) and then purify it by the method of the present invention. In this anion exchange chromatography for preliminary purification, it is preferable to use anion exchangers having DEAE groups (diethylaminoethyl), AH groups (aminohexyl), etc. as anion exchange groups, and Preferably PADIase in
It is adsorbed onto these anion exchangers at pH 6 to 9, washed with an appropriate washing solution, and then washed with a salt solution such as sodium chloride or calcium chloride.
All you have to do is elute PADIase. A specific embodiment of this anion exchange chromatography is also reported in Journal of Biochemistry (J. Biochm.) 94, 1945 (1983). In order to purify an aqueous solution of PADIase according to the present invention, the aqueous solution is first brought into contact with the adsorbent in the presence of calcium ions, and the PADIase in the aqueous solution is
is adsorbed onto the peptide or protein as an adsorption group on the adsorbent. The concentration of calcium ions coexisting is preferably 0.2 mM or more, particularly 0.6 mM or more. As a source of calcium ions,
Water soluble calcium compounds such as calcium chloride, etc. In addition, in order to suppress denaturation or decomposition of PADIase during purification, if necessary,
Reducing agents such as DTT and 2-mercaptoethanol,
It is preferable to coexist with protease inhibitors and the like. Furthermore, this adsorption step is not carried out in the presence of only calcium ions, and other metal ions, such as alkali metal ions, may coexist as long as they do not inhibit the effects of calcium ions. Next, the adsorbent that has adsorbed PADIase is washed with a washing solution containing calcium ions. This washing solution preferably contains 0.2mM or more of calcium ions and is capable of removing substances nonspecifically adsorbed or attached to the adsorbent without eluting PADIase. Apart from the calcium ion source (see above for specific examples), examples of the source include aqueous solutions of sodium chloride, potassium chloride, and the like. This cleaning solution contains calcium ions and therefore may be an aqueous solution of a calcium source (eg calcium chloride) itself. Thereafter, the PADIase adsorbed on the adsorbent is eluted using an aqueous solution of a chelating agent that has the ability to form a chelate with calcium ions. Chelating agents that can be used for this purpose include ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), ethylenediaminetetraacetic acid (EDTA) , citric acid, etc. Further, there is no particular restriction on the concentration of the chelating agent, but it is usually used at a concentration of several mM or more. Furthermore, the aqueous solution of the chelating agent may contain hydrochloric acid such as sodium chloride, potassium chloride, etc., if necessary. The pH of the PADIase solution, washing solution, and aqueous chelating agent solution (eluent) used for purification in the present invention can be appropriately selected as long as it does not deactivate the enzyme, but it should be in the range of 6 to 9 in particular from the viewpoint of enzyme recovery rate. is desirable. Further, the purification operation temperature can be appropriately selected as long as it does not deactivate PADIase, but in order to use the adsorbent repeatedly, it is preferable to operate at a low temperature, particularly 5° C. or lower. There are two possible embodiments of the purification method of the present invention: a batch method and a column chromatography (=affinity chromatography) method, and PADIase can be purified by either method. Since these specific operating procedures themselves may be those commonly used in the field of protein purification, procedures other than those specifically mentioned in the present invention may be carried out according to these commonly used techniques. Experimental Example In the following experimental example, the activity of PADIase was measured using the Journal of Biochemistry (J.
Biochm.), 94 1945 (1983) was used. Reference example (sample preparation) Fresh rabbit skeletal muscle was mixed with 5 times the volume of buffer A (20
The homogenate was homogenized in a homogenizer (mM Tris-HCl PH7.6, 10mM 2-mercaptoethanol, 1mM MEDTA, 0.43mM phenylmethylsulfonyl fluoride), the resulting homogenate was filtered through a nylon gauze, and the furnace solution was heated at 10,000 x g. Centrifuged for 30 minutes. This supernatant was added to "DEAE-Sefacel" which had been equilibrated with buffer A containing 0.1M NaCl.
(manufactured by Pharmacia) column. After washing the column with this buffer until the A280nm of the effluent was 0.05 or less, PADIase was eluted with a linear concentration gradient of NaCl in buffer A (0.1→0.5M). The obtained active fractions were pooled and an equal volume of saturated ammonium sulfate aqueous solution was added to this.
The PADIase was precipitated and stored in suspension in 50% saturated aqueous ammonium sulfate. Example 1 PADIase (approximately 470U,
25U/mg protein) was collected by centrifugation and
Buffer _B containing mMCaCl2 (20mM Tris-HCl (PH: 7.6), 10% glycerol, 0.1M NaCl, 10mM
"STI-Sepharose" dissolved in M2-mercaptoethanol, 10mM MDTT), dialyzed against a large amount of the same buffer, and equilibrated in advance with the same buffer.
Column (1.0 x 12cm) [Journal of Biological Chemistry (j.Biol.Chem.), 245 ,
3059 (1970)]. Next, after washing this column with the same buffer, buffer B containing 10mMEGTA was added.
PADIase was eluted from the column. The elution pattern of protein and enzyme activity at this time is
It was as shown in the attached drawing. In addition, the proteins are Read and Northcote (Read & Northcote).
Northcote method [Analytical Biochem., 166 , 53 (1981)]. The specific activity of PADIase obtained by this operation was 560 U/mg protein, the concentration was 22.4 times, and the yield was 60%. Furthermore, the obtained PADIase showed a single band (electrophoretically homogeneous) on SDS-PAGE. Example 2 Using 3.0 kg of fresh rabbit skeletal muscle, PADIase was purified under the same conditions as in Reference Example and Example 1. The purification results at this time were as shown in the table below. In addition, obtained at this time
PADIase also showed a single band on SDS-PAGE. 【table】

[Brief explanation of the drawing]

The figure is a graph showing the elution pattern of protein and enzyme activity in Example 1.

Claims (1)

[Claims] 1. Protein arginine residue deiminase in a solution is adsorbed in the presence of calcium ions on an adsorbent made of trypsin inhibitor supported on an insoluble carrier, and after washing this adsorbent with a washing solution containing calcium ions, 1. A method for purifying protein arginine residue deiminase, which comprises eluting the protein arginine residue deiminase from this adsorbent using an aqueous solution of a chelating agent having the ability to form a chelate with calcium ions.