JP4025867B2 - Protein elution method by isoelectric focusing. - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a technique for elution and recovery of proteins, particularly enzymes that are susceptible to function depending on pH environment, in a pH stable region under pH control using a pH gradient by isoelectric focusing.
[0002]
[Prior art]
When a voltage is applied to a molecule such as a protein present in a liquid, it has a positive or negative charge depending on the nature and shape of the molecule, the positively charged molecule is in the cathode direction, and the negatively charged molecule is There is a phenomenon of movement in the anode direction. This phenomenon is so-called electrophoresis, and the sample is held on a support such as an acrylamide gel or agarose gel containing a buffer solution, and the molecules are separated while receiving the action of an electric field. That is, required separation is performed based on the difference in mobility of components in the sample. Various apparatuses have been proposed for this purpose.
For example, FIG. 7 is a schematic diagram showing a configuration of a conventional electrophoresis elution apparatus used for protein elution.
In the figure, reference numeral 1 denotes a migration tank filled with a buffer solution, which comprises a cathode tank 2 having a cathode 2a and an anode tank 3 having an anode 3a. A plurality of communication tubes 4, 4, 4 are installed between the cathode cell 2 and the anode cell 3, and flow between the cathode cell 2 and the anode cell 3 by these communication tubes 4, 4, 4. A road is formed.
[0003]
Then, a sample is set in the communication tube 4, and the target protein is eluted from the sample by energizing between the cathode 2a and the anode 3a. That is, in FIG. 7, 5 is a gel containing a protein as a sample locked to a mounting net 6 installed in the communication tube 4, and 7 is a communication tube eluted from the gel 5 as a sample in a buffer solution. 4 is a conductive film such as a cellophane film stretched at the open end of the communication tube 4 on the anode tank 3 side in order to capture and collect the protein moving in the tube 4. In the figure, arrows indicate current.
[0004]
When a voltage is applied between the cathode 2a and the anode 3a, the protein 8 is eluted from the gel 5 and moves in the communicating tube 4 in the anode direction, abuts against the cellophane film 7 and is blocked and concentrated in the vicinity thereof. While the protein in the sample is eluted and migrates, it is diffused and diluted in the buffer solution, so it is recovered using a high salt concentration solution or the like.
[0005]
[Problems to be solved by the invention]
By the way, proteins having functions such as enzymes are influenced by the surrounding pH environment. That is, the enzyme has an optimum pH range where the function can be expressed, and cannot function outside the optimum pH range. In addition, pH stability is one of the characteristics of enzymes, and the pH range in which enzymes can maintain their functions is also unique to each enzyme.
The enzyme may lose its function when exposed to a pH environment deviating from the pH stable region.
Therefore, in order to recover the enzyme without losing its function, it is necessary to maintain the pH at the time of recovery within the pH stable region of the enzyme. However, in the above-described prior art, the pH of the buffer solution that fills the electrophoresis tank cannot be set freely, so that an enzyme that is unstable under the pH of the buffer solution for migration (pH 8.0-pH 9.0) It was difficult to recover while maintaining the activity.
[0006]
[Means for Solving the Problems]
The present invention is
(A) A pH gradient created by a pI marker can be visually recognized on an isoelectric focusing gel provided with cathode and anode electrodes at both ends, and holes are formed at predetermined positions based on the visible pH gradient. Forming step,
(B) filling the pores with an ion exchanger equilibrated with a buffer for capturing the protein;
(C) a step of bringing a sample containing the protein to be eluted into or in contact with the adsorbent,
(D) An electric field is applied to the gel, and the pH value of the ion exchanger is changed to a pH value corresponding to the pH gradient on the gel corresponding to the position of the pores to elute the protein in the sample and Adsorbing to the ion exchanger;
After stirred with buffer pH compatible with the protein to the ion exchanger having a pH value to adapt to the protein of (e) recovering expected, proteins were eluted by filtration, the eluate was desalted and concentrated A step of recovering the protein.
An object of the present invention is to provide a protein elution method by isoelectric focusing comprising the above steps to solve the above conventional problems.
[0007]
In the above, the gel for isoelectric focusing may be constituted by adding an amphoteric carrier.
[0008]
Moreover, in the protein elution method according to any of the above isoelectric focusing methods, the sample containing protein may be constituted by a section of a band containing a single protein purified on a gel by electrophoresis.
[0009]
Further, in the protein elution method according to any one of the isoelectric focusing methods described above, the pores are rectangular and the side length in the migration direction is set to be less than 1/10 of the migration direction length of the gel. In some cases, it is possible to construct an appropriate pH gradient.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The gist of the present invention resides in that a protein having a function such as an enzyme that is easily influenced by the pH environment is recovered without impairing the function of the enzyme by controlling and maintaining the pH environment at the time of recovery in the pH stable region of the enzyme. The pH is controlled using a pH gradient formed by isoelectric focusing.
[0011]
First, a sample containing a protein is added to a gel for electrophoresis, and electrophoresis is performed with an electrophoresis apparatus schematically shown in FIG. 1 to purify it into a band containing a single protein.
In FIG. 1, reference numeral 11 is a support plate for the gel 12 and also serves as a cooling means during voltage application. Reference numerals 13 and 13 are filter papers installed at both ends of the gel 12 and impregnated with an electrode solution as an electrical interface. 14 and 14 are conductive wires connecting the filter papers 13 and 13 and the electrodes. The arrow indicates the current direction.
A small piece of filter paper is placed on the gel-containing sample at an appropriate location near the cathode side on the gel, and the sample solution is added to the small piece. The gel is then energized to generate electrophoresis and each protein is separated into a single band.
[0012]
The separated sample is then collected as a section from the gel. The subsequent procedure will be described with reference to FIG. FIG. 2 is a plan view of the electrophoresis surface of the electrophoresis apparatus shown in FIG. 1, and the gel 12 is an isoelectric focusing gel to which an amphoteric carrier is added. As shown in FIG. By applying an electric field, a predetermined range of pH gradient is formed on the gel. Now, on the surface of the gel 12, a rectangular hole 21 is formed at a predetermined position corresponding to the required pH as shown in FIG.
In addition, if the length in the migration direction (horizontal direction in the figure) is excessive in the hole portion 21, the pH gradient is affected. Therefore, the length is preferably 1 / gel length (migration direction). Set to less than 10.
[0013]
Next, an ion exchanger 22 equilibrated with a buffer solution is prepared as a protein adsorbent, and filled in the holes 21. The ion exchanger 22 is controlled to have a pH corresponding to the position of the hole 21 on the pH gradient produced on the gel 12 when an electric field is applied. Therefore, it is desirable to use a buffer solution having a pH close to that in the equilibration treatment with the buffer solution. As an ion exchanger as an adsorbent, well-known ones such as ion exchange resin, ion exchange cellulose, ion exchange sepharose, ion exchange sephadex can be used. Adoption of suitable ion exchange cellulose, ion exchange sepharose, etc. is desirable.
There are anion exchangers and cation exchangers, and the former adsorbs a protein with a pI (pH) lower than the equilibrated pH, while the latter conversely equilibrated pH. Adsorb higher pI (pH) proteins. From this point of view, the adsorbent is selected appropriately in accordance with the properties of the object.
[0014]
Next, the sample 23 is placed and added into the ion exchanger 22 filled in the hole 21 (FIG. 2C). The position of the sample 23 is not limited to the inside of the ion exchanger 22 as long as there is no problem in recovery. In the above state, a voltage is applied under conditions suitable for the gel 12, and the protein 24 is eluted and adsorbed onto the ion exchanger 22. The adsorbed protein is recovered by a usual method such as forming a solution and desalting and concentrating.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Next, a demonstration test of pH control of an ion exchanger, a demonstration test such as protein adsorption, and one embodiment of the present invention based on these will be described.
Experiment 1 (see FIG. 3 and FIG. 4)
Electrophoresis device used: Multiphor II Electrophoresis System (Pharmacia)
Gel: Ampholine PAG Plate pH3.5-9.5 (Pharmacia)
Ion exchanger: DEAE Sepharose Fast flow (trademark of Pharmacia)
pI marker: Broad pI Kit, pH 3.5-9.3 (trademark of Pharmacia)
1. Insulating kerosene was dropped on the cooling plate of the apparatus, and the gel was placed with the support film.
2. 1M glycine solution was impregnated into the filter paper for the cathode, and 1M phosphoric acid solution was impregnated into the filter paper for the anode, and set on both ends of the gel.
3. As shown in FIG. 3, rectangular holes 21 (length: 7 mm × width: 1 cm) were formed at five locations where the gel 12 was roughly divided into six portions between the two electrodes.
4. The hole 21 was filled with the ion exchanger without any gap.
The ion exchanger was equilibrated with 10 mM acetate buffer pH 5.5 and allowed to settle spontaneously, and adjusted to a slightly loose paste form except for the supernatant.
5. The pI markers 25 and 25 were added to the positions shown on the gel 12 using filter paper for visual confirmation of the pH gradient created in the gel.
6. It was energized for 1 hour under conditions suitable for the gel 12 used (1400V, 25mA, 35W).
7. After the electrophoresis, the ion exchangers in each hole 21 were collected and their pH values were measured with a pH meter.
As a result, as shown in FIG. 4, the pH value of the ion exchanger filled in the hole 21 (the white line encircled portion in the figure) changes according to the distance from the cathode, and is almost the same as the pH value indicated by the pI marker. Match. Therefore, if the pH value at each position on the gel is known in advance using the pI marker, it is confirmed that the ion exchanger pH can be set, that is, the pH can be controlled by the position where the pores 21 are formed.
[0016]
Experiment 2 (see Fig. 5)
The same gel, ion exchanger and other reagents and apparatus as those used in Experiment 1 were used.
1. The steps from installing the gel to forming the hole 21 and filling the ion exchanger are the same as in Experiment 1.
2. A filter paper of 5 mm × 5 mm was set at a position 1.5 cm from the cathode, and a pI marker was added thereto.
3. A 5 mm × 5 mm filter paper was set on the ion exchanger, and a pI marker was added thereto.
4). In the above state, electrophoresis was performed for 40 minutes under the same conditions as in Experiment 1.
5). The ion exchanger was removed from the gel, and the gel was stained with Kumasi Blue R250 staining solution.
In the above, with the pI marker added at a position where there is no ion exchanger, all contained protein bands were detected. On the other hand, in the pI marker added on the ion exchanger, all the protein bands on the cathode side were detected from the positions of the holes not adsorbed on the ion exchanger, but the anode side adsorbed on the ion exchanger was detected. In the protein, no band was formed in the gel. Usually, anion exchangers adsorb proteins with a pI (isoelectric point) lower than the equilibrated pH. Also in the experiment, it was confirmed that a protein having a pI below the pH at which the ion exchanger was equilibrated by the pH produced in the gel was adsorbed.
[0017]
Example 1 (see FIG. 6)
1. A sample containing four partially purified manganese peroxidase isozymes was separated into four bands by isoelectric focusing, and each band was cut from the gel to obtain a gel slice.
2. As in Experiments 1 and 2, a gel was placed in the apparatus, four holes were formed at a position where the pH was 5.5 to 6.0, and ion exchangers were filled. The gel slices were placed in each of these ion exchangers. Electrophoretic elution was performed in the same manner as in Experiment 2 above.
3. After the electrophoresis, the ion exchanger was recovered from each hole.
4). To the collected ion exchanger, 2 ml of 10 mM acetate buffer pH 5.5 containing 0.5 M sodium chloride was added, and after stirring, the protein was eluted by filtration. The eluate was desalted and concentrated to 5 μl.
As described above, as shown in FIG. 6, each protein was detected as a single band at the same position as originally purified from the result of isoelectric focusing of the collected protein.
Therefore, it can be confirmed that all four manganese peroxidase isozymes were eluted and adsorbed on each ion exchange resin from the gel slice. Therefore, it was confirmed that the protein can be eluted and collected from the gel slice of the electrophoresis sample.
[0018]
【The invention's effect】
As described above, the present invention elutes a protein in an ion exchanger whose pH is controlled via a pH gradient formed by isoelectric focusing, so that the functions and characteristics of proteins, particularly enzymes, etc. These elutions can be recovered without impairing the process.
[Brief description of the drawings]
FIG. 1 is a schematic configuration diagram of an electrophoresis apparatus according to an embodiment.
FIG. 2 is a plan view of a migration plane in the apparatus shown in FIG.
FIG. 3 is a plan view showing holes formed on the gel of the electrophoresis apparatus.
4 is a gel plan view showing the correlation between the measured pH of the ion exchanger and the distance from the cathode in each hole after electrophoresis in the setting state of FIG. 3. FIG.
FIG. 5 is a plan view showing the result of electroelution of a pI marker on an isoelectric focusing gel.
FIG. 6 is a gel plan view showing a protein recovery result according to the first example.
FIG. 7 is an explanatory diagram of a conventional technique.

Claims (4)

A protein elution method by isoelectric focusing comprising the following steps.
(A) A pH gradient created by a pI marker can be visually recognized on an isoelectric focusing gel provided with cathode and anode electrodes at both ends, and holes are formed at predetermined positions based on the visible pH gradient. Forming step,
(B) filling the pores with an ion exchanger equilibrated with a buffer for capturing the protein;
(C) a step of bringing a sample containing the protein to be eluted into or in contact with the adsorbent,
(D) An electric field is applied to the gel, and the pH value of the ion exchanger is changed to a pH value corresponding to the pH gradient on the gel corresponding to the position of the pores to elute the protein in the sample and Adsorbing to the ion exchanger;
(E) to the ion exchanger that is adapted to have a pH value adapted to the protein expected to be collected, after stirred with buffer at about the same p H value and pH value of the ion exchanger, filtered to A step of eluting the protein and desalting and concentrating the eluate to recover the protein. The protein elution method according to claim 1, wherein the gel for isoelectric focusing is an amphoteric carrier added thereto. . The protein elution method according to claim 1 or 2, wherein the protein-containing sample is a section of a band containing a single protein purified on a gel by electrophoresis. Protein elution method by isoelectric focusing. The protein elution method according to any one of claims 1 to 3, wherein the hole is rectangular, and the side length in the migration direction is the length of the gel in the migration direction. 1/10 Set to less than in the gel pH A protein elution method by isoelectric focusing, wherein an appropriate gradient can be prepared.

Images