JP4008177B2 - Protein purification method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a protease removal method. More specifically, the invention of this application relates to a method for removing a host-derived protease that degrades a target protein when the protein is expressed in a large amount.
[0002]
[Prior art and its problems]
In general, proteins are obtained by isolating them from human organs, cell lines, etc., preparing peptides by chemical synthesis based on specific amino acid sequences, or recombining using specific DNA fragments. A method obtained by DNA technology is used. In particular, the recombinant DNA technology is useful as a method for industrially synthesizing a large amount of a uniform protein or for synthesizing a fusion protein that does not exist in nature.
[0003]
In recombinant DNA technology, an expression vector having a DNA fragment that synthesizes a target protein is introduced into a host such as Escherichia coli, Bacillus subtilis, yeast, insect cells, mammalian cells, or plant cells to express the target protein in large quantities. be able to.
[0004]
However, E. coli, Bacillus subtilis, and microorganisms such as yeast, animal cells, plant cells, owns various proteolytic enzymes (proteases) in cells thereof, the proteins expressed by recombinant technology and cell disruption During and after extraction, since the protein in which these proteases are expressed is degraded, it is necessary to suppress the activity.
[0005]
The most common method for inhibiting protease activity is to add various protease inhibitors. However, depending on the type of protease inhibitor, it may not be possible to completely inhibit the activity of the protease, and even if the target protein is separated and purified, the degradation of the protein often progressed over time. It is. Furthermore, it has been pointed out that the addition of a protease inhibitor makes purification difficult.
[0006]
Therefore, the invention of this application has been made in view of the circumstances as described above, solves the problems of the prior art, and effectively and simply eliminates the target protein and effectively and easily reduces the host protein mass expression system. It aims at providing the method of removing protease from.
[0007]
[Means for Solving the Problems]
Therefore, the invention of this application provides the following inventions.
[0008]
First of all, the invention of this application relates to a protease removal method characterized in that a host in which a large amount of protein is expressed is crushed in a crushing solution containing at least urea and then purified by ion exchange chromatography. provide.
[0009]
The invention of this application is that, secondly, the crushing method is physical crushing, third, the physical crushing is ultrasonic crushing, and fourth, the concentration of urea. The fifth aspect of the present invention provides an aspect of the method for removing proteases, wherein the ion exchange chromatography is a combination of a cation exchange column and an anion exchange column.
[0010]
Further, the invention of this application, the sixth, the protein mass expression host, it is a bacterium, the seventh, provides that the bacteria is E. coli as aspects thereof.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
The protease removal method of the invention of this application is effective and convenient for purifying a protease derived from a host often problematic when purifying a protein expressed in large quantities by a general genetic engineering technique. It is intended to be removed.
[0012]
The procedure is as follows. First, a disruption solution containing at least urea is added to a host in which a large amount of the target protein is expressed, the cell membrane of the host is disrupted therein, and then the target protein and the substrate are separated by ion exchange chromatography. Isolate the protease.
[0013]
At this time, various hosts generally used in genetic engineering techniques can be applied as a host used as a protein mass expression system. Specific examples include Escherichia coli, Bacillus subtilis, prokaryotic cells, yeast, insect cells, mammalian cells, plant cells, and the like. These are selected according to the structure, properties, use, etc. of the target protein.
[0014]
In order to extract a protein expressed in a large amount from a host, the genetic engineering technique usually involves crushing the host. In the protease removal method of the invention of this application, first, a crushing solution is added. The crushing solution only needs to contain at least urea, and the urea concentration is not particularly limited, but is preferably 6 M or more. Components other than urea are not particularly limited, but may be a buffer whose pH is adjusted according to the properties of the target protein and purification conditions.
[0015]
As a disruption method, various methods used in general gene recombination operations can be applied. Examples include physical disruption methods such as homogenizers, French presses, and ultrasonic waves, and cell membrane lysis with a surfactant. In particular, there is no fear of complicating the purification process by adding chemicals and the like, and simple and highly effective ultrasonic crushing is preferable.
[0016]
Protease is separated and removed from the target protein extracted by crushing the microbial cells as it is, by ion exchange chromatography. The ion exchange chromatography is selected from a wide variety of commonly used ones, and it is particularly effective and preferable to carry out by connecting a cation exchange column and an anion exchange column. In this case, conditions such as column order, type, size, capacity, solution flow rate, and flow rate can be appropriately changed depending on the properties of the target protein and the protease. Preferably, the cation exchange column is first and then the anion exchange column is connected.
[0017]
Since the protease is adsorbed on the cation exchange column, the protease is removed from the extract by passing the extract. On the other hand, the target protein is adsorbed on the anion exchange column. Impurities such as urea that do not adsorb on the cation or anion exchange column flow out and are removed.
[0018]
In the protease removal method of the invention of this application, finally, only the target protein can be recovered by separating the anion exchange column from the cation exchange column and washing the column with an appropriate solvent.
[0019]
The following examples further illustrate the invention of this application in more detail. Of course, the invention of this application is not limited to these examples, and it goes without saying that various modes can be considered.
[0020]
【Example】
In the following Examples and Comparative Examples, protease X refers to a host-derived protease using FlgF as a substrate.
[0021]
Example 1 Effect of Inhibitor and Urea on Protease X Activity FlgF (Salmonella typhimurium flagellar protein), which is one of Salmonella flagellar proteins, is produced in large amounts by the usual genetic engineering technique using Escherichia coli BL21 (DE3) pLysS as a host. Expressed. 8M urea was added, and the cell membrane of E. coli was disrupted by ultrasound, and at the same time, the protease was denatured.
[0022]
This solution was centrifuged to separate the soluble fraction.
[0023]
The obtained soluble fraction was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the presence of FlgF.
[0024]
Comparative Examples 1-6
A TE solution (Comparative Example 1) in which 50 mM Tris-HCl (pH 8.0) and 1 mM EDTA were mixed with E. coli BL21 (DE3) pLysS in which protein was expressed in a large amount in the same manner as in Example 1, A solution containing 2 μg / ml of pepstatin A (Comparative Example 2), a solution containing 2 μg / ml of leupeptin in the TE solution (Comparative Example 3), and 4- (2-aminoethyl) benzenesulfonylfluoride · HCl ( p-ABSF) with 2 mM added (Comparative Example 4), TE solution with 2 mM L-1-tosylamide-2-phenylethylchloromethylketone (TPCK) added (Comparative Example 5), and TE solution with Boehringer Mannheim Using a solution (Comparative Example 6) containing 1 protease inhibitor cocktail tablet / 50 ml manufactured by KK In the same manner as in Example 1, the mixture was sonicated and subjected to centrifugation to obtain a soluble fraction. The components contained in each soluble fraction were analyzed by SDS-PAGE to confirm the presence of FlgF and degraded FlgF.
[0025]
FIG. 1 is an SDS-PAGE profile in Example 1 and Comparative Examples 1-6. From this, the inhibitory effect on protease X can be compared. According to this, in Comparative Examples 1 to 6, it is clear that the protease-degraded FlgF is present in the soluble fraction, but it was crushed and protease-modified in the solution of Example 1 to which urea was added. In the system, no degraded FlgF was seen.
[0026]
From this, it was shown that urea is effective in inhibiting the activity of protease.
[0027]
Example 2, 3 Stability of FlgF after removal of protease As in Example 1, E. coli BL21 (DE3) pLysS in which a large amount of FlgF (Salmonella typhimurium flagellar protein) was expressed was disrupted by ultrasonication in an 8 M urea solution. After that, a soluble fraction was obtained through centrifugation.
[0028]
An ion exchange column connected with HiTrapSP (cation exchange column) and HiTrapQ (anion exchange column) was equilibrated with Tris-HCl (pH 8.0), and the soluble fraction was passed through. After further washing with Tris-HCl (pH 8.0) and removing the cation exchange column, FlgF adsorbed on the anion exchange column was eluted. The FlgF fraction (Example 2) and the one after the FlgF fraction was allowed to stand at 37 ° C. for 1 hour (Example 3) were each subjected to SDS-PAGE to analyze the contained components.
[0029]
Comparative Examples 7-8
In the same manner as in Example 2, the FlgF solution before ion exchange chromatography (Comparative Example 7), protease X eluted from the cation exchange column was added again to the separated FlgF, and the solution was allowed to stand at 37 ° C. for 1 hour ( Comparative Example 8) was subjected to SDS-PAGE, and the components contained therein were analyzed.
[0030]
From the above, it was revealed that immediately after ion-exchange chromatography, only FlgF was present in the soluble fraction, and FlgF was stably present without decomposition even after 1 hour at 37 ° C. On the other hand, in the sample in which only the protease X was added to FlgF after separation and purification, two spots derived from each component appeared clearly.
[0031]
From this, it was shown that the protease can be completely removed from the target protein by the protease removal method of the invention of this application.
[0032]
【The invention's effect】
As described above in detail, the present invention provides a method for removing protease from a host protein mass expression system effectively and simply without degrading the target protein.
[Brief description of the drawings]
FIG. 1 is a diagram showing the results of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of a FlgF solution when various protease inhibitors and urea are added in Examples of the invention of this application.
FIG. 2 is a diagram showing the results of SDS-PAGE of a FlgF solution after removing a protease by the inventive method of the present application in the inventive example of the present application.

Claims (5)

A host in which a large amount of protein is expressed is crushed in a crushing solution containing urea of 6 M concentration or more, and then protease is removed from the crushed material by cation exchange chromatography that adsorbs denatured protease, and then undegraded protein is removed. A protein purification method comprising purifying a protein by adsorbing anion exchange chromatography. The protein purification method according to claim 1, wherein the crushing method is physical crushing. 3. The protein purification method according to claim 2, wherein the crushing method is ultrasonic crushing. The protein purification method according to any one of claims 1 to 3 , wherein the host for protein mass expression is a bacterium. The protein purification method according to claim 4 , wherein the bacterium for mass protein expression is Escherichia coli.

Images