JP2000229995A - Method and apparatus for regenerating protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide both method and apparatus capable of continuously regenerating a protein in a high regeneration efficiency. SOLUTION: A denatured protein solution is fed from a number of points to a reaction tank while flowing a regenerated stream in a plug flow into the interior of the reaction tank so as to make the concentration of the denatured protein rise as a position approaches the downstream side. A protein is regenerated in high efficiency at a part of low concentration of denatured protein. The regenerating apparatus is equipped with an inner cylinder 2 composed of a membrane capable of permeating the denatured protein solution inside an outer cylinder 1 to which the denatured protein solution is fed. The denatured protein solution is permeated through the circumference of the inner cylinder while flowing the regenerated flow in plug flow in the interior of the inner cylinder 2 to carry out regeneration.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method and apparatus for regenerating a protein having an abnormal three-dimensional structure.

[0002]

2. Description of the Related Art The development of genetic engineering has made it possible to produce useful proteins by incorporating genes encoding the proteins into microorganisms and the like. As a production method, there are a method of producing a useful protein in the cells and a method of secreting the useful protein outside the cells. In the former method, useful proteins are sometimes accumulated in the form of inclusion bodies called inclusion bodies in the cells. The amino acid sequence of this inclusion body protein is the same as that of the natural protein, but is inactive because the tertiary structure is different from that of the natural protein. In order to utilize this inactive protein as an active form, it is necessary to solubilize the protein once and then fold it into a correct three-dimensional structure (refolding). This refolding is referred to herein as protein regeneration.

A method for regenerating an inactive protein that has become an inclusion body is disclosed in, for example, JP-A-59-16.
No. 1312 is already known. A frequently used solubilization method is to use a high-concentration urea or guanidine hydrochloride solution, and to solubilize those having a disulfide bond by reducing with a reducing agent such as mercaptoethanol or dithiothreitol. Protein regeneration can be achieved by diluting the solubilized protein (denatured protein) under optimal redox conditions obtained using oxidized and reduced glutathione, or by diluting or dialyzing with an appropriate buffer. It becomes possible.

[0004] A major obstacle to regeneration of the denatured protein is the aggregation of protein molecules. This aggregation means that an intermediate exists before the protein, which has become single-stranded by the denaturing agent, rewinds to an active structure, and the intermediate forms a hydrophobic interaction, a disulfide bond, and an electrostatic interaction. Incorrect aggregates between molecules or within molecules due to, for example, an inert and insoluble aggregate. This aggregation occurs more as the denatured protein concentration is higher. That is, in order to achieve high regeneration efficiency, it is known that it is necessary to lower the concentration of denatured protein (Japanese Patent Application Laid-Open No. Sho 61-5021).
No. 67). However, in order to reduce the concentration, a large amount of processing is required, and additional time and equipment costs are required. Therefore, it is desired to achieve as high a concentration as possible and high regeneration efficiency.

[0005] In addition, as a conventional typical reproducing method,
There are a batch method in which a predetermined amount of a regenerating solution is put in a treatment tank, and a denatured protein solution is added thereto at once, and a reaction is performed, and a semi-batch method in which the denatured protein solution is added in several portions. However, in the batch method, as described above, when the concentration of the denatured protein is increased, the regeneration efficiency is reduced. On the contrary, when the concentration of the denatured protein is reduced, a large processing tank is required.

On the other hand, in the semi-batch method, the regeneration reaction proceeds in a low concentration state until the final denatured protein concentration is reached, so that a higher regeneration efficiency can be obtained than in the batch method. However, the semi-batch method has various inconveniences when applied to industrial processes because continuous processing cannot be performed.

[0007]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a method for regenerating a protein capable of achieving a high regenerating efficiency comparable to the semi-batch method and enabling continuous processing. This is to provide a playback device.

[0008]

According to the method for regenerating a protein of the present invention, which has been made to solve the above-mentioned problems, a denatured protein solution is introduced into a reaction vessel while flowing the regenerating solution inside the reaction vessel. It is characterized in that the concentration is added from a number of points so that the concentration gradually increases toward the downstream side. In addition, the protein regenerating apparatus of the present invention made in order to solve the above-mentioned problem is provided with an inner cylinder made of a membrane through which a denatured protein solution can pass, inside an outer cylinder to which a denatured protein solution is supplied. In addition, a supply port for the regenerating liquid is provided at one end of the inner cylinder, a take-out port is provided at the other end, and a partition plate for flowing the regenerating liquid is disposed inside the inner cylinder.

According to the protein regeneration method of the present invention, the denatured protein solution is added to the regenerating solution flowing inside the reaction tank from a number of points so that the concentration gradually increases toward the downstream side. , High-efficiency regeneration is possible in the low concentration range. Moreover, unlike the conventional method, continuous production becomes possible. According to the apparatus of the present invention, the treatment tank is constituted by an inner cylinder made of a membrane through which a denatured protein solution can pass,
Since the denatured protein solution is allowed to permeate through the inside of the regenerating solution flowing through the plug flow from the surroundings, the denatured protein solution can be continuously added over substantially the entire length of the reaction tank. Therefore, high-efficiency reproduction becomes possible.

[0010]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described below. FIG. 1 is a cross-sectional view showing a regenerating device for carrying out the method for regenerating a protein according to claim 1, wherein 1 is an outer cylinder installed horizontally, and 2 is an inner cylinder serving as a reaction tank arranged at the center thereof. is there. A denatured protein solution is supplied between the outer cylinder 1 and the inner cylinder 2 through a supply port 3 for the denatured protein liquid. A regenerating liquid is supplied into the inner cylinder 2 serving as a reaction tank through a regenerating liquid supply port 4 provided at one end thereof.

The regenerating liquid supplied from the supply port 4 flows through the inside of the inner cylinder 2 toward the outlet port 5 at the other end by a plug flow. This plug flow is also called an extrusion flow, and is a flow in which liquid does not mix in the flow direction. In order to generate this plug flow, a rotating shaft 7 having a partition plate 6 is provided inside the inner cylinder 2.
Since the rotating shaft 7 is provided with the stirring blades 8, the regenerating liquid is uniformly stirred and mixed in the same cross section perpendicular to the flow direction, but is not mixed in the flow direction (horizontal direction) by the partition plate 6. It has become.

The inner cylinder 2 is formed of a membrane through which a denatured protein solution can pass. In this embodiment, a cylindrical ceramic membrane is used, and the denatured protein solution can be injected into the inner cylinder 2 with a uniform flux from a myriad of points on the entire membrane surface. However, it is not necessary to allow the denatured protein solution to permeate the entire membrane surface, and any material can be used as long as the membrane has a large number of pores into which a highly viscous denatured protein solution can be injected without clogging. Can be.

As described above, if the denatured protein solution is permeated from the surroundings while the regenerating solution flows through the inside of the inner tube 2 as a reaction tank by a plug flow, the concentration of the denatured protein inside the inner tube 2 as a reaction tank can be increased. Is low on the inlet side and gradually increases toward the outlet side where the outlet 5 is provided. What is important here is that the concentration of denatured protein in the reaction vessel is low on the inlet side,
Gradually increasing toward the outlet side, it is not always necessary to have one reaction vessel to achieve that,
A form in which several reaction tanks are connected may be used. As a result, similar to the above-mentioned semi-batch method, the protein is regenerated with high efficiency in the portion where the concentration of the denatured protein is low. Then, by appropriately setting the residence time in the reaction tank, the liquid can be mixed while suppressing the aggregation of the intermediate, so that high-efficiency regeneration is possible, and according to this method, continuous processing is also possible. .

[0014]

Examples of the present invention will be described below. First, a denaturing buffer (8 M Urea, 0.1 M Tris-HCl, 1 mM EDTA, 10 mM DTT, pH 8.
After aeration of nitrogen for 5 minutes in 5), lysozyme was added to a predetermined concentration, and the mixture was stirred and incubated at 38 ° C for 2 hours to obtain a denatured protein solution. The denatured protein solution was regenerated using the regenerating device shown in FIG. The inner membrane of this device is a ceramic membrane with an outer diameter of 30 mm, an inner diameter of 22 mm, a length of 120 mm, and a pore diameter of 2 μm, and a regeneration buffer (1.5 M Urea, 3 mM
GSH, 5 mM GSSG, 0.1 M Tris-HCl, 1 mM EDTA, pH 8.0) is flowing through the plug flow. The denatured protein solution was injected through the ceramic membrane into the regeneration buffer from the entire membrane surface with a uniform flux and diluted.

The effluent taken out from the outlet was sampled every 20 minutes for 240 minutes. Thereafter, the sample was incubated for 24 hours with gentle stirring, and the activity and concentration were measured to determine the final regeneration efficiency. The activity was measured using Micrococcus lysodeiktics dried cell as the substrate.
Was determined from the rate of decrease in absorbance at 450 nm. The concentration was measured using a spectrophotometer and determined from the absorption at 280 nm. The regeneration efficiency was determined from the activity ratio to the activity of native lysozyme at the same concentration.

The operating conditions of the apparatus were as follows: a denatured protein solution flow rate of 0.135 mL / min, a regeneration buffer solution flow rate of 1.22 mL / min, and a stirring blade rotation speed of 40 rpm. In a steady state, the denatured protein solution was diluted 10-fold. Was adjusted to The liquid mixing in the apparatus under the same operating conditions was determined by a step response experiment using Blue Dextran 2000 as a tracer.

From the step response experiment, it was found that the number of stages in the tank row model was three. The residence time in the reactor is 34 minutes. FIG. 2 shows the influence of the regeneration time on the time of adding the denatured protein in the semi-batch operation. At this time, the addition time of the semi-batch operation and the residence time of the present method have the same meaning. As a result, a regeneration efficiency of 95% was achieved in 30 minutes or more, indicating that the operation conditions are appropriate residence times. FIG. 3 shows the concentration and the regeneration efficiency of the regenerated protein solution sampled at each time. At this time, the denatured protein solution contains lysozyme
The one at 15 mg / mL was used. After 120 minutes, steady state
An almost uniform regenerated protein solution having a lysozyme concentration of 15 mg / mL and a regenerating efficiency of 95% was obtained.

FIG. 4 shows the concentration dependence of the regeneration efficiency by batch operation, semi-batch operation and continuous operation of the present invention. As shown,
With the continuous operation of the present invention, a regeneration efficiency higher than that of the conventional batch operation and equivalent to that of the semi-batch operation was achieved. It is considered that the continuous operation of the present invention sufficiently satisfies the effect of the half-time operation of performing the regeneration at a low concentration at the expected stage even when the number of the device stages is three.

[0019]

As described above, according to the present invention, protein can be continuously regenerated, and a high regenerating efficiency equivalent to the conventional half-batch operation can be obtained.
For this reason, the present invention is of high value in industrially performing protein regeneration.

[Brief description of the drawings]

FIG. 1 is a sectional view showing an apparatus of the present invention.

FIG. 2 is a graph showing the effect of addition time of a denatured protein solution on regeneration efficiency in Examples.

FIG. 3 is a graph showing the time-dependent changes in the concentration of the regenerated protein solution and the regeneration efficiency in Examples.

FIG. 4 is a graph showing the concentration dependence of the regeneration efficiency in an example.

[Explanation of symbols]

1 outer cylinder, 2 inner cylinder, supply port for denatured protein solution, 4
Regeneration liquid supply port, 5 take-out port, 6 partition plate, 7 rotating shaft, 8 stirring blade

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B050 CC02 GG02 HH01 4H045 AA20 AA40 BA05 DA89 FA67

Claims (5)

[Claims] 1. A method for producing a protein, characterized in that a denatured protein solution is added from a number of points while flowing a regenerating solution inside the reaction tank such that the concentration of the denatured protein solution gradually increases toward the downstream side. Playback method. 2. The method according to claim 1, wherein the denatured protein solution is supplied over the entire length of the reaction vessel.
The method for regenerating a protein according to claim 1, wherein the protein is added in a uniform flux. 3. An inner cylinder made of a membrane through which the denatured protein solution can pass is provided inside the outer cylinder to which the denatured protein solution is supplied, and a supply port for the regenerating solution is provided at one end of the inner cylinder. An apparatus for regenerating a protein, characterized in that a take-out port is provided at an end and a partition plate for flowing a regenerating solution is arranged inside the inner cylinder. 4. The protein regeneration device according to claim 3, wherein the inner cylinder is made of a porous ceramic membrane. 5. The protein regenerating apparatus according to claim 3, wherein a stirring means for stirring the regenerating solution in a direction perpendicular to the flow direction is disposed inside the inner cylinder.