JP2005029531A - Method for activating function of inactive protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for refolding, namely functionally activating an inactive protein, in which a higher-order structure is not formed, produced by Escherichia coli, etc. or a protein in which a three-dimensional structure is changed and deactivated by a certain kind of cause. <P>SOLUTION: The present invention relates to the method for activating the function and the activity which the protein has originally by treating the inactive protein, in which the higher-order structure is not formed, produced by Escherichia coli, etc. or the protein in which three-dimensional structure is changed and deactivated by a certain kind of cause with zeolite beta and a method for producing an active protein by utilizing the method. The present invention provides the method for activating a function of a new protein, having high general purpose property and universality and being simply and readily operatable and inexpensive and enabling repeated use of a function-activating agent, compared with conventional methods. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a method for activating the function of an inactive protein, and more specifically, because a higher-order structure is not formed, an inactive protein or a three-dimensional structure is changed due to a certain cause,
The present invention relates to a function activation method for an inactive protein that can refold an inactivated protein and activate / regenerate the intrinsic function inherent to the protein, and a method for producing an active protein using the method. The present invention, for example, in the technical field of biochemicals and pharmaceutical production, refolds a higher-order structure non-formed protein produced using a gene expression system such as Escherichia coli so that the original function / activity of the protein can be improved. This is useful for providing a function activation method of a new protein that can be activated. Conventionally, in general, proteins obtained from expression systems such as Escherichia coli have a problem in that the three-dimensional structure is disordered, the protein does not have the original functions and activities, and does not exhibit activity. The method is
The present invention provides an innovative protein synthesis technique that activates the function and activity of an inactive protein typified by the above-mentioned protein and makes it possible to regenerate it into a protein having a predetermined function and activity.

It is not a gene that actually acts and works in the body, but a protein made from it. Therefore, elucidation and analysis of protein functions and structures are extremely important, for example, directly related to disease treatment and drug discovery. For this reason, conventionally, various proteins have been synthesized and produced by various methods, their structures have been examined, and their action mechanisms and roles in vivo have been elucidated. Now, it is well known that the functions of proteins are determined not only by the sequence and chain length of amino acids constituting them but also by the ordered three-dimensional structure (higher order structure) taken by them.

Protein synthesis is generally performed using expression systems such as Escherichia coli, insect cells, and mammalian cells. In protein synthesis by insect cells and mammalian cells, the protein obtained is
Higher order structures are controlled, take an ordered three-dimensional structure, and are often soluble. However, these methods have the disadvantages that the operation of separating and purifying the protein is very complicated, and it takes time to obtain the target protein, resulting in an increase in cost and an extremely small amount of protein to be obtained. . In contrast, protein synthesis by E. coli is easy to operate, requires less time to obtain the target protein, and does not cost much. For this reason, at present, a method using E. coli into which a gene code responsible for the synthesis of a target protein is incorporated has become the mainstream of protein synthesis, and its production process is being established.

However, when proteins of higher organisms such as humans are synthesized in an E. coli expression system, the amino acid binding order and number, that is, the amino acid chain length, can be obtained as designed, but the three-dimensional structure is not ordered. Thus, a protein whose higher-order structure is not controlled, that is, an insoluble protein called an inclusion body in which an amino acid chain is entangled is obtained. As a matter of course, the inclusion body of this insoluble protein does not have the desired function and performance and does not exhibit activity. For this reason, in the protein production process by Escherichia coli, it is necessary to unravel the inclusion body, prepare a higher-order structure, and convert it into a soluble protein having an ordered three-dimensional structure, that is, refolding (rewinding) the inclusion body.

This type of refolding is an extremely important technique that can be applied not only to the production of proteins produced by Escherichia coli but also to the regeneration of proteins that have been inactivated due to certain causes such as thermal history.
Therefore, this refolding has been actively studied and various methods have been proposed. Most of them have a low refolding rate and a limited protein (especially a specific protein having a low molecular weight). In many cases, only favorable results were obtained by chance, and this refolding is currently applicable to various proteins.
It is not an efficient method with generality, universality, and high refolding rate.

Dialysis and dilution are used for the refolding operation that has been used most often since the oldest. In the former, the protein is dissolved in an aqueous solution containing a surfactant or denaturant and dialyzed against a buffer solution that does not contain a surfactant or denaturant to reduce the concentration of the surfactant or denaturant. Refolding (typical example: FoldIt kit, manufactured by Hampton Research). On the other hand, the latter is a method in which the protein is dissolved in an aqueous solution containing a surfactant or denaturing agent, and then simply diluted, thereby reducing the concentration of the surfactant or denaturing agent and refolding (typical example: FoldIt
Kit, manufactured by Hampton Research). These are common, but in addition, the surfactant Sodium N-lauroyl sarcosinate solution has glutathione S
-Dissolve the transferase fusion protein and replace it with 1-2% Triton
There are also cases where refolding is performed using a diluent such as a method of diluting with X-100 and rewinding (see Non-Patent Document 1).

Disposable kits are available from Hampton Research for both dialysis and dilution,
In these operations, Ligand binding domains from glutamate and kainate
receptors
, Lysozyme, Carbonic anhydrase B and other limited proteins have been found to cause refolding (see Non-Patent Document 2), but it is no exaggeration to say that they remain in the area of trial and error methods. . Therefore, even if it happens to be successful, it usually does not work well when applied to other proteins.

The use of an adsorption separation column for refolding has also been tried. When a protein denatured with urea / guanidine hydrochloride, thioredoxin is subjected to gel filtration, the protein is unwound during gel filtration (see Non-Patent Document 3). However, refolding is not always sufficient with this method, and other proteins usually do not give satisfactory results. Molecular chaperone GroE, a type of protein that promotes the unwinding of proteins with broken structures
When the protein solubilized with 8M urea is adsorbed to a column fixed with L and eluted with a solution containing 2M each of potassium chloride and urea, the elution of the eluted protein occurs (see Non-Patent Document 4).

However, these are only found in very limited proteins such as Cyclophilin A. Three types of proteins that are thought to be involved in refolding promotion, GroE
L, DsbA (Escherichia coli disulfide
oxidereductase) and PPI (human proline
cis-trans iso
Scorpion toxin, a protein that has been denatured with guanidine hydrochloride on a resin simultaneously fixed with merase)
When Cn5 is mixed, this protein rewinding occurs on the resin (see Non-Patent Document 5).
It has also been reported, but about this, Scorpion
In addition to the disadvantages that can only be applied to specific proteins such as toxin Cn5, there is also the problem that the preparation of a resin in which three types of proteins are immobilized is complicated and expensive.

In some cases, a metal chelate is used instead of protein as a fixed substance on the column. When a His6-tag fusion protein dissolved and modified with an aqueous solution containing guanidine hydrochloride and urea is adsorbed on a resin to which nickel chelate is fixed and washed with a buffer solution containing no denaturant, the fusion protein is unwound (non-patented). Reference 6). However, the application of this method is limited to this protein, and the preparation of the resin is complicated and expensive, as described above.

When β-cyclodextrin or cycloamylase is used as an artificial chaperone and a protein denatured with a surfactant is mixed into this chaperone solution, the surfactant is taken up by the artificial chaperone and the protein is unwound in this process. Report (non-patent literature 7 ~
9). However, this method is only successful with carbonic anhydrase B or the like, and is not a method that can be repeated, but is expensive.

Anal. Biochem. Vol. 210 (1993) 179-187 Protein Sci. Vol. 8 (1999): 1475-83 Biochemistry, Vol. 26 (1987), 3135-3141 Natl. Acad. Sci. USA, Vol. 94 (1997) 3576-3578 Nat. Biotechnol. Vol. 17 (1999) 187-191 Life Science News (Japan Ed.) Vol. 3 (2001) 6-7 J. Am. Chem. Soc. Vol. 117 (1995) 2373-2374 J. Biol. Chem. Vol. 271 (1996) 3478-3487 FEBS Lett. Vol. 486 (2000) 131-135

As described above, various refolding methods have been reported in the past. However, these methods have the above-described problems. For this reason, in this technical field, the chain length is There is an urgent need to develop a low-cost, high-efficiency refolding method that can be applied to various types of high-order structures that are not long or short, and can be applied to denatured and inactivated proteins. It was an issue.

Under such circumstances, the present inventors proceeded with intensive research and development with the goal of developing a new refolding technology capable of solving the above-mentioned problems in view of the above-described conventional technology, We investigated in detail the state of adsorption of biopolymers such as DNA, RNA, and proteins onto metal oxides such as zeolite (Chem. Eur. J. Vol. 7 (2001) 1555-1560), and conducted extensive research on protein separation and purification methods. In the process, proteins that have not been produced in higher-order structures produced by expression systems such as Escherichia coli, or proteins that have been inactivated due to certain causes such as heat history, are treated with zeolite beta. And this method applies the present invention to the refolding of various conformational disordered proteins, including large proteins with molecular weights exceeding 100,000. Kill, generality, found that may be used as a highly universal method, thereby completing the present invention.

The present invention for solving the above-described problems comprises the following technical means.
(1) A method of activating the function of a protein that is inactive due to disordered higher order structure,
A method for activating a function of a protein, wherein the protein is brought into a state capable of expressing an inherent function inherent to the protein by contacting the protein with zeolite beta.
(2) The method according to (1), wherein the protein is contacted with zeolite beta in the presence of a protein denaturant, a surfactant, and / or a refolding buffer.
(3) The method according to (1) above, wherein the protein that is inactive due to disordered higher order structure is a protein produced in an expression system of E. coli.
(4) The method according to (1) above, wherein the protein that is inactive due to disordered higher order structure is a protein that has been inactivated due to thermal history.
(5) The method according to (1) above, wherein the protein is adsorbed to zeolite beta and then desorbed by mixing with a solution containing zeolite beta or injection into a zeolite beta packed column.
(6) A method for altering the main structure of a protein, comprising bringing a protein that is inactive due to disordered higher order structure into contact with zeolite beta to refold the three-dimensional structure of the protein.
(7) By contacting a protein that is inactive due to disordered higher-order structure with zeolite beta and refolding the three-dimensional structure of the protein, the higher-order structure is controlled and the inherent function inherent to the protein is activated. A method for producing an active protein, characterized in that a protein is produced.
(8) The production of the protein according to (7) above, wherein an inactive protein produced by E. coli incorporating a gene code responsible for synthesis of the target protein is contacted with zeolite beta to refold the three-dimensional structure of the protein. Method.

The present invention relates to a function activation method of an inactive protein and the like.
) Refolding of the original functions and activities of proteins that are inactive due to the absence of higher-order structures produced in expression systems such as E. coli, or proteins that have been inactivated due to a change in the three-dimensional structure due to certain causes 2) This method is useful as a method of efficiently refolding inclusion bodies, 3) Generality, universality, and high refolding rate applicable to various proteins Can provide an efficient method 4
) Zeolite beta, a functional activator used in the present invention, is low in cost and can be used repeatedly. 5) This method can be used to modify various conformationally disordered proteins including large proteins with molecular weights exceeding 100,000. 6) For example, by combining the protein synthesis process by the expression system of E. coli and the method of the present invention, production of a new active protein whose protein is produced by controlling the higher-order structure and producing the intrinsic function of the protein The effect is that the process can be constructed.

Next, the present invention will be described in more detail.
In the present invention, the protein to be functionally activated is generally a protein having a disordered three-dimensional structure obtained in an expression system such as Escherichia coli, a so-called inclusion body, or a protein inactivated due to a certain cause such as thermal history. Is used. In the present invention, these proteins are treated with zeolite beta to refold the three-dimensional structure of the protein, thereby activating the intrinsic function inherent to the protein. In the activation operation, usually, the protein is first dispersed and dissolved in a solution containing a denaturant, a surfactant, etc., and then mixed with a solution containing zeolite beta or injected into a zeolite beta-packed column to thereby convert the protein into zeolite. The procedure is performed by adsorbing to beta and then desorbing the protein from zeolite beta. Examples of the zeolite beta used as the functional activator in the present invention include unfired zeolite beta and, for example, calcined zeolite beta obtained by firing synthetic zeolite beta at 300 to 500 ° C. for 3 to 10 hours. It is not a thing, but if it is equivalent to these, it can be used similarly.

As a protein dispersion solvent before adsorption to zeolite beta, it is generally produced in an expression system such as Escherichia coli, and the protein is usually used in an aqueous solution. For example, water is preferably used because it is often present inside. However, the present invention is not necessarily limited to this, and there is basically no problem as long as it does not cause a reaction with the protein and does not change the three-dimensional structure of the protein into an unintentional form. In some cases, these solvents can be used alone or mixed with water. Typical examples of this type of solvent include, but are not limited to, monohydric and polyhydric alcohols.

The adsorption / desorption of the protein is generally performed by using a denaturing agent, a surfactant, p, etc. in order to make it easy to unwind and rewind the entangled protein chain length, such as an inclusion body.
It is carried out in the presence of a reducing agent in order to cleave the S—S bond generated inadvertently in the protein chain length in the presence of an H regulator, a refolding factor or the like. Typical examples of this type of modifier, surfactant, pH adjuster, and refolding factor include, for example, guanidine hydrochloride, trisaminomethane hydrochloride, polyethylene glycol, cyclodextrin,
4- (2-hydroxyethyl) -1-piperazineethanesulfonic
Examples include acid (HEPES), polyphosphoric acid, sucrose, glucose, glycerol, inositol, Dextran T-500 and Ficol1400. .

As a reducing agent that cleaves the unintentionally generated SS bond and returns it to its original structure, 2-mercaptoethanol is usually used because it is inexpensive and easily available, but is not limited to this. Anything that has an effect can be used. Of course, when the protein chain length is easily unraveled or when the S—S bond is not generated unintentionally, it is not always necessary to use a denaturant, a surfactant, and / or an inhibitor. Existence is not always essential, and is appropriately selected according to the situation. Moreover, also when using them, those quantity will be suitably determined according to a condition.

In addition, substitution adsorption is generally used for desorption of the protein, but basically any operation can be applied as long as it does not hinder the function activation after desorption of the protein.
It is not particularly limited. Therefore, pH change, temperature change, etc. can be used, and these can be used in combination with displacement adsorption. As a substance that promotes desorption of the protein by displacement adsorption, a surfactant such as sodium dodecyl sulfate (SDS) or a salt such as an alkali halide is generally used, but is not limited thereto. As long as it does not hinder the function activation after desorption of the protein, various compounds such as those usually used for elution in column chromatography can be used.

Furthermore, in order to promote adsorption of the protein to the silicate or desorption from the protein, various additional operations can be performed in combination with the above operation. Typical examples of such operations include irradiation of ultrasonic waves and microwaves, application of magnetic fields and electric fields, and the like. By the procedures and operations of the present invention described above, refolding occurs in unstructured proteins produced using an expression system such as Escherichia coli, as well as proteins inactivated due to certain causes. The original function that it has is activated quickly. The zeolite beta of the functional activator of the present invention is extremely stable thermally and chemically, is inexpensive, and can be repeatedly used. Therefore, the present invention is useful for, for example, biochemical production and pharmaceutical production. It is extremely useful and its economic effects are immeasurable.

Next, the present invention will be specifically described based on examples and comparative examples, but the present invention is not limited to the following examples.
Examples and Comparative Examples Hereinafter, in this example, functional activation of E. coli expression system production protein and denatured protein will be described, but the present invention is not limited or limited to these examples.
1) Preparation of sample etc. (a) Functional activator As a functional activator, the uncalcined zeolite beta (Na-BEA) shown in Table 1 mentioned later,
Synthetic zeolite beta shown in Table 1, and calcined zeolite beta obtained by calcining it, and Table 1
Comparative products shown in Comparative Examples 1 to 15 were used.
(B) Denatured protein solution RPA70 (derived from yellow Drosophila), P53 (derived from human), etc. having the contents shown in “Proteins” and “Remarks” in Table 1 were used as proteins.
(C) Refolding buffer Using Na-BEA as the zeolite and RPA70 as the protein, the salt concentration of the refolding buffer was examined. Refollowing buffer is 20 mM
Tris HCl pH 7.5, 0.5 M NaCl, 20 mM 2-mercaptoethanol, 2.5 (w / v)% polyethylene glycol 20,000,
A nonionic surfactant is used, and the surfactant is 1 (v / v)% Tween 20, T
Riton X-100 and NP-40 were used. Table 2 to be described later shows details of the refolding buffer actually used in the examples and comparative examples.

2) Refolding operation 100 mg of functional activator is placed in a 1.5 ml Eppendorf tube, 0.5 ml of 6 M guanidine hydrochloride, 20 mM trisaminomethane trihydrochloride (TrisHCl) pH 7
. 5, 0.5 M NaCl and 20 mM 2-mercaptoethanol were added and suspended. 6M guanidine hydrochloride and 20 mM 2-mercaptoethanol were added to this, and the mixture was allowed to stand on ice for 1 hour, and a denatured protein solution (concentration: 0.5 to 1.0 mg / ml) was added to 0.5 m.
l was added. In order to ensure the adsorption of the protein onto the functional activator, this mixed solution was put into a ROTARY CUTURE RCC-100 (IWAKI GLASS) placed in a cold room.
For 1 hour.

Thereafter, the functional activator was precipitated by centrifugation at 10,000 × g for 5 seconds, and the supernatant was removed. Next, in order to completely remove the protein denaturant from the precipitated functional activator, it was washed 4 times with 1 ml of 20 mM TrisHCl pH 7.5, 20 mM 2-mercaptoethanol, and then centrifuged at 10000 × g for 5 seconds. In addition, the resulting supernatant was discarded. 1 ml of refolding buffer (50 mM HEPES pH 7.
5, 0.5 M NaCl, 20 mM 2-mercaptoethanol, refolding factor and nonionic surfactant) were added and suspended.

In order to desorb and elute the protein adsorbed on the functional activator, the suspension was again stirred with ROTARY CUTURE RCC-100 (manufactured by IWAKI GLASS) at low temperature. Thereafter, the functional activator was precipitated by centrifugation at 10,000 × g for 5 seconds, and the supernatant containing the protein was transferred to a new Eppendorf tube and used for activity measurement (assay).
In addition, the method according to the function of the used protein was employ | adopted for the activity measurement. In particular,
Three measurements were performed: gel shift assay, polymerase assay and lysozyme activity measurement.

3) Activity measurement operation (a) Gel shift assay Oligonucleotide DNA labeled with 1 pmol of radioisotope and refolded protein were combined with a composition of 25 mM HEPES pH 7.4, 50 mM KCl, 20%.
glycerol, 0.1% NP-40, 1 mM DTT, and 1 mg / ml bov
Incubate for 30 minutes on ice in a solution of in serum albumin, 4.5
% Polyacrylaminogel was electrophoresed at 4 ° C. using 0.5 × TBE buffer. The results are shown in FIG. 1 and FIG.
If the protein is DNA-binding (ie active), the protein will bind to the DNA, which slows the electrophoresis and shifts the band, so that the activity (
That is, the refolding rate) was determined.

(B) Polymerase assay As template DNA, poly (dA) oligo (dT) _12-18 , or DNase
I-activate calf thymus DNA was used, and the reaction solution had a composition (final concentration) 50 nm M TrisHCl pH 7.5, 1 mM DTT, 15% gly.
Cerol, 5 mM MgCl ₂ , 0.5 μM dTTP (cold) (thymidylic acid triphosphate), [ ³ H] -dTTP (5 mCi / ml: 100-500 cpm / pmol) were used. First, a protein (enzyme) sample solution was added to 10 μl of a double concentration of this reaction solution, suspended, incubated at 37 ° C. for 1 hour, and then placed on ice to stop the reaction.

Thereafter, the reaction solution was dropped onto DE81 paper cut into a square, dried, then transferred into a beaker, and washed to dissolve and remove unreacted dTTP. Washing was first performed 3 times with 5% aqueous solution of disodium hydrogen phosphate, then 3 times with distilled water, 2 times with ethanol, and then dried. The dry DE81 paper thus obtained was placed in a vial containing a scintillator and the radioactivity (cpm) was measured with a scintillation counter. The stronger the activity of the enzyme sample, the more dTTP labeled with a radioisotope is incorporated into the DNA synthesized with it, and the radioactivity becomes higher. Thus, the activity of the protein was determined. FIG. 2 shows the recovery rate of the protein refolded using Tween 20 and the recovery rate of the activity.

(C) Activity measurement of lysozyme Bacteria M. Lysodeikticus was selected and suspended in 50 mM phosphate buffer to prepare a substrate solution having a concentration of 0.16 mg / ml. Add 480 μl of this substrate solution to 2
0 μl of protein (enzyme lysozyme) solution was added and incubated at room temperature for 30 minutes. Thereafter, the absorbance at a wavelength of 450 nm was measured.
Since lysozyme has the ability to break down bacterial cell walls, the higher its ability, ie activity, the lower the absorbance. Lysozyme activity 1 unit was defined as a 0.001 decrease in absorbance at 450 nm per minute.

The activity (refolding rate) and protein recovery rate, which are the results of this example obtained by the above procedures and operations, are summarized in Table 1 together with the results of the comparative examples. Table 2 shows the refolding buffers used in the examples and comparative examples. As shown in the Examples, it was found that the original activity of the protein such as DNA binding activity can be obtained by refolding. The present invention
It is useful as a general and universal refolding method that can be applied to various high-order structure unformed and denatured / inactivated proteins, and its application is not limited to the proteins shown in the Examples. It can be applied to any protein.

As described in detail above, the present invention relates to a method for activating the function of an inactive protein, etc., and according to the present invention, it is not possible because 1) higher-order structures produced in an expression system such as Escherichia coli are not formed. The original function / activity of an active protein or a protein deactivated due to a change in the three-dimensional structure due to a certain cause can be activated by refolding. 2) This method is useful as a method for efficiently refolding an inclusion body. 3) It is possible to provide an efficient method that is applicable to various proteins and has generality, universality, and high refolding rate. 4) Zeolite beta, a functional activator used in the present invention, is low in cost and can be used repeatedly. 5) This method can be applied to the refolding of various conformationally disordered proteins including large proteins with molecular weights exceeding 100,000. 6)
For example, by combining the protein synthesis process by the expression system of Escherichia coli and the method of the present invention, it is possible to construct a process for producing a new active protein in which a higher-order structure is controlled and the original function inherent to the protein is produced. .

The result of the gel shift assay by electrophoresis is shown. The recovery rate of the refolded protein and the recovery rate of activity are shown. The result of the gel shift assay by electrophoresis is shown.

Claims (8)

A method of activating the function of a protein that is inactive due to disorder of the higher-order structure, characterized by bringing the protein into contact with zeolite beta so that the inherent function inherent to the protein can be expressed. Protein functional activation method. The method according to claim 1, wherein the protein is contacted with zeolite beta in the presence of a protein denaturant, a surfactant and / or a refolding buffer. The method according to claim 1, wherein the protein that is inactive due to disordered higher order structure is a protein produced in an E. coli expression system. The method according to claim 1, wherein the protein that is inactive due to disordered higher order structure is a protein that has been inactivated due to thermal history. The method of claim 1, wherein the protein is adsorbed to zeolite beta and then desorbed by mixing with a solution containing zeolite beta or by injection into a zeolite beta packed column. A method for altering the main structure of a protein, comprising bringing a protein that is inactive due to disordered higher order structure into contact with zeolite beta to refold the three-dimensional structure of the protein. By contacting a protein that is inactive due to disordered higher-order structure with zeolite beta and refolding the three-dimensional structure of the protein, a protein whose original function inherent to the protein is activated by controlling the higher-order structure is obtained. A method for producing an active protein, which comprises producing the active protein. The method for producing a protein according to claim 7, wherein an inactive protein produced by E. coli incorporating a gene code responsible for synthesis of the target protein is contacted with zeolite beta to refold the three-dimensional structure of the protein.