JP4273205B2 - Functional activation method of inactive protein - Google Patents

Description

The present invention relates to a method for activating the function of an inactive protein, and more specifically, an inactive protein because a higher-order structure is not formed, or a protein that has been inactivated due to a change in the three-dimensional structure due to a certain cause. was Rifo over Rudingu, functional activation method inert protein that allows to activated and playback the original function of the protein-specific, and to a manufacturing method of the active protein by using the method. The present invention is, for example, biochemical products, in the art of pharmaceutical production, conformation unformed protein produced using gene expression systems such as Escherichia coli, was Rifo over Rudingu, the original function-activity of the protein It is useful as a method for activating the function of a new protein capable of activating protein. 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 provides an innovative protein synthesis technique that activates the function / activity of an inactive protein typified by the above-described protein and enables regeneration to a protein having a predetermined function / 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. Therefore, in the protein production process by E. coli, unravel the inclusion body, established a conformation operation is converted into a soluble protein having a three-dimensional ordered structure, i.e. Rifo chromatography Rudingu (unwinding) of the inclusion body is required .

Rifo over Rudingu of this kind not only proteins produced by E. coli, can also be applied to the regeneration of proteins inactivated in some causes, such as thermal history, it is very important technique. Therefore, conventionally, the Rifo over Rudingu is actively studied, although various methods have been proposed, most of them, on top Rifo over Rudingu rate is low, limited protein (particularly, lower molecular weight many that only accidental favorable results for a particular protein) obtained now the Rifo over Rudingu is applicable to a variety of proteins, generality, a universal, yet Rifo over Rudingu It is not a highly efficient method.

The oldest of often used its dependent Rifo over Rudingu operation, it is used dialysis or dilution. 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. which Rifo over Rudingu (typical example: Foldit kit, Hampton Research Corporation) is. The latter is a protein was dissolved in an aqueous solution containing a surfactant or modifier, which simply reduce the concentration of surface active agent or denaturant by going diluted one which Rifo over Rudingu (typical example: FoldIt kit, manufactured by Hampton Research). These are common, but other methods include dissolving the glutathione S-transferase fusion protein in a solution of the detergent Sodium N-lauroyl sarcosinate, diluting it with 1-2% Triton X-100 and unwinding. (non-Patent Document 1), etc., in some cases to Rifo over Rudingu out using diluents.

For both dialysis and dilution, are commercially available disposable kit from Hampton Research Inc., in these procedures, Ligand binding domains from glutamate and kainate receptors, Lysozyme, Carbonic anhydrase Rifo over in very limited proteins, such as B Rudingu It is not an exaggeration to say that this is merely a trial-and-error method. Therefore, even if it happens to be successful, it usually does not work well when applied to other proteins.

It is also tried to use an adsorption separation column Rifo over Rudingu. 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, in this method, Rifo over Rudingu is not always sufficient, which is normally not satisfactory results are obtained with other proteins. When a protein that has been solubilized with 8M urea is adsorbed onto a column immobilized with molecular chaperone GroEL, which is a type of protein that promotes unwinding of a protein with a broken structure, and eluted with a solution containing 2M each of potassium chloride and urea, Rewinding of the eluted protein occurs (see Non-Patent Document 4).

However, these are only found in very limited proteins such as Cyclophilin A. Further, the protein three believed to be involved in Rifo over Rudingu promoting, GroEL, the DsbA (E. coli disulfide oxidereductase) and PPI (human proline cis-trans isomerase ) simultaneously fixing resin, protein Scorpion toxin CN5 modified with guanidine hydrochloride It has also been reported that the unwinding of this protein occurs on the resin when mixed with (see Non-Patent Document 5), but in addition to the disadvantage that can only be applied to specific proteins such as Scorpion toxin Cn5, There is also a problem that the preparation of the resin in which the three types are fixed 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

Thus, conventionally, have been reported methods of various Rifo over Rudingu, these methods are circumstances that there are problems as described above, therefore, in the art, chain length applicable to a variety regardless of length of conformation unformed and denaturation and inactivation protein, generality, high universality, yet to be developed repeatedly available low-cost high efficiency Rifo over Rudingu method It was an urgent issue.

Under such circumstances, the present inventors have found that the in light of the prior art, together with advances intensive research with the goal of developing a new Rifo over Rudingu technique capable of solving the problems described above Investigate in detail the state of adsorption of biopolymers such as DNA, RNA and protein onto metal oxides such as zeolite (Chem. Eur. J. Vol. 7 (2001) 1555-1560) In the process of treatment, if the unstructured protein produced in an expression system such as Escherichia coli or a protein deactivated due to a certain cause such as heat history is treated with zeolite beta, these proteins are it is shown the activity and the method of the present invention, the Rifo over Rudingu various conformational disordered proteins containing large proteins exceeding a molecular weight of 100,000 Can use, generality, it 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 for activating the function of a protein that is inactive due to disordered higher order structure, and contacting the protein with zeolite beta in the presence of a protein denaturant, a surfactant, and a refolding buffer By this, the function activation method of the protein characterized by making it into the state which can express the original function intrinsic | native to this protein.
( 2 ) 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.
( 3 ) The method according to (1), wherein the protein that is inactive due to disordered higher order structure is a protein that has been inactivated due to thermal history.
( 4 ) The method according to (1) above, wherein the protein is adsorbed on the zeolite beta and then desorbed by mixing with a solution containing the zeolite beta or injection into a zeolite beta packed column.
( 5 ) refolding the protein's three-dimensional structure by contacting a protein that is inactive due to disordered higher-order structure with zeolite beta in the presence of a protein denaturant, a surfactant, and a refolding buffer. A method for modifying the main structure of a characteristic protein.
( 6 ) By refolding the three-dimensional structure of the protein by contacting a protein that is inactive due to disorder of the higher order structure with zeolite beta in the presence of a protein denaturant, a surfactant, and a refolding buffer. A method for producing an active protein, characterized by producing a protein in which a higher-order structure is controlled and an original function inherent to the protein is activated.
( 7 ) The protein production according to ( 6 ), 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 method for activating the function of an inactive protein, etc. According to the present invention, 1) a protein that is inactive because a higher-order structure produced in an expression system such as Escherichia coli is not formed, or a certain kind cause in can be activated the original function-activity of the protein conformation is deactivated changed by Rifo over Rudingu of 2) this method is useful for inclusion body as efficiently Rifo over Rudingu method of , 3) applicable to a variety of proteins, generality, a universal, yet can provide a high efficient method of Rifo over Rudingu rate, 4) the zeolite beta function activator used in the present invention, a low-cost 5) This method can be used for various three-dimensional disordered proteins including large proteins with molecular weights exceeding 100,000. Of applicable to Rifo over Rudingu, 6) for example, by combining the methods of protein synthesis process and the present invention by expression system of E. coli, a new activity controlled original function of the protein-specific and conformation to produce a protein There is an effect that a protein production 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, by Rifo over Rudingu the three-dimensional structure of the protein by these proteins were treated with zeolite beta, it performs activation of the protein-specific original function. 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. The zeolite beta used as a functional activator in the present invention, uncalcined zeolite beta, and for example, although the firing zeolite beta was 3~10h fired synthetic zeolite beta at 300 to 500 ° C. is illustrated, are these limits 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.

Adsorption and desorption of the protein, generally, such inclusion bodies, to facilitate unfolding of the protein chain length tangled tangling, also in order to facilitate unwinding, modifier or a surfactant, pH adjusting agents, Rifo over Rudingu factors such And / or in the presence of certain reducing agents in order to cleave S—S bonds formed inadvertently during the protein chain length. A typical example of this type of denaturing agent or a surfactant, pH adjusting agents, Rifo over Rudingu factors, for example, guanidine hydrochloride, Tris aminomethane hydrochloride, polyethylene glycol, cyclodextrins, 4- (2-hydroxyethyl) -1- Examples include piperazineethanesulfonic acid (HEPES), polyphosphoric acid, sucrose, glucose, glycerol, inositol, Dextran T-500 and Ficol1400. is there.

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. Thus the procedure and operation of the present invention have been described, conformation unformed protein was produced using an expression system such as E. coli, and the proteins inactivated the cause of certain, occurs Rifo over Rudingu, the proteins The original function of 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 Samples, etc. (a) Functional Activator As a functional activator, uncalcined zeolite beta (Na-BEA) shown in Table 1 described later, synthetic zeolite beta shown in Table 1, and calcined zeolite obtained by calcining it Beta and comparative products shown in Comparative Examples 1-15 in Table 1 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) Rifo Lumpur loading buffer zeolite as Na-BEA, with RPA70 as protein, was examined the salt concentration of the refolding buffer. Li Four Ludington buffer, 20mM Tris HCl pH7.5,0.5M NaCl, 20mM 2- mercaptoethanol, 2.5 (w / v)% polyethylene glycol 20,000, nonionic surfactants used, surfactants As the agent, 1 (v / v)% Tween 20, Triton X-100, and NP-40 were used. Table 2 below shows the details of Rifo Lumpur loading buffer actually used in Examples and Comparative Examples.

2) Rifo over Rudingu the operation 1.5ml Eppendorf tube was placed a function activator of 100 mg, 6M guanidine hydrochloride 0.5 ml, 20mM Tris aminomethane trihydrochloride (TrisHCl) pH 7.5, 0.5 M NaCl, and 20mM 2-mercaptoethanol was added and suspended. To this, 6M guanidine hydrochloride and 20 mM 2-mercaptoethanol were added, allowed to stand on ice for 1 hour, and 0.5 ml of denatured protein solution (concentration: 0.5 to 1.0 mg / ml) was added. In order to ensure the adsorption of the protein onto the functional activator, this mixed solution was stirred with ROTARY CUTURE RCC-100 (manufactured by IWAKI GLASS) 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. The remaining function activator, Rifo Lumpur loading buffer 1ml of (50mM HEPES pH7.5,0.5M NaCl, 20mM 2- mercaptoethanol, consists refolding agent and a nonionic surfactant) was 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 activity measurement. In particular,
Three measurements were performed: gel shift assay, polymerase assay and lysozyme activity measurement.

3) activity measurement operation (a) a gel shift assay 1pmol radioisotope labeled oligonucleotide DNA and Rifo over Rudingu protein composition 25mM HEPES pH7.4,50mM KCl, 20% glycerol , 0.1% NP-40 The solution was incubated for 30 minutes on ice in a solution of 1 mM DTT and 1 mg / ml bovine serum albumin, and electrophoresed at 4 ° C. using a 0.5% TBE buffer in 4.5% polyacrylaminogel. The results are shown in FIG. 1 and FIG.
If protein has DNA-binding (that is, active) protein binds to the DNA, thereby, electrophoresis slows, the band is shifted, thereby to determine the activity (i.e. Rifo over Rudingu rate) .

(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 results of this example obtained by the above procedure and operation, activity (Rifo over Rudingu rate), the protein recovery, in Table 1, summarizes together with the results of Comparative Example. Incidentally, showing the Rifo Lumpur loading buffer used in the Examples and Comparative Examples in Table 2. As shown in the examples, by Rifo over Rudingu it was found that the protein intrinsic activity of DNA-binding activity, etc. is obtained. The present invention is applicable to various conformation unformed and denaturation and inactivation protein, generality is useful as a highly universal Rifo over Rudingu method, its application is limited to a protein 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 E. coli are not formed. active protein, or the original function-activity of certain causes a protein conformation was deactivated by changing can be activated by Rifo over Rudingu. 2) This method is useful for inclusion body as efficiently Rifo over Rudingu method of. 3) applicable to a variety of proteins, generality, a universal, yet can provide a high efficient method of Rifo over Rudingu rate. 4) Zeolite beta, a functional activator used in the present invention, is low in cost and can be used repeatedly. 5) The method is applicable to Rifo over Rudingu various conformational disordered proteins containing large protein a molecular weight of more than 100,000. 6) For example, by combining the methods of protein synthesis process and the present invention by expression system of E. coli, the new active protein to produce a protein inherent function of the protein-specific conformation is controlled is exerted manufacturing process Can be built.

The result of the gel shift assay by electrophoresis is shown. Recovery of Rifo over Rudingu protein, and shows the recovery rate of the active. The result of the gel shift assay by electrophoresis is shown.

Claims (7)

A method of activating the function of a protein that is inactive due to disordered conformation, wherein the protein is contacted with zeolite beta in the presence of a protein denaturant, a surfactant and a refolding buffer , A method for activating a function of a protein, wherein the protein is activated so that the intrinsic function inherent to the protein can be expressed.   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 disorder of the 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. Proteins that are inactive due to disordered conformation are contacted with zeolite beta in the presence of a protein denaturant, a surfactant, and a refolding buffer to refold the three-dimensional structure of the protein. A method for modifying the main structure of a protein. Proteins that are inactive due to disordered conformation are brought into contact with zeolite beta in the presence of a protein denaturant, a surfactant and a refolding buffer to refold the conformation of the protein. A method for producing an active protein, characterized by producing a protein whose structure is controlled and whose intrinsic function inherent to the protein is activated. The method for producing a protein according to claim 6 , 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.