JP5692678B2 - Protein refolding condition setting kit - Google Patents

Description

  The present invention relates to a protein refolding screening method using zeolite (High-throughput protein) for easily and quickly setting the use conditions of reagents, substances, materials, etc. used in the function activation operation / process of an inactive protein. The present invention relates to a protein refolding screening system using a refolding screening method (ie, a “protein refolding condition setting kit”), and more particularly, a protein that is inactive due to the absence of higher-order structure, or certain types of proteins. Used in operations / processes that refold proteins that have been inactivated due to three-dimensional structure changes and activate / regenerate the intrinsic functions of the protein For use conditions such as drug-Materials, to a "protein refolding condition setting kit" as protein refolding screening system which allows to set easily and quickly the optimum or suitable refolding conditions. In the present specification, a high-throughput protein refolding screening method and system using zeolite is referred to as a protein refolding condition setting kit.

  The present invention, for example, in the technical fields of biochemicals and pharmaceutical production, refolds a higher-order unformed protein produced using a gene expression system such as Escherichia coli so that the original function / activity of the protein is improved. Protein refolding condition setting kit, which is a new high-efficiency protein refolding screening system capable of easily and quickly setting optimal or suitable conditions for activation, and new function activation using the conditions set thereby It provides new technology and new products.

  It is not a gene that actually acts and works in vivo, but a protein made from them. 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, various proteins are synthesized and produced by various methods, their structures are examined, and action mechanisms and roles in the living body are elucidated. Now, it is well known that the functions of proteins are determined not only by the sequence and chain length of the 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 the synthesis by insect cells and mammalian cells, the resulting protein is often soluble, with a higher order structure controlled, an ordered three-dimensional structure. However, these methods have a drawback that the operation of separation and purification 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 a production process is being established.

  However, when proteins of higher organisms such as humans are synthesized in the E. coli expression system, the protein as designed is obtained with respect to the binding order and number of amino acids, that is, the amino acid chain length, but the three-dimensional structure is not ordered, A protein whose higher-order structure is not controlled, that is, an insoluble protein called an inclusion body in which amino acid chains are entangled is obtained.

  This insoluble protein inclusion body naturally does not have the desired function and performance and does not exhibit activity. For this reason, in the production process using E. coli, it is necessary to unwind 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 by E. coli but also to the regeneration of proteins that have been inactivated due to certain causes such as thermal history. Therefore, conventionally, this refolding has been actively researched and various methods have been proposed.

  However, most of them are batch-based methods, and in addition to low refolding rates, they have only been able to give good results for certain limited proteins, especially for low molecular weight specific proteins. Often, this refolding is not an efficient and economical method with generality, universality, and high refolding rate applicable to various proteins.

  Under such circumstances, the present inventors have studied the selective adsorption phenomenon of biopolymers by inorganic oxides such as zeolite (see Non-Patent Document 1), and in this process, the adsorption and desorption to zeolite beta is inactive. It discovered that protein activated and came to develop the function activation method (refer patent document 1-4 and nonpatent literature 2) of the inactive protein using zeolite beta.

  However, in the methods described in Patent Documents 1-4 and Non-Patent Document 2, after a protein that has been denatured and solubilized by a denaturant is adsorbed on a zeolite having a BEA structure in the denaturant, After removing the denaturant and washing, there is diversity in the solution component called refolding buffer that dissociates the protein adsorbed in a denatured state on the BEA structure zeolite into the solution, Each individual protein has a problem that optimum or preferred conditions, that is, optimum or preferred values of solution components are different.

  In addition to the pure water component, the active component in the refolding buffer includes a pH adjustor as an essential component, and further, as a refolding control factor, a protein SS cross-linking formation control agent, a surfactant, a polyvalent It has been found that there are salts, protein denaturants, metal ions, etc., such as alcohols, sugar alcohols, chelating agents, arginine, NaCl. However, the examination of the optimum or suitable conditions including the difference in the concentration of each of these components takes a great deal of time and effort, and as a result, there is a problem that the range that can be studied is practically limited. there were.

Furthermore, the refolding agent made of a BEA-structured zeolite also changes the efficiency of refolding depending on whether the ratio of silicon to aluminum is changed or whether the zeolite is made into NH ₄ ⁺ or Na ⁺ form. It has been clarified, and the examination of the optimum or suitable conditions also takes time and effort, and as a result, there is a problem that the range that can be examined is practically limited.

  As described above, the refolding method using the BEA-structured zeolite is an effective method and can be applied as a refolding method for a wide variety of proteins. Time was a big problem.

  Therefore, in this technical field, regardless of the length of the chain, it can be applied to various high-order structure unformed and denatured / inactivated proteins, generality, high universality, low cost, high efficiency, Protein refolding screening system that can easily and quickly set the optimum or preferred conditions in a refolding method using a high-performance BEA structure zeolite that is easy to scale up, ie, a protein refolding condition setting method or kit Developing has become an urgent issue.

JP-A-2005-29531 JP 2005-192452 A Japanese Patent Laid-Open No. 2005-220121 JP 2005-281267 A

Chem. Eur. J. et al. , Vol. 7 (2001) 1555-1560 Anal. Biochem. , Vol. 348 (2006) 307-314

  In such a situation, in view of the above-mentioned prior art, the present inventors have arranged a higher-order structure of a protein that is inactive because the higher-order structure is disordered to obtain an active protein. Protein refolding condition setting kit as a protein refolding screening system that can easily and quickly set the optimum or preferred conditions of the refolding method, particularly the optimum or preferred conditions of the refolding buffer in the refolding method using zeolite. The present invention has been completed.

  An object of the present invention is to provide a protein refolding condition setting kit capable of easily and quickly setting optimum or suitable conditions for a refolding method using a BEA-structured zeolite. In addition, the present invention is applicable to various high-order structure unformed and denatured / inactivated proteins regardless of the length of the chain, and has generality, generality, low cost, high efficiency, and scale. It is intended to provide a new technology for setting protein refolding conditions as a high-throughput protein refolding screening system that can be applied to refolding methods using high-performance, high-performance BEA-structured zeolites. is there.

The present invention for solving the above-described problems comprises the following technical means.
(1) When carrying out protein refolding operations / steps for preparing a protein that is inactive due to disordered higher-order structure to make it active, a suitable refolding condition is derived easily and quickly. A protein refolding condition setting kit capable of
A refolding agent composed of a BEA structure zeolite that functions as a carrier adsorbed in a denaturant in a denatured state by a protein denaturant is an essential constituent,
Refolding agent made of the BEA structure zeolite, proteins adsorbed in a denatured state, a solution component to dissociate in solution, other pH adjusting agents of pure water component as essential components, further, Li folding regulators and / or surfactants, protein S-S bridge formation control agent consists of a combination comprising salts, proteins denaturing agents, or one or more metal ions,
Further includes integral test tube having a space of the plurality of divided same volume, or test plate, as a component, concurrently, Ri Kits Product der for testing by multiple criteria,
1) A BEA-structured zeolite is a zeolite in NH ₄ ⁺ or Na ⁺ form or a framework-substituted zeolite containing NH ₄ ⁺ or Na ⁺ , or a CP 811E-75, CP811E-150 or CP811E-300 zeolite in H ⁺ form. Yes,
2) The BEA-structured zeolite is granulated in a powdered state or particles,
3) The BEA-structured zeolite is immobilized in a powdered or particulate form in an integrated test tube or test plate having a plurality of divided spaces of the same volume,
4) The glue for granulating the BEA structured zeolite or the glue for immobilizing it on an integral test tube or test plate is a material that does not adversely affect the protein,
5) The integrated test tube or test plate having a plurality of divided spaces having the same volume is a 96-well microplate or a microplate having more holes, and each test tube or plate has a hole on it. respect, by the solution of each different configurations are injected, Ru can be tested by concurrently multiple conditions,
A protein refolding condition setting kit.
(2) The framework structure of the BEA structure zeolite is composed of oxygen and one or more other elements, or composed of only silicon and oxygen, or silicon, aluminum, and oxygen. Protein refolding condition setting kit.
( 3 ) The protein refolding condition setting kit according to (1) or (2) , wherein the protein denaturant is guanidine hydrochloride or urea.
( 4 ) The pH adjusting agent is trisaminomethane trihydrochloride (Tris-HCl), 3- (N-morpholino) propanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), or N-2-hydroxy The protein refolding condition setting kit according to any one of (1) to ( 3 ), wherein the kit is ethylpiperazine-N′-ginethanesulfonic acid (HEPES).
( 5 ) The protein ligand according to any one of (1) to ( 4 ), wherein the protein SS crosslinking formation control agent is cystine, cysteine, 2-mercaptoethanol, dithiothreitol, or thiophenol. Folding condition setting kit.
( 6 ) Refolding regulator and / or surfactant is polyethylene glycol (PEG20K, PEG3350, PEG800, PEG200), Ficol170, Ficol1400, arginine, EDTA, NaCl, polyphosphate, sodium dodecyl sulfate (SDS), sucrose, glucose , glycerol, inositol, cyclodextrin, amylose, Dextran T-500, Tween20, Tween40, Tween60, NP-40, SB3-14, is composed of SB12, CTAB, or one or more of the Triton X-100 The protein refolding condition setting kit according to any one of (1) to ( 5 ).
( 7 ) The protein refolding condition setting kit according to any one of (1) to ( 6 ), wherein the kit is an automatic protein refolding condition setting device that performs a series of operations automatically or semi-automatically.
( 8 ) Any of the above (1) to ( 7 ), wherein the basic components of the solution component, the microplate on which zeolite is dispensed or fixed, and the instruction manual describing the operating method are combined as a set of packages The protein refolding condition setting kit according to one item .

Next, the present invention will be described in more detail.
The present inventors have previously developed a versatile protein refolding technique utilizing the adsorption performance of zeolite having a BEA structure (Patent Documents 1-4 and Non-Patent Document 2). As disclosed in patent documents and non-patent documents related to this technique, this technique has an advantage that the processing time is short and the processing method does not depend on the difference in protein. In this method, the amino acid chain is once stretched and adsorbed on zeolite, and then refolding is caused by using a release agent. It is possible to deal with various proteins only by adjusting the buffer composition used for the release agent, and this can be said to be obvious from the disclosure of the above-mentioned patent documents and non-patent documents. Therefore, the protein refolding condition setting kit of the present invention also has an advantage that it does not depend on the difference in protein, and is capable of dealing with various proteins without being limited by the type of protein.

  In this method, for example, when the target protein is forcibly expressed in E. coli, most of the “expressed” (expression step) protein is accumulated as an insoluble inactive protein. After disrupting the cells, the insoluble protein is “recovered” (recovery step) with high purity by centrifuging. Next, the recovered inclusion body is “solubilized” (solubilization step) with a buffer containing guanidine hydrochloride or the like, which is a protein denaturing agent, and the protein solution containing the solubilized protein is mixed with the zeolite having the BEA structure. Proteins are “adsorbed” (adsorption step) on a BEA structured zeolite.

  Since zeolite is a solid, guanidine hydrochloride, etc. can be easily removed by simply “washing” (washing step) the protein-adsorbed zeolite with a small amount of buffer. Mix with buffer containing additives. As the protein is “eluted” (elution step), refolding occurs and an active protein can be obtained. FIG. 3 shows the refolding procedure of inert protein with zeolite.

  In order to impart versatility to this method, it is important to ensure applicability to various proteins. Therefore, in the present invention, a screening method and system capable of setting refolding conditions simply and quickly have been successfully constructed. Specifically, the properties of the buffer containing the elution additive are important, and a screening technique and system have been established that can easily and quickly set the concentration and pH setting conditions of polyethylene glycol, which is a basic component. In addition, a screening technique and system that can columnize the zeolite carrier and continuously process it were constructed. The present invention makes it possible to develop a protein function elucidation research as an infrastructure technology and construct a protein mass production system by using such screening methods and systems (FIG. 4).

  The protein that is the target of the protein refolding condition setting kit that searches for the optimal or suitable conditions for the functional activation of the inactive protein of the present invention is that the protein refolding method itself with the BEA structure zeolite does not depend on the difference in protein. Because of its advantages, it is an inactive protein that does not have a higher order structure. Generally, there is a protein with a disordered three-dimensional structure obtained in an expression system such as Escherichia coli, so-called inclusion body, or thermal history. Protein inactivated due to species.

  The kit of the present invention activates / provides the original function of the protein by refolding the three-dimensional structure of the protein in the process of adsorption / desorption to the refolding agent composed of the zeolite having the BEA structure. It is. The ability of the refolding agent is usually exhibited by the following operation, in other words, the function of the inactive protein is activated by the following operation.

  First, the protein is dispersed and dissolved in a solution containing a denaturing agent, and then mixed with the solution containing the refolding agent or injected into the column packed with the refolding agent to convert the protein into the refolding agent. The procedure is performed by adsorbing and then desorbing the protein from the refolding agent.

  As a protein dispersion solvent before adsorption to the refolding agent, it is generally produced in an expression system such as Escherichia coli, and the protein is usually used in an aqueous solution. Water is preferably used because it is often in an aqueous solution.

  However, the present invention is not necessarily limited to this, and there is basically no problem as long as the solvent does not react with the protein and the three-dimensional structure of the protein is not changed into an unintentional form. In some cases, these solvents can be used alone or mixed with water. As typical examples of this type of solvent, monohydric and polyhydric alcohols and saccharides can be exemplified, but are not limited thereto.

  The adsorption / desorption of the protein is generally performed by a denaturing agent, a surfactant, a pH adjuster, a refolding factor, etc. in order to make it easy to unravel the entangled protein chain length of the inclusion body, etc. Presence of certain protein S—S cross-linking regulators in order to cleave S—S bonds (cross-links) that are inadvertently generated in the presence and / or protein chain length, or to form appropriate cross-links Done below.

  Therefore, the kit of the present invention includes a refolding agent composed of a zeolite having a BEA structure, a denaturing agent and a pH adjusting agent as essential components, and in addition to these, the protein desorption promotion from the refolding agent is promoted. And a refolding factor and / or a surfactant for preventing re-inclusion body formation after refolding, and an SS crosslinking control agent.

  Typical examples of the denaturing agent in the kit of the present invention include guanidine hydrochloride and urea. Typical examples of the pH adjusting agent include, for example, trisaminomethane trihydrochloride (Tris-HCl), 3- (N-morpholino) propanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), and N- Examples include 2-hydroxyethylpiperazine-N′-ginethanesulfonic acid (HEPES). However, the present invention is not limited to these, and can be used in the same manner as long as they have similar actions.

  Typical examples of the refolding factor and surfactant in the kit of the present invention include polyethylene glycol (PEG20K, PEG3350, PEG800, PEG200), Ficol170, Ficol1400, arginine, EDTA, NaCl, polyphosphoric acid, sodium dodecyl sulfate ( SDS), sucrose, glucose, glycerol, inositol, cyclodextrin, amylose, Dextran T-500, Tween 20, Tween 40, Tween 60, NP-40, SB3-14, SB12, CTAB, Triton X-100 and the like. However, the present invention is not limited to these, and can be used in the same manner as long as they have similar actions.

  As an S—S cross-linking formation control agent that cleaves an unintentionally generated S—S cross-link and returns it to its original structure, it is usually easy to obtain at low cost, so usually 2-mercaptoethanol, dithiothreitol, cystine, Cysteine or thiophenol is used. However, the present invention is not limited to these, and can be used in the same manner as long as they have similar actions.

  If the protein chain length is easily unraveled or if S—S crosslinks are not generated unintentionally, of course, it is not always necessary to use a denaturant, a surfactant, and / or an inhibitor. It is not always essential, and it is appropriately selected according to the situation and additionally used as a kit. Moreover, the amount of the components constituting the kit is appropriately determined according to the situation when using the kit.

  The kit of the present invention usually comprises a refolding agent comprising a zeolite having a BEA structure, guanidine hydrochloride (modifier), trisaminomethane trihydrochloride (Tris-HCl), 2-morpholinoethanesulfonic acid (MES) (pH adjustment) Agent). Further, among the above-described refolding factor protein SS cross-linking control agent, surfactant, polyhydric alcohol, sugar alcohol, chelating agent, salts such as arginine, NaCl, protein denaturant, metal ions, necessary ones Can be additionally added.

  In general, in the protein desorption process during the refolding operation / step by this kit, substitution adsorption by a reagent in the kit, that is, a folding factor or a surfactant is usually performed. However, basically, any operation is applicable as long as it does not hinder the function activation after desorption of the protein, and is not particularly limited. Therefore, pH change, temperature change, etc. can be used, and these and substitution adsorption can also be used together.

  By the way, as a substance that promotes desorption of the protein during substitution adsorption, conventionally, salts such as alkali halides have been frequently used in column chromatography elution. May be. In addition, arginine is used as a substance that has an effect of preventing refolding of the protein from causing aggregation again, and there are cases where a remarkable effect can be obtained by using these in combination.

  The zeolite having a BEA structure (commonly known as zeolite beta, Zeorite Beta) that constitutes the protein refolding condition setting kit of the present invention is also called β-zeolite. Zeolites, Vol. 11 (1991) 202), BEA-structured zeolites synthesized and prepared in-house, and BEA-structured zeolites obtained by calcining them.

  Furthermore, a zeolite having a BEA structure having ammonium or various aliphatic and / or aromatic ammonium in the space of the zeolite, and a framework-substituted BEA structure in which a part of the framework silicon forming the zeolite is changed to another metal Examples of the zeolite include the above-mentioned ammonium-containing skeleton-substituted BEA structure zeolite. Basically, if it has the framework structure of the zeolite of BEA structure, it has the function and ability and can be used, and the refolding column packing material and the zeolite of the BEA structure constituting the column However, the manufacturing method and properties are not particularly limited.

  However, the function / capability of the zeolite having the BEA structure constituting the refolding column packing material and the column of the present invention is exhibited by inert and contact of the modified protein with the zeolite, that is, adsorption / desorption. The affinity between the zeolite surface of the BEA structure and the target protein is important.

  In addition, the adsorption / desorption of the protein is often affected by the dispersion solvent, the denaturing agent or surfactant in the dispersion solvent, the refolding factor, and the pH of the dispersion solvent. The refolding performance of the protein refolding condition setting kit of the present invention may vary somewhat depending on the various zeolites having the BEA structure constituting the protein, depending on the component status of the protein and the solution containing the protein.

Generally speaking, a refolding column packing composed of a BEA-structured zeolite containing ammoniums or Na ⁺ exhibits higher refolding performance than those not containing it. Accordingly, examples of the refolding column filler and column, those constituted by the skeleton substituted BEA structure zeolite containing zeolite and ammonium compounds or Na ⁺ of BEA structure including ammoniums or Na ⁺ are preferred.

  As the ammoniums to be contained in the zeolite having the BEA structure of the protein refolding condition setting kit of the present invention, ammoniums that are likely to remain in the space of the zeolite, for example, ammonium ions, alkyl groups are methyl, ethyl, Mono, di, tri and tetraalkylammonium ions such as propyl and butyl, as well as ammonium ions of 5-membered, 6-membered and 7-membered aliphatic amines and aromatic amines.

  Specific examples include piperidium ions, alkyl piperidium ions, pyridinium ions, alkylpyridium ions, aniline ions, N-alkylaniline ions, and the like. Basically, any ammonium may be used as long as it can enter the pores of the BEA structure zeolite, and the ammonium is not limited to those exemplified here.

  The elements that form the framework of the BEA structure zeolite in the protein refolding condition setting kit of the present invention are generally silicon and oxygen, or silicon, oxygen, and aluminum. The substituted BEA structured zeolite and the substituted BEA structured zeolite containing the ammoniums in the pores also basically have an activity activation function of the inactive protein. Typical examples of the BEA-structured zeolite that can be replaced with skeleton silicon or aluminum include boron, phosphorus, gallium, tin, titanium, iron, cobalt, copper, nickel, zinc, chromium, vanadium, zirconium, hafnium, and the like. be able to.

  However, the present invention is not limited to these, and basically any one may be used as long as it does not destroy the structure of the zeolite having the BEA structure. Further, regarding the substitution amount, the substitution amount may be any amount as long as it does not destroy the structure of the zeolite having the BEA structure, and the substituted BEA structure zeolite has an inactive or defolding function of the modified protein. It can be a protein refolding condition setting kit to be exhibited.

  As described above, in the refolding method using the BEA structure zeolite, the solution components used for eluting the protein adsorbed on the BEA structure zeolite in the modified state also in the BEA structure zeolite as the refolding agent. In addition, various conditions can be set. The protein refolding condition setting kit of the present invention is often a great convenience in order to easily and quickly set these optimum or preferred conditions.

  In the protein refolding condition setting kit of the present invention, in addition to a pure water component, a solution component that dissociates a protein adsorbed in a denatured state into a refolding agent composed of a zeolite having a BEA structure into a solution, A pH regulator is an essential constituent, and further, protein SS cross-linking formation regulator as a refolding regulator, surfactant, polyhydric alcohol, sugar alcohol, chelating agent, arginine, NaCl, etc., salts, protein denaturation It is set as the combination containing 1 or more types among an agent and a metal ion.

  Furthermore, in order to set the optimum or preferred conditions from among the condition settings whose concentration has been changed, an integrated test tube having a plurality of spaces of the same volume divided in advance or prior to evaluation, or a test For plates, eg, 96-well microplates, or microplates with more holes, or similar or functionally similar, each test tube or solution of different configuration for each hole on the plate By injecting, a test under a plurality of conditions can be performed concurrently.

  In the protein refolding condition setting kit of the present invention, when there is no particular problem or trouble such as a reaction between kit constituent reagents, some of the kit constituent reagents may be combined in advance and used as one reagent. it can. For example, a solution consisting of Tris-HCl, alkali halide, i.e., sodium chloride, refolding factor, e.g., one of the polyethylene glycols PEG20K, PEG3350, PEG800, PEG200 and arginine, is religated. It is also possible to collect them as a folding buffer and to be one of the kit constituent reagents of the present invention.

  Therefore, in addition to the pure water component injected into an integrated test tube or a test plate having a plurality of divided spaces of the same volume, for example, a 96-well microplate, a pH adjuster is an essential component, and Protein SS cross-linking formation regulator as a folding regulator, surfactant, polyhydric alcohol, sugar alcohol, chelating agent, arginine, NaCl, etc., a combination containing at least one of salts, protein denaturants, metal ions And Furthermore, various solutions with different concentrations can be prepared and attached in advance, or solutions for each component can be mixed by a predetermined operation and prepared before evaluation. is there.

In the protein refolding condition setting kit of the present invention, the ratio of silicon and aluminum of the refolding agent made of a zeolite having a BEA structure is changed, or the condition setting is changed to a zeolite with NH ₄ ⁺ or Na ⁺ form. It is possible to use an integrated test tube or a test plate having a plurality of divided spaces having the same volume, which are set in advance or before the evaluation.

  For example, for 96-well microplates, or microplates with more holes, or similar or similar functions, for each test tube or hole on the plate, a differently configured BEA structure By injecting or fixing a refolding agent made of zeolite, it becomes possible to perform tests under a plurality of conditions simultaneously.

  Dispensing a refolding agent composed of a BEA-structured zeolite into a plurality of divided test tubes or test plates having the same volume of space, for example, 96-well microplates, is a relatively inefficient operation. For this reason, it is preferable to inject or immobilize a refolding agent made of a zeolite having a different BEA structure into each test tube or hole on the plate in advance.

  For example, a refolding agent composed of a BEA-structured zeolite used in the protein refolding condition setting kit of the present invention is formed into a single-piece test tube or test plate having a plurality of divided spaces of the same volume, which are formed into particles. If dispensed, the optimum or suitable conditions can be examined simultaneously in parallel without performing an operation such as centrifugation.

  In addition, the refolding agent comprising a zeolite having a BEA structure used in the protein refolding condition setting kit of the present invention is immobilized in a powdered state in an integrated test tube or a test plate having a plurality of divided spaces of the same volume. If so, the optimum or preferred conditions can be examined simultaneously in parallel without performing an operation such as centrifugation.

  The function of the protein refolding condition setting kit of the present invention can be used as an automatic protein refolding condition setting device capable of automatically performing work on the kit.

  In order to improve convenience or operability, the protein refolding condition setting kit of the present invention includes basic constituent materials of solution components such as buffers, a microplate on which zeolite has already been dispensed or fixed, and the like. It is possible to supply instruction manuals, manuals, etc. describing operation methods in a refolding kit form as a set of packages, and to optimize or optimize the refolding conditions with high throughput, and the BEA structure Thus, it is possible to easily use the refolding method comprising zeolite of (Fig. 5).

  The protein refolding condition setting kit of the present invention can easily and quickly set the optimum or preferred conditions when applying an efficient refolding method comprising a BEA structure zeolite. Due to the synergistic effect with the ease of scale-up possessed by the refolding method, it is possible to activate the original functions of a large amount of a wide range of inactive proteins regardless of the type of protein.

  In the present invention, for example, due to the synergistic effect of the protein synthesis process by the expression system of E. coli and the protein refolding condition setting kit of the present invention, and the ease of scale-up possessed by the refolding method comprising a BEA-structured zeolite, Since it is possible to propose and establish a novel active protein production process in which a higher-order structure is controlled and a protein having an intrinsic function inherent to the protein can be proposed, the kit of the present invention and the function activation method of a denatured protein are For example, it is extremely useful for biochemical product manufacturing and pharmaceutical manufacturing, and its technical and economic effects are immeasurable.

  In the present invention, in the refolding method using a BEA-structured zeolite that is effective as a method for activating the function of an inactive protein, the use conditions of reagents, substances, materials, etc. are optimally set quickly or simply by a simple operation. It is possible to do. The present invention relates to an operation for refolding a protein that is inactive because a higher-order structure has not been formed, or a protein that has been inactivated due to a change in a three-dimensional structure, and that activates and regenerates the inherent function inherent to the protein. -It is possible to efficiently set usage conditions for reagents, substances, materials, etc. used in the process, and to set optimal or suitable refolding conditions simply and quickly. About this technique, Table 7 mentioned later shows the comparison with the conventional method in protein refolding technique.

  Conventionally, proteins obtained by expression systems such as Escherichia coli generally have a problem in that the three-dimensional structure is disordered, the original functions and physical properties of the protein are not present, and the activity is not exhibited. , A predetermined function / activity that makes it possible to easily and quickly set optimum or preferred conditions in a refolding method using a BEA-structured zeolite that activates the function / activity of an inactive protein typified by the above protein. It enables efficient operation of innovative protein production and production technology for regenerating protein having a protein content.

The present invention has the following effects.
(1) Optimal or suitable refolding conditions when applying a refolding method with a BEA-structured zeolite (zeolite beta / β-zeolite) that activates the original functions of a wide range of inactive proteins A refolding condition setting kit can be provided as a high-throughput protein refolding screening method and system for simple and rapid setting.
(2) For proteins that are inactive because the higher-order structure produced in an expression system such as Escherichia coli is not formed, or for proteins that have been inactivated due to a change in steric structure due to a certain cause, their original functions It is possible to select optimum or suitable conditions when applying a refolding method using a BEA-structured zeolite that activates.
(3) Optimum or suitable conditions can be selected when the refolding method using BEA-structured zeolite is applied to the inclusion body protein.
(4) By changing the ratio of silicon and aluminum in the refolding agent made of BEA structure zeolite or changing to NH ₄ ⁺ or Na ⁺ form zeolite, the refolding method using the BEA structure zeolite is applied. With regard to the conditions of the refolding agent, the optimum or preferred conditions can be studied in parallel.

(5) As a refolding control factor in addition to a pure water component and a pH adjuster, a solution component that dissociates the protein adsorbed in a denatured state into a refolding agent composed of a BEA structure zeolite into the solution. Optimal or suitable conditions for the contained components can be studied in parallel.
(6) An integrated test tube having a plurality of divided spaces having the same volume, or a test plate, for example, a 96-well microplate, or a microplate having more holes, or similar or similar functions As for the conditions, the optimum or suitable conditions can be studied in parallel.
(7) Centrifugation, etc., by refolding the refolding agent composed of BEA-structured zeolite into a powdered or particulate form and dispensing it into a plurality of divided integrated test tubes or test plates having the same volume The operation of can be omitted.

(8) The kit of the present invention can be used as an automatic protein refolding condition setting system and apparatus capable of performing operations automatically.
(9) To provide a kit in the form of a package consisting of a basic component material of a solution component, a microplate on which zeolite has already been dispensed or fixed, and an instruction manual describing the operating method. be able to.
(10) Due to the synergistic effect with the ease of scale-up of the refolding method using BEA-structured zeolite, their original functions can be activated for a wide range of inactive proteins, regardless of the type of protein. It is possible to establish a novel active protein production process in which a higher-order structure is controlled to produce a protein having an intrinsic function unique to the protein.

The result of having measured the fluorescence intensity is shown. The result of having measured enzyme activity is shown. The explanatory drawing of the refolding procedure by the zeolite of an inactive protein is shown. The explanatory drawing of an example of the protein refolding condition setting kit and apparatus by the zeolite of a BEA structure is shown. An explanatory view of an example of a refolding kit is shown. The created refolding matrix is shown. The created refolding matrix is shown. The created refolding matrix is shown. The influence by the kind of zeolite is shown.

  Next, the present invention will be specifically described based on examples, but the present invention is not limited by the following examples.

  In this example, the refolding conditions of GFP (Green Fluorescent Protein) under 96 conditions were examined using a protein refolding condition setting kit using a 96-well deep well plate.

(1) Configuration of Kit Zeolite of 930 NHA (manufactured by Tosoh); NH ₄ type (NH ₄ ⁺ form) was used for the kit. 30 mg of this zeolite was dispensed into each hole of a 96-well deep well plate, and a 96-well deep well plate cap was used as an accessory.

  Table 1 shows the buffer components affecting the studied refolding. The protein refolding condition setting kit used in this example was constructed in order to examine the influence of each component shown in Table 1. Specifically, due to the difference in pH adjuster, it is possible to simultaneously examine four conditions of 6.5, 7.5, 8.0, and 8.5 for pH, and for salts, the presence or absence of NaCl 500 mM. The additive was configured as a requirement that glycerol 20% (v / v) and guanidine hydrochloride 1M can be examined for the presence or absence of addition.

  Moreover, about surfactant, it can consider about the presence or absence of addition of Tween20 0.2% (v / v), about SS bridge | crosslinking formation control agent (Redox reagent), 2-mercaptoethanol 10 mM and cysteine 10 mM. + Cystine It was configured as a requirement that the presence or absence of addition of 1 mM could be examined simultaneously.

The basic solution (buffer) structure used in this example was as follows.
1. Denaturation buffer: 20 mM Tris-HCl pH 7.5, 6 M guanidine hydrochloride, 0.5 M NaCl, 10 mM 2-mercaptoethanol Washing buffer: 1 mM Tris-HCl pH 7.5

3. pH 6.5 basic buffer: 125 mM MES-Na (pH 6.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 2.5 mM EDTA
4). pH 7.5 basic buffer: 125 mM Tris-HCl (pH 7.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 2.5 mM EDTA

5. pH 8.0 basic buffer: 125 mM Tris-HCl (pH 8.0), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 2.5 mM EDTA
6). pH 8.5 basic buffer: 125 mM Tris-HCl (pH 8.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 2.5 mM EDTA

  The composition of the salts, additives, surfactant, and S—S cross-linking formation control agent consisted of 8M guanidine hydrochloride, 5M NaCl, 66.7% (v / v) glycerol, 10% (v / v) Tween 20, 200 mM L- Cysteine-HCl, 200 mM cystine, 182 mM 2-mercaptoethanol.

(2) Instrument to be used The composition of the instrument to be used was an 8 pipette (20 μl), an 8 pipette (300 μl), an 8 pipette (1250 μl), a 12 pipette (300 μl), and a reservoir. The reservoir can be set as an accessory of the kit.

(3) Method First, 96 conditions of refolding buffer were prepared. Various buffers were dispensed into a 96-well deep well plate for preparing a refolding buffer by the following procedure. Table 2 shows the buffer components of each hole when a 96-well microplate is used. According to the following procedure, 96 different refolding buffers as shown in Table 2 were prepared, and 96 refolding conditions were simultaneously examined using this.

1. 110 μl each of ddH ₂ O, using 8 pipettes (300 μl), A, B, C, D, E, F, G in rows 1, 2, 3, 4, 5, 6 of 96-well deep well plate , H row.
2. 22 μl each of ddH ₂ O using 12 pipettes (300 μl), 1, 2, 3, 4, 5, 6, 7, 8, in rows A, C, E, G of 96-well deep well plate Added to 9, 10, 11, 12 rows.

3. 138 μL of ddH ₂ O was added to each of A, B, C, D, and E in 96-well deep well plates on rows 1, 3, 4, 5, 7, 9, 10, and 12 using an 8-unit pipette (300 μl). , F, G, H rows.
4). 330 μl each of ddH ₂ O, using 8 pipettes (300 μl), 1, 2, 4, 5, 7, 8, 10, 11 rows A, B, C, D, E of 96-well deep well plate , F, G, H.

5. Add 440 μl of the pH 6.5 basic buffer of 3 above to 6 pipettes (1250 μl) and attach 6 tips to 96-well deep well plates A, B rows 1, 2, 3, 4, 5, 6 rows And A, B columns 7, 8, 9, 10, 11, 12 rows.
6). 440 μl each of the above pH 7.5 basic buffer of 4 with 6 tips attached to an 8 pipette (1250 μl), 1, 2, 3, 4, 5, 6 rows in C and D columns of a 96-well deep well plate And C, D columns 7, 8, 9, 10, 11, 12 rows.

7). Add 440 μl of pH 8.0 basic buffer of 5 above to 6 pipettes (1250 μL) to 8 pipettes, and 1, 2, 3, 4, 5, 6 in E and F rows of 96-well deep well plate. Added to row and rows 7, 8, 9, 10, 11, 12 of E, F columns.
8). Attach 6 tips of pH 8.5 basic buffer of 6 above in 440 μl, 8 pipettes (1250 μl), G, H rows 1, 2, 3, 4, 5, 6 of 96 well deep well plate. Added to rows and 7, 8, 9, 10, 11, 12 rows of G, H columns.

9. 110 μl each of the above 8 5M NaCl, using 8 pipettes (300 μl), A, B, C, D, E, F in rows 7, 8, 9, 10, 11, 12 of 96-well deep well plate , G, H row.
10. 22 μl each of the above 10% 10% (v / v) Tween 20 using a 12-well pipette (300 μl), 1, 2, 3, 4, 5 in B, D, F, H rows of a 96-well deep well plate , 6, 7, 8, 9, 10, 11, 12 lines.

11. 138 μl of each of the above 7 8M guanidine hydrochloride using an 8 pipette (300 μl), A, B, C, D, E, F, G, A, B, C, D, E, F, G of 96-well deep well plate Added to row H.
12 330 μl each of the above 96.7% (v / v) glycerol, using 8 pipettes (1250 μl), A, B, C, D in rows 3, 6, 9, 12 of a 96-well deep well plate , E, F, G, H rows.

13. 55 μl each of the above-mentioned 11 200 mM L-cysteine-HCl using an 8 pipette (300 μl), A, B, C, D in rows 4, 5, 6, 10, 11, 12 of a 96-well deep well plate , E, F, G, H rows.
14 5.5 μl of each of the above-mentioned 12 200 mM cystines using an 8 pipette (20 μl), A, B, C, D, E in rows 4, 5, 6, 10, 11, 12 of a 96-well deep well plate , F, G, H rows.

15. 60.5 μl of each of the above 13 182 mM 2-mercaptoethanol was added to the 1, 2, 3, 7, 8, 9 rows of A, B, C, Added to D, E, F, G, H.
16. After capping, tumbling and mixing, the mixture was centrifuged at 4 ° C. and 3000 rpm for 1 minute.

  Next, as for GFP to be evaluated, 96 mL or more was prepared in order to inject 1 mL of each GFP solution, which was denatured and dissolved in 6 M guanidine hydrochloride, at a concentration of 1 mg / mL into 96 wells. 30 mg of zeolite was dispensed into each well, and 1 ml of the above 1 denaturing buffer was added to each well of a 96-well deep well plate to equilibrate.

  Centrifugation was performed at 3,000 rpm for 5 minutes, and the supernatant was discarded. Add 1 mL of 1 mg / mL denatured GFP to each hole, cover the 96-well deep well plate, turn it upside down, fully suspend the zeolite, and rotate it with a rotator for 1 hour. GFP was adsorbed on the zeolite.

  After centrifuging a 96-well deep well plate at 3,000 rpm for 5 minutes, discarding the supernatant, washing with 1 mL of the above washing buffer 5 times in total, and removing the supernatant by the fifth wash Further, the remaining liquid was removed as much as possible by centrifuging at 3,000 rpm for 1 minute. In this state, the wet zeolite and the modified GFP adsorbed thereon remained, and this was used as a 96-well deep well plate for studying conditions.

  From each hole of the 96-well deep well plate for preparing the refolding buffer containing 96 kinds of refolding buffers prepared in advance, 1 mL of the refolding buffer is added to the 96-well deep well plate for condition examination. Added to move. This operation was performed with 12 pipettes or 8 pipettes. Each hole was capped and vortexed. Refolded by rotating overnight at 4 ° C. Centrifugation was performed at 3,000 rpm for 5 minutes, and the activity of the supernatant was measured.

  For the activity measurement, fluorescence intensity was measured at Ex: 486 nm and Em: 511 nm using a CORONA Microplate Reader SH-8000. A soluble GFP solution was diluted with a refolding buffer, and the fluorescence intensity was measured at 0, 1.875, 3.75, 7.5, 15, 30, 60 μg / mL, and a calibration curve was prepared.

  Refolding sample was measured as it was. After refolding sample and standard (denatured GFP) having high activity were precipitated by TCA, the protein concentration was measured by the BCA method, and the recovery rate and the activity recovery rate were calculated.

(4) Results The results are shown in FIG. FIG. 1 summarizes the results of measuring the fluorescence intensity. Table 3 shows the numerical results obtained by measuring the fluorescence intensity. Table 4 shows the results of measuring the protein concentrations of C1, C7, E1, E4, E7, E10, G1, G4, G7, G10, H3, and H9 having high activity, and calculating the recovery rate and the activity recovery rate. Show.

  The recovery rate and activity recovery rate of E7 and E10 were the best. E7 and E10 have substantially the same conditions, and E10 is obtained by changing 2-mercaptoethanol, which is the redox reagent of the E7 buffer, to Cysteine / Cystein.

  GFP may have no S—S bond, but it can be seen that there is no difference in the effect of 2-mercaptoethanol 10 mM and cysteine 10 mM + cystine 1 mM with respect to the S—S cross-linking formation regulator (Redox reagent). . The optimum conditions for GFP refolding were as follows, the recovery rate was 38.8%, and the activity recovery rate was 26.9%.

(GFP refolding conditions)
pH 8.0
0.5M Arginine 0.5% Polyethylene glycol 20000
0.5M NaCl
10 mM 2-mercaptoethanol or 10 mM cysteine + 1 mM cystine 2.5 mM EDTA

  In this example, the refolding conditions of GK (glucokinase) under 96 conditions were examined using a protein refolding condition setting kit using a 96-well deep well plate.

(1) Structure of kit Zeolite fixed to 930 NHA (manufactured by Tosoh); NH ₄ type (NH ₄ ⁺ form) was used for the kit. 30 mg of this zeolite was dispensed into each hole of a 96-well deep well plate. A 96-well deep well plate cap was set as an accessory.

  Table 5 shows the buffer components affecting the studied refolding. The protein refolding condition setting kit used in this example was constructed in order to examine the influence of each component shown in Table 5. Specifically, due to the difference in pH adjuster, it is possible to simultaneously examine four conditions of 6.5, 7.5, 8.0, and 8.5 for pH, and for salts, the presence or absence of NaCl 500 mM. The additive was configured as a requirement that glycerol 20% (v / v) and guanidine hydrochloride 1M can be examined for the presence or absence of addition.

  For surfactants, it is possible to examine the presence or absence of addition of Tween 20 0.2% (v / v), and for SS crosslinking formation control agent (Redox reagent), DTT 1 mM and cysteine 10 mM + cystine 1 mM are added. As a requirement, it is possible to examine whether or not there is a problem at the same time.

The configuration of basic solutions (buffers) used in this example was as follows.
1. Denaturation buffer: 20 mM Tris-HCl pH 7.5, 6 M guanidine hydrochloride, 0.5 M NaCl, 10 mM 2-mercaptoethanol Washing buffer: 1 mM Tris-HCl pH 7.5

3. pH 6.5 basic buffer: 125 mM MES-Na (pH 6.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 5 mM MgCl 2
4). pH 7.5 basic buffer: 125 mM Tris-HCl (pH 7.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 5 mM MgCl ₂

5. pH 8.0 basic buffer: 125 mM Tris-HCl (pH 8.0), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 5 mM MgCl ₂
6). pH 8.5 basic buffer: 125 mM Tris-HCl (pH 8.5), 1.25% (w / v) polyethylene glycol 20000, 1.25 M arginine, 5 mM MgCl ₂

  The composition of the salts, additives, surfactant, and S—S cross-linking formation control agent consisted of 8M guanidine hydrochloride, 5M NaCl, 66.7% (v / v) glycerol, 10% (v / v) Tween 20, 200 mM L- Cysteine-HCl, 200 mM cystine, 18.2 mM DTT.

(2) Instrument to be used The composition of the instrument to be used was an 8 pipette (20 μl), an 8 pipette (300 μl), an 8 pipette (1250 μl), a 12 pipette (300 μl), and a reservoir. The reservoir can be set as an accessory of the kit.

(3) Method First, 96 conditions of refolding buffer were prepared. Various buffers were dispensed into a 96-well deep well plate for preparing a refolding buffer by the following procedure. Table 6 shows the buffer components in each hole when a 96-well microplate is used. According to this procedure, 96 different refolding buffers as shown in Table 6 were prepared, and 96 refolding conditions were simultaneously examined using this.

1. 110 μl each of ddH ₂ O, using 8 pipettes (300 μl), A, B, C, D, E, F, G in rows 1, 2, 3, 4, 5, 6 of 96-well deep well plate , H row.
2. 22 μl each of ddH ₂ O using 12 pipettes (300 μl), 1, 2, 3, 4, 5, 6, 7, 8, in rows A, C, E, G of 96-well deep well plate Added to 9, 10, 11, 12 rows.

3. 138 μL of ddH ₂ O was added to each of A, B, C, D, and E in 96-well deep well plates on rows 1, 3, 4, 5, 7, 9, 10, and 12 using an 8-unit pipette (300 μl). , F, G, H rows.
4). 330 μl each of ddH ₂ O, using 8 pipettes (300 μl), 1, 2, 4, 5, 7, 8, 10, 11 rows A, B, C, D, E of 96-well deep well plate , F, G, H.

5. Add 440 μl of the pH 6.5 basic buffer of 3 above to 6 pipettes (1250 μl) and attach 6 tips to 96-well deep well plates A, B rows 1, 2, 3, 4, 5, 6 rows And A, B columns 7, 8, 9, 10, 11, 12 rows.
6). 440 μl each of the above pH 7.5 basic buffer of 4 with 6 tips attached to an 8 pipette (1250 μl), 1, 2, 3, 4, 5, 6 rows in C and D columns of a 96-well deep well plate And C, D columns 7, 8, 9, 10, 11, 12 rows.

7). Add 440 μl of pH 8.0 basic buffer of 5 above to 6 pipettes (1250 μL) to 8 pipettes, and 1, 2, 3, 4, 5, 6 in E and F rows of 96-well deep well plate. Added to row and rows 7, 8, 9, 10, 11, 12 of E, F columns.
8). Attach 6 tips of pH 8.5 basic buffer of 6 above in 440 μl, 8 pipettes (1250 μl), G, H rows 1, 2, 3, 4, 5, 6 of 96 well deep well plate. Added to rows and 7, 8, 9, 10, 11, 12 rows of G, H columns.

9. 110 μl each of the above 8 5M NaCl, using 8 pipettes (300 μl), A, B, C, D, E, F in rows 7, 8, 9, 10, 11, 12 of 96-well deep well plate , G, H row.

10. 22 μl each of the above 10% 10% (v / v) Tween 20 using a 12-well pipette (300 μl), 1, 2, 3, 4, 5 in B, D, F, H rows of a 96-well deep well plate , 6, 7, 8, 9, 10, 11, 12 lines.

11. 138 μl of each of the above 7 8M guanidine hydrochloride using an 8 pipette (300 μl), A, B, C, D, E, F, G, A, B, C, D, E, F, G of 96-well deep well plate Added to row H.
12 330 μl each of the above 96.7% (v / v) glycerol, using 8 pipettes (1250 μl), A, B, C, D in rows 3, 6, 9, 12 of a 96-well deep well plate , E, F, G, H rows.

13. 55 μl each of the above-mentioned 11 200 mM L-cysteine-HCl using an 8 pipette (300 μl), A, B, C, D in rows 4, 5, 6, 10, 11, 12 of a 96-well deep well plate , E, F, G, H rows.
14 5.5 μl of each of the above-mentioned 12 200 mM cystines using an 8 pipette (20 μl), A, B, C, D, E in rows 4, 5, 6, 10, 11, 12 of a 96-well deep well plate , F, G, H rows.

15. 60.5 μl of each of the above 13 182 mM 2-mercaptoethanol was added to the 1, 2, 3, 7, 8, 9 rows of A, B, C, Added to D, E, F, G, H.
16. After capping, tumbling and mixing, the mixture was centrifuged at 4 ° C. and 3000 rpm for 1 minute.

  Next, as for GK to be evaluated, 96 mL or more was prepared in order to inject 1 mL of each GK solution denatured with 6 M guanidine hydrochloride or the like at a concentration of 1 mg / mL into each 96-well. 30 mg of zeolite was dispensed into each well, and 1 ml of the above 1 denaturing buffer was added to each well of a 96-well deep well plate to equilibrate.

  Centrifugation was performed at 3,000 rpm for 5 minutes, and the supernatant was discarded. Add 1 mL of 1 mg / mL denatured GK to each hole, cover the 96-well deep well plate well, invert it upside down, fully suspend the zeolite, rotate it with a rotator for 1 hour, GK was adsorbed on the zeolite.

  After centrifuging a 96-well deep well plate at 3,000 rpm for 5 minutes, discarding the supernatant, washing with 1 mL of the above washing buffer 5 times in total, and removing the supernatant by the fifth wash Further, the remaining liquid was removed as much as possible by centrifuging at 3,000 rpm for 1 minute. In this state, wet zeolite and modified GK adsorbed on the zeolite remained, and this was used as a 96-well deep well plate for studying conditions.

  From each hole of the 96-well deep well plate for preparing the refolding buffer containing 96 kinds of refolding buffers prepared in advance, 1 mL of the refolding buffer is added to the 96-well deep well plate for condition examination. Added to move. This operation was performed with 12 pipettes or 8 pipettes. Each hole was capped and vortexed. Refolded by rotating overnight at 4 ° C. After centrifuging at 3,000 rpm for 5 minutes, the activity of the supernatant was measured.

The activity was measured according to the following procedure. First, the reaction substrate, which is a mixture of D-glucose and ATP, was evaluated by a reaction that becomes D-glucose 6-phosphate and ADP by GK, and the following substrate mixed solution for activity measurement was prepared.
(Substrate mixed solution)
100 mM Tris-HCl pH 7.0
2 mM ATP
10 mM MgCl ₂
1 mM DTT
500μM NADP
50 mM D-glucose

The following procedure was followed.
1. The refolded sample was diluted 10 times.
2. The diluted sample was dispensed into a Corning UV transparent 96-well plate.
3. 200 μl each of the substrate mixed solution was dispensed into a 96-well plate, and OD340 was measured for 10 minutes every minute using a CORONA Microplate Reader SH-8000 (24 samples were measured).

The procedure for measuring 24 samples was as follows. 1st (A, B) (1-12), 2nd (C, D) (1-12), 3rd (E, F) (1-12), 4th (G, H) (1-12 ).
4). Activity was calculated by determining the slope of the graph.

The calculation formula is shown below.
Units / mL enzyme = (ΔA340 nm / min Sample−ΔA340 nm / min Blank) (0.21 mL) (df) / (6.22) (0.01)
Here, 6.22 = Milli molar extension of NADPH at 340 nm, 0.01 = Volume (mL) of enzyme used.

(4) Results The results are shown in FIG. It can be seen that the activity was high under the conditions of C2, D2, E2, F2, C8, D8, E8, and F8.

  The recovery rate and activity recovery rate were the best under the conditions of F8, but as a common term for the conditions of C2, D2, E2, F2, C8, D8, E8, and F8, the modifier guanidine hydrochloride It can be seen that 2-mercaptoethanol, which is an S—S crosslink formation control agent, is highly effective. The preferred conditions for GK refolding were as follows, with a recovery rate of 52.0% and an activity recovery rate of 17%.

(GK refolding conditions)
pH 8.0
0.5M Arginine 0.5% Polyethylene glycol 20,000
0.5M NaCl
0.2% Tween20
1M guanidine hydrochloride 5 mM MgCl ₂
1 mM DTT

In this example, creatine kinase was refolded.
(1) Cloning and expression of creatine kinase An EST clone (clone ID: MHS1011-60128) containing a human creatine kinase gene was purchased from Open biosystems. Using this EST clone as a template, the creatine kinase gene was amplified using iProof High-Fidelity DNA polymerase. In addition, the following sequences were used as primers.
Forward primer: aaaaaaCATATGCTGGTCCCTTCTCCC
Reverse primer: aaaaaaGTCGCACATGCTGTGTGTGGATGACAGGG

  After the amplified gene was cloned into the pBluescript II (SK +) vector, the DNA sequence was confirmed. The DNA containing the correct sequence was cleaved with restriction enzymes NdeI and XhoI and subcloned into the pET21a vector (Novagen) to prepare an expression clone. This expression clone was transformed into the BL21star (DE3) strain.

  The creatine kinase expression construct was cultured overnight at 37 ° C. in 30 mL of LB-1.5% glucose-100 μg / mL ampicillin medium. 20 mL of the preculture was transferred to 2 L of turbo broth-1.5% glucose-100 μg / mL ampicillin medium and cultured at 37 ° C. until OD600 was 0.6. IPTG was added so that the final concentration was 100 μM, and further cultured at 37 ° C. for 4.5 hours.

  The cells were collected by centrifugation, and further washed with TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). This was suspended in 80 mL of 0 M imidazole buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl), immersed in liquid nitrogen, frozen, and stored at −80 ° C.

(2) Purification of creatine kinase After the frozen cells were dissolved, the following reagents were added and incubated at 4 ° C for 30 to 60 minutes.
(reagent)
100 mM PMSF 800 μL
1mg / mL Pepstatin A 80μL
1M MgCl ₂ 100 μL
2U / μL DNaseI 100μL
50 mg / mL Lysozyme 400 μL

  After sonication, the mixture was centrifuged at 28000 × g, 4 ° C. for 15 minutes, and separated into a supernatant (soluble fraction) and a precipitate (inclusion body).

(3) Purification of soluble fraction The soluble fraction was filtered through a 0.45 µm filter, and 500 mM imidazole buffer (0 M imidazole buffer + 500 mM imidazole) was added to a final concentration of 5 mM. Two His-Trap HP columns (5 mL) were loaded onto AKTA10S linked and eluted with a 5-500 mM imidazole gradient. The peak fraction of creatine kinase was collected, concentrated with Amicon Ultra-15 (MWCF = 10,000), and dialyzed against TBS at 4 ° C. overnight. The dialyzed sample was centrifuged, filtered through a 0.45 μm filter, and the protein concentration was measured.

(4) Purification of denatured creatine kinase The inclusion body was washed with 30 mL of 0 M imidazole buffer + 0.5% Triton X-100. 25 mL of denatured 0M imidazole buffer (0M imidazole buffer + 6M guanidine hydrochloride) was added, and the mixture was incubated at room temperature overnight to be denatured and dissolved. Centrifugation was performed at 100000 × g and 4 ° C. for 45 minutes to remove insoluble substances, and then filtered through a 0.45 μm filter.

  Two His-Trap HP columns (5 mL) were loaded onto AKTA10S linked and eluted with a 0-500 mM imidazole gradient. The peak fraction of creatine kinase was collected, concentrated with Amicon Ultra-15 (MWCF = 10,000), and dialyzed overnight at 4 ° C. against denatured 0M imidazole buffer + 20 mM 2-mercaptoethanol. The dialyzed sample was centrifuged, filtered through a 0.45 μm filter, and the protein concentration was measured.

(5) Examination of refolding conditions A refolding condition examination matrix was prepared and buffer conditions were examined. FIG. 6 shows the created matrix. The buffer conditions examined are shown in the following table.

(6) Reagents The following reagents were used as reagents.
(reagent)
Zerolite: HSZ930-NHA 30mg / well
pH 7.5 basic buffer [500 mM Tris-HCl (pH 7.5), 5% (w / v) PEG 20000, 10 mM EDTA]
pH 8.5 basic buffer [500 mM Tris-HCl (pH 8.5), 5% (w / v) PEG 20000, 510 mM EDTA]

1% Tween20
1M NDSB256
5M guanidine hydrochloride (Gdn-HCl)
Salt (3M NaCl, 1M KCl)
20 mM DTT
2M arginine (Arg) (pH 7.5, pH 8.5)

(7) Preparation of refolding buffer A reagent was prepared in a 96-well deep well plate as follows, and the reagent was dispensed.
1) 100 μL each of ddH ₂ O was added to A, B, C, D, E, and F columns of rows 1 and 4 of a 96-well deep well plate, using an 8 pipette (300 μL).
2) 500 μL each of ddH ₂ O was added to rows 1, 2, 3, 4, 5, and 6 in rows A and D of a 96-well deep well plate using an 8 pipette (1250 μL).

3) 250 μL each of ddH ₂ O was added to A, B, C columns of 1, 2, 3, 4, 5, 6 rows of a 96-well deep well plate using an 8 pipette (300 μL).
4) 100 μL each of pH 7.5 basic buffer was added to 1, 2, 3 rows A, B, C, D, E, F columns of a 96-well deep well plate using an 8 pipette (300 μL). .

5) 100 μL each of pH 8.5 basic buffer, using 8 pipettes (300 μL), A, B, C, D, E, F, G, H of 4, 5 and 6 rows of 96-well deep well plate Added to the column.
6) Add 500 μL each of 1% (v / v) Tween 20 to the B, E columns of rows 1, 2, 3, 4, 5, 6 of the 96-well deep well plate using an 8 pipette (1250 μL). It was.

7) 500 μL each of 1M NDSB 256 was added to the C, F columns of rows 1, 2, 3, 4, 5, 6 of a 96-well deep well plate using an 8 pipette (1250 μL).
8) Add 100 μL each of Salt (3 M NaCl, 1 M KCl) to the A, B, C, D, E, and F columns of 2, 5 rows of a 96-well deep well plate using an 8 pipette (300 μL). It was.

9) 100 μL each of 5M guanidine hydrochloride was added to 3rd and 6th rows of A, B, C, D, E, and F columns of a 96-well deep well plate using an 8 pipette (300 μL).
10) 250 μL each of 2 M arginine (pH 7.5) was added to D, E, F columns of rows 1, 2, and 3 of a 96-well deep well plate using an 8 pipette (300 μL).

11) 250 μL of 2M arginine (pH 8.5) was added to columns 4, 5, and 6 rows D, E, and F of a 96-well deep well plate using an 8-unit pipette (300 μL).
12) 50 μL each of 20 mM DTT was added to A, B, C, D, E, and F rows of 1, 2, 3, 4, 5, and 6 rows of a 96-well deep well plate using an 8 pipette. .

(8) Method About 30 mg of zeolite (HSZ930 NHA) was added to the deep well plate. 500 μL of denatured 0M imidazole buffer was added and equilibrated. 0.3 mg of modified creatine kinase was added, and the mixture was shaken for 1 hour to be adsorbed on the zeolite. The deep well plate was centrifuged at 2150 × g and 4 ° C. for 5 minutes to precipitate the zeolite.

  The supernatant was discarded and 1 mL of 5 mM Tris-HCl, pH 7.5 was added to resuspend the zeolite. Further, the zeolite was washed by repeating centrifugation and resuspension 6 times. After removing the supernatant by centrifugation, 0.6 mL of refolding buffer was added, and after resuspension, the suspension was shaken overnight at 4 ° C. The deep well plate was centrifuged at 2150 × g for 5 minutes at 4 ° C. to precipitate the zeolite, and the supernatant was transferred to a new deep well plate. The collected samples were assayed for activity and protein quantification.

(9) Activity measurement CKII-Test Wako (Wako Pure Chemical Industries, Ltd.) was used for activity measurement. 5 μl of the refolded sample was dispensed into a 96-well assay plate. The activity was determined by dispensing 150 μL of substrate and measuring the change in absorbance at 570 nm using a plate reader (Model 680 XR, Bio-Rad).

(10) Results The measurement results are shown in the following table. The recovery was 14% under the conditions of pH 7.5 (50 mM Tris-HCl), 500 mM arginine, 0.5% (v / v) Tween 20, 500 mM guanidine hydrochloride. Furthermore, when the specific activity was compared with that of soluble creatine kinase, the activity recovery rate was 51.1%.

In this example, MMP-3 was refolded.
(1) Expression of MMP-3 The human MMP-3 expression construct used was transferred from Mr. Matsuura (AIST, Tohoku Center). The MMP-3 expression construct was cultured overnight at 37 ° C. in 30 mL of LB-1.5% glucose-100 μg / mL ampicillin medium. 20 mL of the preculture was transferred to 2 L of turbo broth-1.5% glucose-100 μg / mL ampicillin medium and cultured at 37 ° C. until OD600 was 0.6. IPTG was added so that the final concentration was 100 μM, and further cultured at 37 ° C. for 4.5 hours.

  The cells were collected by centrifugation, and further washed with TBS (20 mM Tris-HCl, pH 7.5, 150 mM NaCl). The suspension was suspended in 80 mL of 0 M imidazole buffer (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl), immersed in liquid nitrogen, frozen, and stored at −80 ° C.

(2) Purification of modified MMP-3 After the frozen cells were dissolved, the following reagents were added and incubated at 4 ° C. for 30 to 60 minutes.
(reagent)
100 mM PMSF 800 μL
1mg / mL Pepstatin A 80μL
1M MgCl ₂ 100 μL
2U / μL DNaseI 100μL
50 mg / mL Lysozyme 400 μL

  After sonication, the mixture was centrifuged at 28000 × g and 4 ° C. for 15 minutes to obtain a precipitate (inclusion body). The inclusion body was washed with 30 mL of 0M imidazole buffer + 0.5% Triton X-100. 25 mL of denatured 0M imidazole buffer (0M imidazole buffer + 6M guanidine hydrochloride) was added, and the mixture was incubated at room temperature overnight to be denatured and dissolved. Centrifugation was performed at 100000 × g and 4 ° C. for 45 minutes to remove insoluble substances, and then filtered through a 0.45 μm filter.

  Two His-Trap HP columns (5 mL) were loaded onto AKTA10S linked and eluted with a 0-500 mM imidazole gradient. The peak fractions were collected, concentrated with Amicon Ultra-15 (MWCF = 10,000), and DTT was added to 0.1M. Dialyzed against denatured 0M imidazole buffer at 4 ° C. overnight. The dialyzed sample was centrifuged, filtered through a 0.45 μm filter, and the protein concentration was measured.

(3) Examination of refolding conditions A refolding condition examination matrix was created and buffer conditions were examined. FIG. 7 shows the created matrix. The buffer conditions studied are shown in the table below.

(4) Reagents The following reagents were used as reagents.
(reagent)
Zeolite (HSZ930-NHA) 30mg / well
pH 6.0 basic buffer [500 mM MES-Na (pH 6.5), 5% (w / v) PEG 20000, 50 mM CaCl ₂ , 5 mM Zn-Acetate]

pH 7.5 basic buffer [500 mM Tris-HCl (pH 7.5), 5% (w / v) PEG 20000, 50 mM CaCl ₂ , 5 mM Zn-Acetate]
pH 8.5 basic buffer [500 mM Tris-HCl (pH 8.5), 5% (w / v) PEG 20000, 50 mM CaCl ₂ , 5 mM Zn-Acetate]

1% Brij-35
1% Tween20
1M NDSB256
5M guanidine hydrochloride (Gdn-HCl)
Salt (3M NaCl, 1M KCl)
20 mM DTT
100 mM reduced glutathione (GSH), 20 mM oxidized glutathione (GSSG)
2M arginine (Arg) (pH 6.0, pH 7.5, pH 8.5)

(5) Preparation of refolding buffer A reagent was prepared in a 96-well deep well plate as follows, and the reagent was dispensed.
1) 750 μL each of ddH ₂ O using an 8 pipette (1250 μL), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 rows of a 96-well deep well plate Of A and B.
2) 375 μL each of ddH 2 O, using 8 pipettes (1250 μL), A, B, C, D, E, F, G in rows 1, 3, 5, 7, 9, 11 of 96-well deep well plate , H row.

3) Using pH 6.5 basic buffer of 150 μL each, 8 pipettes (300 μL), A, B, C, D, E, F, G of 1, 2, 3, 4 rows of 96-well deep well plate , H row.
4) 150 μL each of pH 7.5 basic buffer, using 8 pipettes (300 μL), A, B, C, D, E, F, G in rows 5, 6, 7, 8 of 96-well deep well plate , H row.

5) Using a 8.5-well pipette (300 μL) with 150 μL each of pH 8.5 basic buffer, 9, 10, 11, 12 rows A, B, C, D, E, F, G of 96-well deep well plate , H row.
6) 1% (v / v) Tween 20 of 750 μL each was added to 96-well deep well plate 1, 2, 3, 4, 5, 6, 7, 8, 9, 1011 using an 8 pipette (1250 μL). , Added to C, D columns of 12 rows.

7) 1% (w / v) Brig-35 of 750 μL each was added to 1, 2, 3, 4, 5, 6, 7, 8, 9 of a 96-well deep well plate using an 8 pipette (1250 μL). , 1011, 12 rows E, F columns.
8) Using 1M NDSB256 of 750 μL each, 8 pipettes (1250 μL), 96-well deep well plates 1, 2, 3, 4, 5, 6, 7, 8, 9, 1011 and 12 rows G, Added to row H.

9) 150 μL each of Salt (2.4 M NaCl, 0.8 M KCl) using an 8 pipette (300 μL), 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12 rows, A, C, E, G columns.
10) 150 μL each of 5M guanidine hydrochloride using 96-well deep well plates 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 rows using 8 pipettes (300 μL) Of B, D, F, and H.

11) 375 μL each of 2M arginine (pH 6.5), using 8 pipettes (1250 μL), A, B, C, D, E, F, G, H in 2 and 4 rows of 96-well deep well plate Added to the column.
12) Using 375 μL each of 2M arginine (pH 7.5), using 8 pipettes (1250 μL), A, B, C, D, E, F, G, H in 6 and 8 rows of a 96-well deep well plate Added to the column.

13) Using 375 μL of 2M arginine (pH 8.5), A, B, C, D, E, F, G, H in 10 and 12 rows of a 96-well deep well plate using an 8 pipette (1250 μL). Added to the column.
14) 75 μL each of 20 mM DTT, using 8 pipettes, 1, 2, 5, 6, 9, 10 rows A, B, C, D, E, F, G, H of 96-well deep well plate Added to the column.
15) 75 μL each of 100 mM reduced glutathione and 20 mM oxidized glutathione using an 8 pipette (300 μL), A, B, C in rows 3, 4, 7, 8, 11, 12 of a 96-well deep well plate , D, E, F, G, H rows.

(6) Method About 30 mg of zeolite was added to a 96-well deep well plate. 500 μL of denatured 0M imidazole buffer was added and equilibrated. 0.25 mg of modified MMP-3 was added and shaken for 1 hour to adsorb onto the zeolite. The deep well plate was centrifuged at 2150 × g, 4 ° C. for 5 minutes to precipitate the zeolite.

  The supernatant was discarded and 1 mL of 5 mM Tris-HCl, pH 7.5 was added to resuspend the zeolite. Further, the zeolite was washed by repeating centrifugation and resuspension 6 times. Centrifuge to remove the supernatant, add 1 mL of refolding buffer, and resuspend. Shake overnight at 4 ° C. Centrifugation was performed at 2150 × g and 4 ° C. for 5 minutes to precipitate the zeolite, and the supernatant was transferred to a new deep well plate. The collected samples were assayed for activity and protein quantification.

(7) Activity measurement Activity measurement used 490 MMP-3 Assay Kit (SensoLyte). 50 μL of MMP-3 substrate attached to the kit was added to 10 mL of assay buffer to prepare an assay solution. 100 μL each was dispensed into a 96-well black assay plate. The refolded sample was diluted 2-fold with ddH2O, 5 μL of which was added. The activity was determined by measuring an excitation wavelength of 340 nm and a radiation wavelength of 490 nm with an SH-8000 microplate reader (Corona Electric Co., Ltd.).

(8) Results The measurement results are shown in the following table. Conditions of pH 8.5 (50 mM Tris-HCl), 500 mM arginine, 5 mM CaCl ₂ , 0.5 mM Zn-acetate, 0.5% (w / v) PEG 20000, 300 mM NaCl, 100 mM KCl, 500 mM guanidine hydrochloride, 500 mM NDSB256 Below, maximum activity and 21.8% recovery were obtained. The specific activity was 3.7 times higher than the value obtained by the dilution method (Rosenfeld et al., Gene, 1994, 139, p281-286).

In this example, the anti-human albumin single chain variable region fragment was refolded.
(1) Cloning and expression of anti-human albumin single chain variable region fragment (HSAscFv) The gene sequence of the mouse IgG variable region that recognizes human albumin was referred to the literature (P2000-139460A). The amino acid sequences of the H chain and L chain shown in this document were synthesized as an artificial gene, which is a DNA sequence matched to the codons of Escherichia coli by connecting the sequences connected by a linker (GlyGlyGlyGlySer × 3). The synthesis of artificial genes was requested from Operon Biotechnology Co., Ltd.

  The obtained gene was cleaved with restriction enzymes NdeI and XhoI and subcloned into the pET28a vector (Novagen) to prepare an expression clone having a 6 × histidine tag at the amino terminus. This expression clone was transformed into the BL21star (DE3) strain.

  The HSAscFv expression construct was cultured overnight at 30 ° C. in 50 mL of LB-100 μg / mL kanamycin medium. 20 mL of the preculture was transferred to 2 L of turbo broth-100 μg / mL kanamycin medium and cultured at 37 ° C. until OD600 was 0.6. IPTG was added so that the final concentration would be 1 mM, and further cultured at 37 ° C. for 4.5 hours. The cells were collected by centrifugation, and further washed with TBS (20 mM Tris-HCl, pH 7.5, 150 mM NhaCl). The suspension was suspended in 80 mL of 0 M imidazole buffer (20 mM Tris-HCl pH 7.5, 0.5 M NaCl), immersed in liquid nitrogen, frozen, and stored at −80 ° C.

(2) Purification of denatured HSAscFv After the frozen cells were dissolved, the following reagents were added and incubated at 4 ° C. for 30 to 60 minutes.
(reagent)
100 mM PMSF 800 μL
1mg / mL Pepstatin A 80μL
1M MgCl ₂ 100 μL
2U / μL DNaseI 100μL
50 mg / mL Lysozyme 400 μL

  After sonication, the mixture was centrifuged to obtain a precipitate (inclusion body). The inclusion body was washed with 30 mL of 0M imidazole buffer + 0.5% (w / v) Triton X-100. 25 mL of denatured 0M imidazole buffer (0M imidazole buffer + 6M guanidine hydrochloride) was added and dissolved by incubation overnight at room temperature. Centrifugation was performed at 100000 × g and 4 ° C. for 45 minutes to remove insoluble substances, and then filtered through a 0.45 μm filter.

  Two His-Trap HP columns (5 mL) were loaded onto AKTA10S linked and eluted with a 0-500 mM imidazole gradient. The peak fractions were collected and concentrated with Amicon Ultra-15 (MWCF = 10,000). Dialyzed against denatured 0M imidazole buffer + 20 mM 2-mercaptoethanol at 4 ° C. overnight. The dialyzed sample was centrifuged, filtered through a 0.45 μm filter, and then the protein concentration was measured.

(3) Examination of refolding conditions A refolding condition examination matrix was created and buffer conditions were examined. FIG. 8 shows a refolding matrix. The buffer conditions examined are shown in the table below.

(4) Reagents The following reagents were used as reagents.
(reagent)
Zeolite (HSZ930-NHA) 50mg / well
Buffer composition pH 6.0 basic buffer [200 mM MES-Na (pH 6.0), 2% (w / v) PEG 20000]
pH 7.5 basic buffer [200 mM Tris-HCl (pH 7.5), 2% (w / v) PEG 20000]
pH 8.5 basic buffer [200 mM Tris-HCl (pH 8.5), 2% (w / v) PEG 20000]

4M guanidine hydrochloride Salt (2.4M NaCl, 0.8M KCl)
8% (v / v) Tween20
160 mM cysteine, 16 mM cystine 2 M arginine (pH 6.0, pH 7.5, pH 8.5)

(5) Method A reagent was prepared as shown below, and the buffer was dispensed into a 96-well deep well plate.
1) A, B, C, D, E, F of 1, 2, 5, 6, 9, 10 rows of a 96-well deep well plate with 100 μL of ddH ₂ O using an 8 pipette (300 μL) , G, H row.
2) A, B, C, D, E, F in rows 1, 3, 5, 7, 9, and 11 of a 96-well deep well plate using 100 μL each of ddH ₂ O using an 8 pipette (300 μL) , G, H row.

3) Rows 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 of a 96-well deep well plate using 200 μL of ddH ₂ O using 12 pipettes (300 μL) Of A, B, C, and D.
4) 400 μL each of ddH ₂ O using 96-well deep well plates 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 rows using an 8 pipette (1250 μL) Of A, B, E, and F.

5) 400 μL each of ddH ₂ O using 96-well deep well plates 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 using an 8 pipette (1250 μL) Of A, C, E, and G.
6) 400 μL each of pH 6.0 basic buffer, using 8 series pipettes (1250 μL), A, B, C, D, E, F, G of 1, 2, 3, 4 rows of 96-well deep well plate , H row.

7) 400 μL each of pH 7.5 basic buffer, using 8 pipettes (1250 μL), A, B, C, D, E, F, G in rows 5, 6, 7, 8 of 96-well deep well plate , H row.
8) 400 μL each of pH 8.5 basic buffer, using an 8 pipette (1250 μL), 9, 10, 11, 12 rows A, B, C, D, E, F, G of 96-well deep well plate , H row.

9) Using 100% of 8% (v / v) Tween 20 in 100 μL each, A, B, C, A, B, C, Rows 3, 4, 7, 8, 11, 12 of 96-well deep well plate using an 8-unit pipette (300 μL). Added to rows D, E, F, G, H.
10) 200 μL each of Salt (2.4 M NaCl, 0.8 M KCl) was added to 96-well deep well plates 1, 2, 3, 4, 5, 6, 7, 8 using an 8 pipette (300 μL). , 9, 10, 11, 12 rows, E, F, G, H columns.

11) 400 μL each of 2M arginine (pH 6.0) was added to C, D, G, and H columns of 1, 2, 3, and 4 rows of a 96-well deep well plate using an 8 pipette (1250 μL). .
12) 400 μL each of 2 M arginine (pH 7.5) was added to the C, D, G, H columns of the 5, 6, 7, 8 rows of a 96-well deep well plate using an 8 pipette (1250 μL).

13) 400 μL each of 2M arginine (pH 8.5) was added to C, D, G, and H columns of rows 9, 10, 11, and 12 of a 96-well deep well plate using an 8-unit pipette (1250 μL). .
14) 1, 4, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 rows of a 96-well deep well plate using 400 μL of 4M guanidine hydrochloride using an 8-unit pipette (1250 μL) Of B, D, F, and H.

15) 100 μL each of 160 mM cysteine, 16 mM cystine, using 8 pipettes (300 μL), A, B, C, D, E in rows 2, 4, 6, 8, 10, 12 of 96-well deep well plate , F, G, H rows.

(6) Method About 50 mg of zeolite was added to a 96-well deep well plate. 500 μL of denatured 0M imidazole buffer was added and equilibrated. 0.2 mg of modified HSAscFv was added and shaken for 1 hour to adsorb onto the zeolite. The deep well plate was centrifuged at 2150 × g and 4 ° C. for 5 minutes to precipitate the zeolite.

  The supernatant was discarded and 1 mL of 5 mM Tris-HCl, pH 7.5 was added and the zeolite was resuspended. Further, the zeolite was washed by repeating centrifugation and resuspension five times. After centrifuging to remove the supernatant, 1 mL of refolding buffer was added, and after resuspension, the suspension was shaken overnight at 4 ° C. Centrifugation was performed at 2150 × g, 4 ° C. for 5 minutes to precipitate the zeolite, and the supernatant was transferred to a new deep well plate. The collected samples were subjected to ELISA and protein quantification.

(7) Activity measurement Using an ELISA plate, the following reagents were used.
Human serum albumin (Sigma Aldrich Japan Co., Ltd.)
Block Ace (DS Pharma Biomedical Co., Ltd.)
Anti-histag antibody peroxidase conjugate

  50 μL of 10 μg / mL human serum albumin was added to the ELISA plate and allowed to bind overnight at 4 ° C. After washing the wells with PBS, 100 μL of 1% (w / v) Block Ace was added and incubated at room temperature for 2 hours. 50 μL of refolded HSAscFv diluted 20-fold with 0.4% (w / v) Block Ace was added and incubated at room temperature for 1 hour. After washing the wells with PBS, 50 μL of anti-Histag antibody peroxidase conjugate diluted 2000 times with 0.4% (w / v) Block Ace was added and incubated at room temperature for 1 hour.

  After washing the wells with PBS, 100 μL of a color developing solution (0.4 mg / mL orthophenylresin diamine, citrate-phosphate buffer, 0.3 μL / mL hydrogen peroxide) was added, and color was allowed to develop for 10 minutes. 50 μL of 2M sulfuric acid was added to complete the color development. Absorbance at a wavelength of 490 nm was measured using a plate reader (Model 680XL, Bio-Rad Laboratories, Inc.).

(8) Results The measurement results are shown in the following table. The absorbance was highest under the conditions of pH 8.5 (50 mM Tris-HCl), 500 mM arginine, 0.5% (w / v) Tween 20, and 0.5% (w / v) PEG 20000.

(9) Influence of the type of zeolite 1) Zeolite used The zeolite used is shown in the following table.

2) Method 20 mg of each type of zeolite was dispensed into a 2 mL tube. 500 μL of denatured 0M imidazole buffer was added and equilibrated. 0.2 mg of modified HSAscFv was added and shaken for 1 hour to adsorb onto the zeolite. Centrifugation was performed at 21000 × g and 20 ° C. for 1 minute to precipitate the zeolite. The supernatant was discarded and 1 mL of 5 mM Tris-HCl, pH 7.5 was added to resuspend the zeolite.

  Further, the zeolite was washed by repeating centrifugation and resuspension five times. After removing the supernatant by centrifugation, 1 mL of optimal refolding buffer was added, and after resuspension, the suspension was shaken overnight at 4 ° C. Centrifugation was performed at 21000 × g and 4 ° C. for 10 minutes to precipitate the zeolite, and the supernatant was transferred to a new 1.5 mL tube. The collected samples were subjected to ELISA and protein quantification.

(10) Results The results are shown in FIG. CP811E-150 was the maximum for both recovery and ELISA.

  As described above in detail, the present invention relates to a protein refolding condition setting kit used in the protein functional activation / recovery method, and according to the present invention, the refolding method using a BEA structured zeolite is optimal. Suitable conditions can be set easily and quickly, and the synergistic effect of the ease of scale-up of the refolding method using a BEA-structured zeolite makes it possible to produce a large amount of inactive proteins regardless of the type of protein. On the other hand, their original functions can be activated. According to the present invention, it is possible to provide a protein refolding condition setting kit for obtaining an active protein having a correct three-dimensional structure and activity from a protein having no activity as an inclusion body. In addition, the present invention can easily and quickly set the optimum or preferred conditions when applying the refolding method of an inactive protein with BEA structure zeolite that can be refolded with high efficiency compared to other methods. It is useful for providing a protein refolding condition setting kit that can be used.

Claims (8)

It is possible to easily and quickly derive suitable refolding conditions when performing protein refolding operations / processes that are inactive due to the disordered conformation of the protein that is inactive. A protein refolding condition setting kit,
A refolding agent composed of a BEA structure zeolite that functions as a carrier adsorbed in a denaturant in a denatured state by a protein denaturant is an essential constituent,
Refolding agent made of the BEA structure zeolite, proteins adsorbed in a denatured state, a solution component to dissociate in solution, other pH adjusting agents of pure water component as essential components, further, Li folding regulators and / or surfactants, protein S-S bridge formation control agent consists of a combination comprising salts, proteins denaturing agents, or one or more metal ions,
Further includes integral test tube having a space of the plurality of divided same volume, or test plate, as a component, concurrently, Ri Kits Product der for testing by multiple criteria,
1) A BEA structured zeolite is a zeolite in NH ₄ ⁺ or Na ⁺ form or a framework-substituted zeolite containing NH ₄ ⁺ or Na ⁺ , or a CP 811E-75, CP811E-150 or CP811E-300 zeolite in H ⁺ form. Yes,
2) The BEA-structured zeolite is granulated in a powdered state or particles,
3) The BEA-structured zeolite is immobilized in a powdered or particulate form in an integrated test tube or test plate having a plurality of divided spaces of the same volume,
4) The glue for granulating the BEA structured zeolite or the glue for immobilizing it on an integral test tube or test plate is a material that does not adversely affect the protein,
5) The integrated test tube or test plate having a plurality of divided spaces having the same volume is a 96-well microplate or a microplate having more holes, and each test tube or plate has a hole on it. respect, by the solution of each different configurations are injected, Ru can be tested by concurrently multiple conditions,
A protein refolding condition setting kit.   2. The protein refolding condition according to claim 1, wherein the framework structure of the BEA structured zeolite is composed of oxygen and one or more other elements, or composed of silicon and oxygen or silicon, aluminum and oxygen only. Setting kit. The protein refolding condition setting kit according to claim 1 or 2 , wherein the protein denaturant is guanidine hydrochloride or urea. The pH adjuster is trisaminomethane trihydrochloride (Tris-HCl), 3- (N-morpholino) propanesulfonic acid (MOPS), 2-morpholinoethanesulfonic acid (MES), or N-2-hydroxyethylpiperazine- The protein refolding condition setting kit according to any one of claims 1 to 3 , which is N'-ginethanesulfonic acid (HEPES). The protein refolding condition setting kit according to any one of claims 1 to 4 , wherein the protein SS crosslinking formation control agent is cystine, cysteine, 2-mercaptoethanol, dithiothreitol, or thiophenol. Refolding regulators and / or surfactants are polyethylene glycol (PEG 20K, PEG 3350, PEG 800, PEG 200), Ficol 170, Ficol 1400, arginine, EDTA, NaCl, polyphosphate, sodium dodecyl sulfate (SDS), sucrose, glucose, glycerol, inositol, cyclodextrin, amylose, Dextran T-500, Tween20, Tween40, Tween60, NP-40, SB3-14, SB12, CTAB, or composed of one or more of the Triton X-100, billing Item 6. The protein refolding condition setting kit according to any one of Items 1 to 5 . The protein refolding condition setting kit according to any one of claims 1 to 6 , wherein the kit is an automatic protein refolding condition setting device that performs a series of operations automatically or semi-automatically. The protein according to any one of claims 1 to 7 , comprising a combination of a basic component of a solution component, a microplate on which a zeolite is dispensed or fixed, and an instruction manual describing an operation method as a set of packages. Refolding condition setting kit.