JP2005343766A - Agent for controlling formation of protein crystal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for controlling the formation of a protein crystal capable of effectively forming a protein crystal in crystallizing the protein and its utilization method. <P>SOLUTION: The agent for controlling the formation of a protein crystal consists of a layered silicate and exhibits the power to control and govern the formation of a protein crystal by contacting the solution dissolving the protein, which is applicable to a wide variety of known and unknown proteins. The method for crystallizing and purifying a protein by utilizing it is also provided. Compared with conventional methods, the agent for controlling the formation of a protein crystal and the method for crystallizing and purifying a protein using it are high in flexibility and generality, simple and easy in handling, and low in cost. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a protein crystal formation control agent used in protein crystallization, and more specifically, promotes or suppresses the rate and / or precipitates the protein during protein crystallization. A protein crystal formation control agent composed of a substance / material having the ability to control and control so-called crystal formation that directly affects the shape and form of the crystal, and a new protein using a protein crystal formation control agent composed of the substance / material This is related to the refining and manufacturing / production technology. The present invention does not require the use of a conventional precipitating agent that causes protein crystal contamination, for example, in the technical fields of protein biochemicals and pharmaceutical production. A novel protein that can efficiently produce purified protein crystals that can determine the three-dimensional structure and immunoassay, which are essential to know the mechanism of action, as well as to obtain crystals in the concentration range and protein species. Since the protein crystal formation control agent of the present invention provides a crystallization technique and is thermally and chemically stable and inexpensive, it is one of the core techniques in protein production and production. It is highly expected that

  It is not a gene that actually acts and works in vivo, but a protein made from them. Therefore, analysis and elucidation of the three-dimensional structure of proteins and the functions that appear in association with them are directly linked to, for example, disease treatment and drug discovery, and are therefore an extremely large and important issue for the post-genome. The elucidation and determination of the three-dimensional structure of a protein is usually performed by spectrum analysis such as NMR, IR, and UV, and X-ray crystal structure analysis, and X-ray crystal structure analysis is the most effective. However, in X-ray crystal structure analysis, in order to perform X-ray diffraction with high accuracy, it is necessary to crystallize the protein extracted or synthesized, purified and isolated into an appropriate shape and form.

  The amount of protein that can be obtained is usually very small, even if it is extracted from a natural product or expressed in large quantities using Escherichia coli or the like. Therefore, it is no exaggeration to say that protein purification, isolation, and crystallization are far more difficult than those of ordinary chemical synthesis. For this reason, various crystallization methods have been studied and proposed. However, the basic principle is to manipulate the solubility of proteins, that is, to create a supersaturated state and crystallize them. This supersaturated state is basically made up of a method of transferring the temperature from the high solubility side to the low solubility side, a method of adding a precipitant to make the protein difficult to dissolve, and a method of evaporating and diffusing only the solvent. Although there are three types, these methods have a major problem that the amount handled is small anyway. For this reason, various kinds of fine contrivances are applied to the three types of operations, and various crystallization methods are proposed. Has reached.

  Typical of these are dialysis, vapor diffusion, batch, free interface diffusion, concentration, temperature gradient method, etc. (see Non-Patent Documents 1-3) for smoothing these crystallization operations. Devices and solution sets (such as those from Hampton Research and Emerald BioStructures) are commercially available (see Non-Patent Document 3). However, there is no versatile method that can be applied to any protein, and it takes a lot of time and effort to change the method used or combine several methods on a trial and error basis, depending on the target protein. The fact is that it is finally crystallized. Therefore, in this technical field, substances and materials effective for protein crystallization and crystallization methods using the same are always in great demand, whether improved or completely new. There is a great demand for a simple and versatile crystallization method.

“PNE Monograph Protein X-ray Analysis”, Kyoritsu Shuppan Co., Ltd., 30-35 (1998) "X-ray crystallographic analysis understood by this", Kagaku Dojin, 66-69 (2000) “Post-Sequence Protein Experimental Method 3 Basics of Structural and Functional Analysis”, Tokyo Chemical Doujin, 2-7 (2002)

  Under such circumstances, the present inventors, in view of the above-mentioned prior art, in the process of searching for a simpler and more effective protein crystallization method, clay minerals adsorb proteins well. Therefore, in recent years, it has been used in facial cosmetics that adsorb and remove proteins, and that the surface of inorganic oxide crystals often works extremely effectively as a place to induce self-organization of organic molecules. Based on this idea, he researched the idea of using clay minerals for protein crystallization. As a result, silicate compounds such as clay minerals promote or suppress the rate of protein crystallization and / or directly affect the shape and form of crystals that crystallize. Recognizing that it has the ability to control and control so-called crystal formation, the present invention, that is, the formation of a protein crystal composed of a silicate compound, which is simple, general, universal, and capable of handling even a small amount It came to complete the invention which concerns on a control agent. An object of the present invention is to provide a novel protein crystal formation control agent capable of controlling protein crystal formation.

The present invention for solving the above-described problems comprises the following technical means.
(1) A reagent for controlling protein crystal formation, which contains a silicate compound as an active ingredient, and has a function of increasing or decreasing the rate of protein crystallization, and / or the shape of crystals precipitated. A protein crystal formation control agent characterized by having a function of controlling morphology.
(2) The protein crystal formation control agent according to (1), wherein the silicate compound exerts its protein crystal formation control function by contacting the protein dissolved in the solution.
(3) The protein crystal formation control agent according to (2), wherein the silicate compound exerts its protein crystal formation control function by contacting with a protein dissolved in a buffer solution. .
(4) The protein crystal formation control agent according to (3), wherein the solvent vapor of the buffer solution is diffused.
(5) The protein crystal formation control agent according to (1), wherein a clay mineral is used as the silicate compound.
(6) The protein crystal formation control agent according to (3) or (4), wherein the solvent of the buffer solution is water.
(7) The protein crystal formation according to the above (5), wherein the clay mineral is composed of a phyllosilicate that expands its basic skeleton chain —Si—O— two-dimensionally and takes a layered structure. Control agent.
(8) The phyllosilicate is kaolin (kaolinite), halloysite, chrysotile, talc, smectite, montmorillonite, vermiculite mica (mica), calcite, gyoganite, ryokdeite (chlorite), pyrophyllite, antigogo The protein crystal formation control agent according to (7) above, which is one or more selected from wright, enderite, and saponite.

Next, the present invention will be described in more detail.
The protein crystal formation control agent comprising the silicate compound of the present invention can be used for crystallization of all proteins, whether known or unknown. The protein crystal formation control agent of the present invention, in principle, has the ability to adsorb protein to it, induce and control protein crystal nucleation, and control and control protein crystal growth and shape and morphology formation processes. This ability is exhibited by the following operation. In other words, protein crystals are formed by the following operation. It consists of a very simple and easy operation / procedure of first mixing a solution in which the protein of interest is dissolved with the crystal formation control agent of the present invention or dropping it on the solution and then leaving it for a certain period of time. Is. During this standing, the solvent gradually permeates and diffuses between the silicate layers of the silicate compound constituting the crystal formation controlling agent of the present invention, and the concentration of the protein in the solution increases. Therefore, the protein concentration effect and the crystal nucleation attraction / dominance action on the silicate surface act synergistically to cause the protein to crystallize.

  As a protein dissolution solvent used in this operation, protein is dissolved in water in the living body and functions in an original form, so that water is most often used, and this use is preferable. Furthermore, since proteins are sensitive to the pH of the solution, it is desirable to use a protein solution as a buffer. There are no particular restrictions on the buffer solution, and any buffer solution that does not adversely affect the protein can be used. For example, a small amount of sodium acetate, sodium carbonate, phosphoric acid can be used. Examples include, but are not limited to, aqueous solutions of sodium, potassium phosphate, sodium citrate, sodium azide, imidazole, and the like.

As mentioned above, the solvent for protein is usually water, but basically any solvent that does not adversely affect the protein, specifically organic solvents, excludes its use. Instead, these solvents can be used alone or mixed with water. Examples of this type of organic solvent include monovalent and polyhydric alcohols such as ethanol, acetone, methanol, dioxane, 2-methyl-2,4-pentanediol, and ethylene glycol, ethers, and ketones. Although it is exemplified as a typical one, it is not limited to these, and anything that does not adversely affect proteins such as denaturation or can be used without causing adverse effects Can also be used. In particular, if it is an appropriate amount, specifically, adding an organic solvent to an aqueous solvent, etc., will be welcomed because it will also manipulate the solubility and promote crystallization.

  Similar to the addition of the organic solvent described above, a precipitation agent has been conventionally used to lower the solubility of the target protein during crystallization. In the present invention, the crystal formation control agent of the present invention is used. It is also possible to use this kind of precipitant together. For example, as in the prior art, by adding a precipitating agent to a protein solution and further mixing and contacting this solution with the crystal formation controlling agent of the present invention, the formation and shape of protein crystals can be controlled and controlled. Such precipitating agents typically include ammonium sulfate (ammonium sulfate), magnesium sulfate, sodium sulfate, sodium phosphate, PEG (polyethylene glycol) 400, 4000, 6000 and 8000, lithium chloride, sodium chloride, potassium chloride. However, the present invention is not limited to these, and inorganic salts may be used as long as they do not adversely affect the essence of the target protein, such as the three-dimensional structure and activity of the target protein, and merely reduce its solubility. , Organic salts, surfactants and the like can be used as precipitating agents. The amount to be used is appropriately determined according to the type of the target protein and the amount dissolved in the solution.

  Furthermore, it is also possible to use an organic solvent (additive) and a precipitant in combination with the crystal formation control agent of the present invention. However, the heavy use of organic solvents and / or precipitating agents may cause unintentional contamination of proteins by them, and therefore it is preferable to avoid using the crystal formation controlling agent of the present invention if possible. Preferably, the crystal formation controlling agent of the present invention has a great advantage that it is not always necessary to use them together with a precipitating agent. Therefore, by using the present crystal formation controlling agent, it is possible to provide a protein purification method that does not require a troublesome purification operation such as dialysis or desalting as in the case of using a precipitant.

  As the silicate compound constituting the crystal formation controlling agent of the present invention having the ability to control and control protein crystal formation described above, first, clay minerals are mentioned. There are roughly three types of clay minerals: amorphous (allophane and imogolite), crystalline chain structure (sepiolite and palygorskite), and crystalline layered structure. A crystal formation controlling agent can be constituted.

  In general, from a comprehensive viewpoint such as availability, quality, price, ease of use, ability as a crystal formation control agent, etc., the basic skeleton chain -Si-O- is expanded two-dimensionally to take a layered structure. So-called phyllosilicates, more specifically, kaolin (kaolinite), halloysite, chrysotile, talc, smectite, montmorillonite, vermiculite, mica (mica), calcite, gyoganite, ryokdeite (chlorite), pyro More preferably, the crystal formation controlling agent is composed of one or more of phylite, antigolite, enderite and the like. However, the present invention is not limited to these specific examples, and any of those composed of a silicate stack in which the skeleton chain -Si-O- is spread two-dimensionally is desirable. Even if it is substituted with the above metal, there is no problem in constituting the present crystal formation controlling agent, but an additional effect can be expected.

  In order to neutralize the negative charge of the silicate layer, there are usually sodium, potassium, magnesium, calcium and the like between the layers of the layered silicate constituting the crystal formation controlling agent of the present invention. Ions can not only adjust the layer spacing but also help the selective penetration and diffusion of protein-dissolving solvents between layers by preferential coordination of solvent molecules to those cations. Thus, the crystal formation controlling agent of the present invention adsorbs proteins on the surface of the silicate layer, regulates the nucleation to the crystallization, controls and controls the crystal growth, that is, the shape and form of the crystal, and the silicate layer Since this microspace serves as a container for solvent molecules and regulates the concentration of the protein in the solution, the present crystal formation control agent has an optimal mechanism for protein crystallization. Because of this mechanism, the present crystal formation control agent is characterized not only by high-concentration protein solutions but also by exhibiting remarkable efficacy in crystallization of proteins from low-concentration solutions.

  Since the layered silicate constituting the protein crystal formation control agent of the present invention is excellent in thermal stability and chemical stability, and is abundant and inexpensive, the present invention and a protein crystallization method using the same Is very useful not only for providing samples for elucidating the three-dimensional structure, properties, and functions of proteins, but also as a simple protein purification method, for example, for biochemical and pharmaceutical production. There is something immeasurable.

  The present invention has the ability to control or control so-called crystal formation, which accelerates or suppresses the rate of protein crystallization and / or directly affects the shape and form of crystallized crystals. The present invention relates to a protein crystal formation control agent comprising a substance / material, and a protein purification, production / production technology using the substance / material. According to the present invention, it is possible to provide 1) a universal substance / material for obtaining crystals of a wide variety of proteins, regardless of types, that is, a crystal formation control agent comprising a layered silicate. 2) This crystal formation Crystals can be obtained by bringing the target protein solution into contact with the control agent regardless of its concentration. 3) Since the present crystal formation control agent does not require the use of a precipitant, It is also useful as an easy and efficient purification method for various proteins that does not require cumbersome dialysis and desalting operations. 4) The method of bringing a protein into contact with this crystal formation control agent to crystallize the protein is as follows: It is a general, universal, and efficient method with high crystallization rate that can be applied to various proteins regardless of the chain length / sequence of amino acids that constitute the protein. The phyllosilicate constituting the agent is inexpensive and can produce and provide the present crystal formation control agent at a low cost. 6) The phyllosilicate constituting the present crystal formation control agent includes a method using a precipitant. 7) Protein crystals can be produced even from a low-concentration solution in which protein crystallization was difficult by the conventional method. In combination with the protein crystallization method according to the above, a special effect that a novel protein production process for purifying and producing a protein can be proposed and established.

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

Example 1
In this example, protein crystallization is performed by applying the present crystal formation control agent to the operation of the sitting drop (see Non-Patent Document 2) of the vapor diffusion method that has been widely used for protein crystallization. I tried to make it.

  A glass rod (manufactured by Hampton Research) treated with siliconization is placed on a 24-well plate, and as a reservoir, 0.2 M sodium acetate buffer (pH 4.7) is added to the well as sodium chloride as a precipitant. And 1 ml of the solution adjusted to a concentration of 6% was placed. Next, a 0.2 mg sodium acetate buffer solution containing no sodium chloride was used to prepare a 50 mg / ml solution of egg white lysozyme, and 10 μl of this adjusted protein solution was added to a layered silicate solution having the same composition as the above reservoir. 10 μl of a solution in which a crystal formation control agent composed of mica, which is a kind of the above, is mixed on a rod (the mica concentration is 10% w / v), the plate is sealed with a plate seal, kept at 24 ° C., I left it alone. As a result, after standing, microcrystals appeared in 6 hours, and in 12 hours, crystals were obtained in a size obtained one day after the conventional method.

Comparative Example 1
Except for mica alone, the same experiment as in Example 1 was performed. As a result, the crystal growth rate was about half that of Example 1.

Example 2-12
Mica concentration from 10% to 7.5%, 5.0%, 2.5%, 1.25%, 0.5%, 0.375%, 0.25%, 0.125%, The protein was crystallized in exactly the same way as in Example 1 except that it was changed to 0.05%, 0.025% and 0.005%. As a result, the crystallization promoting effect was recognized in all of Comparative Example 1. That is, when there is no crystal of the same size in the presence of a crystal formation control agent composed of mica (Comparative Example 1; in this example, there is no crystal formation control agent and only the precipitation agent is used as a control). As shown below, the same applies to the above), and when the crystal size was almost the same, a large amount of crystals were obtained in the same time.

Examples 13-57
The protein is changed from lysozyme to trypsin, catalase, concanavalin A, thaumatin, and the concentration of crystal formation control agent (mica) is changed as in Example 1-12. Instead, the same experiment as in Example 1 was performed. The experimental conditions and results are summarized in Tables 1 and 2, with the addition of those of Examples 1-12. Table 1 shows the composition of the protein solution placed on the rod, and Table 2 shows the results at each crystal formation control agent concentration.

Comparative Example 2-5
For trypsin, catalase, concanavalin A, and thaumatin proteins, crystallization was performed without using the same crystal formation control agent (mica) as in Comparative Example 1 for comparison and reference. These and Comparative Example 1 are shown in the control column of Table 2 (there is no crystal formation controlling agent and only the precipitating agent is used).

  In any protein, in the presence of the crystal formation control agent composed of mica, the promoting effect of increasing the size of the crystal and / or significantly increasing the number of crystals in the same time was recognized. Some of these results are shown in FIG. 1 for reference.

Examples 58-60
The experiment was performed in exactly the same manner as in Example 1 except that the crystal formation controlling agent was changed from mica to smectite, kaolin and talc. In either case, the same lysozyme crystallization promoting effect as described above was observed.

Example 61
Crystallization was performed in the same manner as in Example 1 except that the crystal formation controlling agent comprising chlorite was used. When left for 1 day, crystals did not appear, and on the 2nd day, crystals of the same size and quality were obtained as in the conventional (control) method using a precipitant without using the crystal formation control agent of the present invention. . It can be seen that the crystal formation control agent comprising chlorite has a delay effect on the crystal formation. Therefore, it can be said that it is suitable for obtaining a good-quality moderate-sized crystal.

Comparative Example 6-11
The crystal formation control agent was changed to silica prepared by hydrolysis from tetraethoxysilane, and lysozyme, trypsin, catalase, concanavalin A, thaumatin and HSA (see Table 1 for each concentration) were used as proteins, respectively. Crystallization was performed in exactly the same procedure as in Example 1. As a result, no crystal formation was observed with trypsin, catalase, concanavalin A and HSA, and crystallization was observed with lysozyme and thaumatin, but there was no crystal formation control agent (so-called control using a normal precipitation agent). ) Compared with the case, the results were significantly inferior in crystal size and quantity (see FIG. 2). It can be seen that those having a similar skeleton chain —Si—O— but not having a layered structure do not have the ability to control and control protein crystal formation.

Examples 62-64
Crystallization was carried out in exactly the same way as in Example 1, except that the initial concentration of lysozyme egg white was 20 mg / ml, 10 mg / ml and 5 mg / ml. As a result, no crystals were observed at the lowest concentration, but crystals were observed after one and a half days in the former two.

Comparative Example 12-14
The initial concentration of egg white lysozyme was set to 20 mg / ml, 10 mg / ml and 5 mg / ml, and the same as in Example 1 (so-called control) except that the protein crystal formation control agent consisting of layered silicate was not used. Crystallization was performed. As a result, at the highest concentration, crystals were finally observed on the second day, but no crystal formation occurred in the latter two at lower concentrations.

Examples 65-68
Crystallization similar to that of Example 1 was carried out for each protein of catalase (15 mg / ml, 7.5 mg / m), concanavalin A (4 mg / ml) and thaumatin (8 mg / ml) at different initial concentrations. As a result, there was no crystal formation control agent composed of mica, and catalase and thaumatin showed almost the same or higher effects than the case of only the precipitant (control). With concanavalin A, crystals were obtained after a day and a half in the presence of a crystal formation control agent, whereas crystallization did not occur only with a precipitant. That is, according to the conventional method, it was found that the crystal formation controlling agent has the ability to crystallize the protein even in a low concentration region where the protein does not crystallize.

Examples 69-155
In Example 1, when a protein solution and a crystal formation control agent consisting of mica were mixed, the same operation as in Example 1 was used except that a buffer solution containing no precipitant was used as a dispersion solution of the crystal formation control agent. Thus, crystallization was performed at various concentrations of the crystal formation control agent with respect to various concentrations of the four types of proteins, lysozyme, catalase, concanavalin A and trypsin. The results are shown in Table 3. It can be seen that the crystallization promoting effect of the crystal formation control agent is remarkably exhibited at any protein concentration as compared with the conventional method (control) in which there is no crystal formation control agent and only the precipitant. More specifically, it has been found that, in catalase, concanavalin A and trypsin, the present crystal formation control agent can crystallize these proteins even in a low concentration region where crystals cannot be obtained by conventional methods.

Examples 156-161
In Example 1, as a crystal formation controlling agent, a material composed of montmorillonite, saponite, talc, mica, kaolin and chlorite was used, and when the protein solution and the crystal formation controlling agent were mixed, The lysozyme was crystallized in the same manner as in Example 1 except that a buffer solution containing no precipitant was used. The result is shown in FIG. For montmorillonite, saponite, talc, mica and kaolin, it is a crystal obtained after one day, and for chlorite, it is obtained after three days. As can be seen from the figure, the formation / controlling effect of lysozyme crystals was observed in any of the crystal formation controlling agents.

  As shown in the above-mentioned Examples, the crystal formation control agent composed of phyllosilicates including clay minerals, so-called layered silicates, has the ability to control and control protein crystal formation. By using a crystal formation controlling agent, protein crystals of a desired size and shape can be obtained. The crystal formation control agent of the present invention is useful as a general and universal crystal formation control agent that can be applied to various known and unknown proteins, and its application is limited to the proteins shown in the Examples. It can be applied to any protein.

  The present invention relates to a crystal formation control agent having the ability to control and control protein crystal formation, and to protein purification, production and production techniques using the same. According to the present invention, crystals can be obtained for a wide range of proteins regardless of the chain length and sequence of amino acids constituting the protein. That is, the present invention provides an efficient method having generality, universality, and high crystallization rate, and provides an efficient protein crystallization / purification method at low cost. An easy and easy-to-work protein crystallization / purification method that does not require troublesome dialysis and desalting operations can be provided. In the conventional method, the protein can be crystallized from a low-concentration protein solution from which protein crystals could not be obtained. For example, it is possible to combine a protein synthesis process using an E. coli expression system and a protein crystallization method using the crystal formation control agent of the present invention, and a novel protein production process for purifying and producing a protein is proposed and established. Therefore, the present invention is particularly useful as a technology for purifying and producing a novel protein in the fields of biochemical production and pharmaceutical production.

The crystallization promotion effect by a crystal formation control agent (mica) is shown. The result (after 2 days) of having performed protein crystallization using the silica of a comparative example is shown. The crystal form (all lysozyme results) when crystal formation is performed without a precipitant is shown.

Claims (8)

  A reagent for controlling protein crystal formation, which contains a silicate compound as an active ingredient, and has the function of increasing or decreasing the rate of protein crystallization, and / or the shape and form of crystals precipitated. A protein crystal formation control agent characterized by having a function of controlling.   The protein crystal formation control agent according to claim 1, wherein the silicate compound exerts its protein crystal formation control function by contacting the protein dissolved in the solution.   The protein crystal formation control agent according to claim 2, wherein the silicate compound exerts its protein crystal formation control function by contacting the protein dissolved in the buffer solution.   The protein crystal formation control agent according to claim 3, wherein solvent vapor of the buffer solution is diffused.   The protein crystal formation control agent according to claim 1, wherein clay mineral is used as the silicate compound.   The protein crystal formation control agent according to claim 3 or 4, wherein a solvent of the buffer solution is water.   6. The protein crystal formation control agent according to claim 5, wherein the clay mineral is composed of a phyllosilicate that expands its basic skeleton chain —Si—O— two-dimensionally and takes a layered structure. The above phyllosilicates are kaolin (kaolinite), halloysite, chrysotile, talc, smectite, montmorillonite, vermiculite mica (mica), calcite, gyoganite, ryokdeite (chlorite), pyrophyllite, antigolite, ende The protein crystal formation control agent according to claim 7, wherein the protein crystal formation control agent is one or more selected from light and saponite.