JP2005224653A - Method for crystallizing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for crystallizing a protein, by which a high-quality protein crystal suitable for X-ray analysis, or the like, can be obtained. <P>SOLUTION: This method for crystallizing the protein comprises a step (a) of adding a protein-containing sample to a gelated polymer keeping a protein crystallizing agent to make a protein solution formed by adding the protein-containing sample contact with the protein crystallizing agent and a step (b) of furthermore stratifying an oil phase on the protein-containing sample. The protein crystallizing agent is migrated slowly to the portion of the protein-containing sample from the gelated polymer and the protein-containing sample is migrated slowly to the gelated polymer keeping the protein crystallizing agent to cause a crystallization reaction little by little. Simultaneously, moisture is migrated to the oil phase to concentrate the protein solution gradually. Since the interaction, which is a crystallizing mechanism, between the protein and the surrounding environment (such as the presence of the protein crystallizing agent, the protein concentration and the pH) is advanced gradually by these two effects from two directions, the generation of a high-quality crystal nucleus can be promoted in a short time and the high-quality protein crystal can be obtained. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for precipitating protein crystals from a protein-containing sample.

  In recent years, a so-called structural genomics science has been activated to comprehensively analyze protein structures and to search for the mechanism of life phenomena based on the analysis. In structural genomics, it is necessary to further save labor and speed up the structural analysis process. To this end, it is hoped to develop a method for screening crystallization conditions quickly and efficiently using a smaller amount of protein sample. It is rare.

  In order to analyze a three-dimensional structure of a protein, a good crystal is required, and a large amount of sample and a long time are currently a subject for searching for conditions for preparing the protein. That is, the existing methods require very complicated operations and are inefficient and require many protein samples.

  For this purpose, crystallization screening reagent kits have been developed, and conditions related to the preparation time of screening reagents necessary for screening crystallization conditions have been improved. For example, an immobilization reagent for protein crystallization screening in which a plurality of types of protein crystallization agents are immobilized on a solid support has been proposed (Patent Document 1). However, the screening of the conventional crystallization conditions including this is performed with a solution amount of several microliters per condition, and the search time for the crystallization conditions ranges from several hours to several days. For this reason, further miniaturization of the sample and shortening of the search time for the crystallization conditions are desired.

  On the other hand, various methods have been proposed as protein crystallization methods from the above background. Examples thereof include a batch method, a vapor diffusion method, and a liquid-liquid diffusion method (or dialysis method).

  In the “batch method”, a solution containing a crystallization agent is added little by little to a protein solution, and insoluble matters are removed by centrifugation in a slightly cloudy state. The supernatant is placed in a small test tube and sealed, and then allowed to stand. It is a method to do. Although this method is simple in operation, it is difficult to obtain crystals. Furthermore, there is a problem that the quality of the crystal is not good. It is also necessary to actively control the crystallization conditions.

  “Vapor diffusion method” is a method in which a droplet of a protein solution containing a protein crystallization agent is placed in a container containing a buffer solution (external) containing a higher concentration crystallization agent, and then left to stand after sealing. . A distinction is made between the hanging drop method and the sitting drop method, depending on how the droplets are placed. The hanging drop method is a method in which a small droplet of a protein solution is placed on a cover glass, the cover glass is inverted on the solution reservoir, and sealed. The sitting drop method is a method in which a droplet base is placed inside a solution reservoir, a small drop of protein solution is placed on the droplet reservoir, and the droplet reservoir is sealed with a cover glass or the like. In the vapor diffusion method, the concentration of the protein solution and the crystallization agent changes with time. Further, since a small amount of protein is used, it is used for screening of a wide range of crystallization conditions (Patent Document 2). In the vapor diffusion method, a large number of conditions can be examined with a smaller amount of protein, but in that case, an expensive apparatus is required.

  “Liquid-liquid diffusion method” or “dialysis method” is a method in which a protein solution is brought into contact with a buffer solution containing a crystallization agent at the interface of both solutions or a gel or a semipermeable membrane, respectively. In this method, the concentration of the crystallization agent is gradually increased. These methods have the advantages of the vapor diffusion method and the batch method, and are suitable for obtaining high-quality protein crystals. However, these methods use a relatively large amount of protein solution, and it is difficult to reduce the amount of the protein solution. Also, since the operation is complicated and time consuming, it is difficult to say that it is simple.

  In addition, based on the above background art, the present inventor used a protein-containing sample for a gel polymer holding a protein crystallization agent, and was suitable for X-ray crystal structure analysis by contacting the protein solution with the crystallization agent. A method for easily obtaining protein crystals has been proposed.

However, the above method is excellent in that the crystal grows slowly, but a large number of energy changes (thermal fluctuations) such as temperature and pressure are required to generate appropriate nuclei. Therefore, there are some parts that are difficult to say.
JP-A-9-89898 WO00 / 60345

  As a result of intensive studies to solve the above problems, the present inventors have provided a protein-containing sample to a gel polymer holding a protein crystallization agent in a method for crystallizing a protein, and the protein In addition to the step of bringing the solution into contact with the crystallization agent, the addition of a step of layering the oil phase on the protein-containing sample promotes the generation of high-quality nuclei in a short time, and further, the protein suitable for X-ray crystal structure analysis The inventors have found that crystals can be easily obtained, and have completed the present invention.

That is, the present invention
A method for crystallizing a protein is a method for crystallizing a protein, comprising the following steps.

(a) A step of subjecting the protein-containing sample to a gel polymer holding a protein crystallization agent and bringing the protein solution into contact with the crystallization agent.

(b) A step of overlaying an oil phase on the protein-containing sample.

  According to the present invention, the protein crystallization agent slowly moves from the gel into the protein-containing sample portion, or the protein-containing sample slowly moves into the gel holding the protein crystallization agent, and the crystallization reaction occurs little by little. At the same time, water moves to the oil phase and the protein solution is gradually concentrated. Because of these two effects, the interaction between the protein and the surrounding environment (presence of protein crystallization agent, protein concentration, pH, etc.), which is the mechanism of crystallization, gradually proceeds from two directions, so that the quality is improved in a short time. Nucleation is promoted and high quality crystals can be obtained.

  The term “protein” as used herein includes natural or synthetic peptides, polypeptides, proteins and protein complexes. These substances are extracted and isolated from natural or synthetic materials, or produced by genetic engineering techniques or chemical synthesis techniques, and then combined with conventional purification methods such as solvent extraction, column chromatography, liquid chromatography, etc. It can be purified by use.

  The term “crystallization” as used herein refers to obtaining crystals by growing or precipitating crystals from a protein solution.

  As used herein, the term “protein-containing sample” refers to a sample containing a protein to be crystallized or a protein to be crystallized for which crystallization conditions are to be specified.

  As used herein, the term “protein crystallizing agent” means a compound that acts to lower the solubility of a protein, and includes a precipitating agent, a pH buffering agent, and other additives.

  As used herein, the term “polymer gel” refers to a polymerized monomer or solution thereof that has been polymerized or gelated by a polymerization reaction, or the fluidity of a solution by the addition of a synthetic polymer or a natural polymer. It means the thing which lost.

  The present invention is a protein crystallization method characterized by including the following steps in a method for crystallizing a protein.

(a) A step of subjecting the protein-containing sample to a gel polymer holding a protein crystallization agent and bringing the protein solution into contact with the crystallization agent.

(b) A step of overlaying an oil phase on the protein-containing sample.

  Various interactions have a complex influence on protein crystallization. In order to gradually and slowly change these various interactions, the present invention uses a diffusion phenomenon in a gel to slowly bring a protein into contact with a protein crystallizing agent, the interaction between them and others. Create a state that gradually changes the interaction. Furthermore, by layering the oil phase on the protein-containing sample, the moisture of the protein solution moves to the oil phase portion, and the protein solution is concentrated. As a result, the present protein crystallization method can promote the generation of high-quality nuclei in a short time, and can precipitate a high-quality protein crystal simply and efficiently.

  In step (a) of the present protein crystallization method, the protein-containing sample is subjected to a polymer gel holding a protein crystallization agent. Here, the protein-containing sample may further contain a protein solubilizing agent that helps dissolve the protein, a stabilizing agent such as a reducing agent, in addition to the protein to be crystallized. Examples of protein solubilizers include surfactants that dissolve membrane proteins. Here, the polymer gel holding the protein crystallizing agent can be prepared as follows.

  The type of polymer gel that can be used in the present invention is not particularly limited. For example, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, Multi-functionality of one or more monomers such as N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, allyl dextrin and methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate, etc. For example, a gel obtained by copolymerizing monomers in an aqueous medium can be used. Other polymer gels that can be used in the present invention include, for example, gels such as agarose, alginic acid, dextran, polyvinyl alcohol, and polyethylene glycol, or gels obtained by crosslinking these.

  In order to fill a gel in an arbitrary container (for example, a microarray), a liquid containing a monomer such as acrylamide as a gel component, a polyfunctional monomer, and an initiator is injected into the container, and polymerization and gelation are performed. You can do it. Gelation may be carried out using a crosslinking agent after copolymerization in the absence of a polyfunctional monomer, in addition to a method of copolymerization in the presence of a polyfunctional monomer. In the case of an agarose gel, gelation may be performed by a temperature drop.

  The method for retaining the protein crystallizing agent on the polymer gel is not particularly limited. Here, “retaining” means immobilizing the protein crystallizing agent on the polymer gel. For example, the protein crystallization agent and the polymerizable monomer can be mixed and introduced in advance into an appropriate container, and then a polymer gel can be formed through a polymerization process to immobilize the protein crystallization agent. The protein crystallization agent to be retained in the polymer gel varies depending on the type and concentration of the protein to be crystallized, but those skilled in the art will select an appropriate protein crystallization agent, set the concentration and amount appropriately, It can be held on a polymer gel.

  Alternatively, it is possible to impregnate a porous particle or the like with a protein crystallizing agent and include the particle in a polymer gel.

As the protein crystallizing agent, for example, conditions similar to those commercially available such as Grid Screen ^™ , Crystal Screen ^™ I & II, Wizard ^™ I & II can be used.

  Here, the polymer gel holding the protein crystallization agent is preferably transparent so that the state of crystallization can be observed over time with a microscope or the like. The term “transparent” does not necessarily mean that the light transmittance is 100%, and it is sufficient that the crystallinity can be observed.

  For example, when NaCl is held in a gel, the gel is preferably prepared from acrylamide, 2-acrylamido-2-methylpropanesulfonic acid, and dimethylaminomethyl chloride methacrylate. In addition, a polymer gel holding a transparent protein crystallization agent can be obtained by appropriately selecting the above-described monomers and the like according to the type of the protein crystallization agent.

  A protein-containing sample is applied to the polymer gel holding the protein crystallizing agent prepared as described above. The application of the protein-containing sample can be performed by any method. For example, the protein-containing sample is dropped on a polymer gel holding a protein crystallization agent, manually or mechanically filled with a syringe, or the polymer The protein-containing sample is applied by immersing the gel in the protein-containing sample. The amount of the protein-containing sample to be applied is set in consideration of protein crystallization conditions and the like.

  Next, in the step (b), any oil phase may be used as long as it can be separated from water without affecting the protein such as denaturation. For example, liquid paraffin is used. The oil phase is appropriately selected in consideration of the type of protein to be crystallized and the partition coefficient.

  This oil phase is provided so as to be overlaid on the liquid surface of the protein-containing solution. The amount is appropriately selected.

  As described above, after the protein-containing sample is applied to the polymer gel holding the protein crystallization agent, the protein and the protein crystallization agent are brought into contact with each other by overlaying the oil phase on the liquid surface of the protein-containing solution. Furthermore, crystallization proceeds as the protein solution is gradually concentrated. In this protein crystallization method, the protein crystallization agent slowly moves from the gel to the portion of the protein-containing sample, or the protein-containing sample slowly moves into the gel holding the protein crystallization agent, and the crystallization reaction is gradually performed. Occur. At the same time, water moves to the oil phase and the protein solution is gradually concentrated. Because of these two effects, the interaction between the protein and the surrounding environment (presence of protein crystallization agent, protein concentration, pH, etc.), which is the mechanism of crystallization, gradually proceeds from two directions, so that high-quality crystals are precipitated. Will do.

  After applying the protein-containing sample, the polymer gel is allowed to stand in an airtight state or in the atmosphere under an appropriate temperature condition for a time sufficient for the protein to precipitate.

  The time sufficient for the protein to precipitate is approximately 1 hour to 10 days, although it varies depending on the specific protein, concentration, crystallization conditions, and the like. Appropriate temperature conditions also vary depending on the specific protein, concentration, crystallization conditions, etc., but are about 4 ° C. to 30 ° C.

  After a sufficient time has elapsed for the protein to precipitate, the state of protein crystal precipitation is observed, for example, with an optical microscope, an X-ray diffractometer or the like. In this protein crystallization method, a known system for monitoring protein crystallization in a polymer gel can be used in combination. For example, it is possible to determine the success or failure of crystallization at high speed by photographing and recording the state of crystal precipitation with a CCD camera mounted on a microscope and performing image processing.

As described above, in the present protein crystallization method, a protein-containing sample and a protein crystallization agent are gradually contacted and reacted using diffusion in the gel to crystallize the protein. Therefore, in the present invention, protein crystals of good quality suitable for X-ray analysis can be obtained by simply applying a protein-containing sample.

Claims (1)

A method for crystallizing a protein, comprising the following steps.
(a) A step of subjecting the protein-containing sample to a gel polymer holding a protein crystallization agent and bringing the protein solution into contact with the crystallization agent.
(b) A step of overlaying an oil phase on the protein-containing sample.