JP2007055931A - Crystallizing method for protein using zeolite - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a crystallizing method for protein without necessitating crystallization in various crystallizing condition, easily causing change of crystal shape and space group. <P>SOLUTION: The invention relates to the method for crystallizing protein in the presence of a zeolite. The invention also relates to a kit for crystallization of protein comprising zeolite as a constituting component. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel method for protein crystallization. Specifically, the present invention relates to a protein crystallization method that can easily change the shape and space group of protein crystals by performing protein crystallization in the presence of zeolite.

Protein X-ray structural analysis is one of the most effective methods for determining protein structure at present. In order to determine the protein structure, the process of determining the phase is indispensable. As a method for determining the phase, there are a molecular substitution method, a heavy atom substitution method, and the like, but sometimes the phase cannot be determined. In such cases, phase determination is often attempted using crystals of different space groups. In addition, when it is desired to increase the resolution in X-ray diffraction of a protein crystal, the resolution may be greatly improved by changing the space group of the crystal. However, in general, if you want to change the space group of crystals, try to crystallize using many crystallization solutions of different compositions (different types of crystallization agents and additives, their concentrations, pH, etc.) (See Non-Patent Document 1 etc.), it is not easy to precipitate a desired crystal. Therefore, the time and expense spent preparing and crystallizing many different crystallization reagents required becomes enormous as the protein sample increases.
N. Kunishima, Yukuhiko Asada, Mayumi Sugahara, Jun Ishijima, Yuichi Nodake, Mitsuaki Sugahara, Masashi Miyano, Seiki Kuramitsu, Shigeyuki Yokoyama and Michihiro Sugahara, 'A Novel Induced-fit Reaction Mechanism of Asymmetric Hot Dog Thioesterase PaaI' Journal of Molecular Biology (Available online from July 2005) (Printing)

  The solution of the present invention is to develop a protein crystallization method that can change the shape and space group of crystals without having to attempt crystallization under many crystallization conditions.

  The present inventors have conducted intensive research in view of the above circumstances, and when protein is crystallized in the presence of zeolite, the shape of the protein crystal can be easily changed, and crystals of different space groups can be obtained. As a result, the present invention has been completed.

That is, the present invention
(1) A protein crystallization method characterized by crystallizing a protein in the presence of zeolite;
(2) The method according to (1), wherein the zeolite is a molecular sieve;
(3) a protein crystallization kit containing zeolite as a constituent;
(4) The kit according to (3), wherein the zeolite is a molecular sieve.

  According to the present invention, the shape and space group of a protein crystal can be changed by a simple operation. Therefore, when it is desired to change the space group of the crystal, it is not necessary to attempt crystallization under many crystallization conditions.

  The present invention relates to a protein crystallization method characterized by crystallizing a protein in the presence of zeolite. If this invention is used, the shape and space group of a protein crystal can be changed easily. Zeolite is a porous crystal mainly composed of aluminum and silicon, and includes natural zeolite and artificial zeolite. In the present invention, both natural zeolite and artificial zeolite can be used, but artificial zeolite is preferable from the viewpoints of abundant variety, constant composition and shape, and good reproducibility of results.

  The type, characteristics, shape, and the like of the zeolite used in the present invention are not particularly limited, and various types can be used depending on factors such as the type of protein, the desired crystal shape, and the space group. A typical example of an artificial zeolite is a molecular sieve. Molecular sieve is a material having adsorption characteristics similar to those of natural zeolite and is used for adsorption of various molecules. In addition, since molecular sieves are obtained by synthesis, various types and shapes are manufactured. For example, there are types of molecular sieves such as 3A, 4A, 5A, and 13X (adsorption diameters increase in this order) and are commercially available. However, there is no other example of using a molecular sieve as an additive for protein crystallization rather than a molecular adsorbent. Any type of molecular sieve can be used in the present invention.

  As described above, in the present invention, protein is crystallized in the presence of zeolite. That is, zeolite is used as a protein crystallization additive. “In the presence” of zeolite means that the zeolite is added throughout or in part of the protein crystallization process. Thus, for example, the zeolite may first be placed in a container, then the crystallization reagent (often in solution) and protein may be added to the container to crystallize the protein, and the protein is dissolved in the crystallization reagent. Then, the protein may be crystallized by adding zeolite to the solution. According to the present invention, since the crystallized protein is precipitated from the zeolite surface, it can be easily obtained.

  The amount of zeolite added during protein crystallization can be appropriately selected according to factors such as the amount of protein and the volume of the crystallization reagent. The size of the zeolite to be used can be selected according to the size of the apparatus or instrument used for crystallization. As will be described later, the zeolite may be crushed to an appropriate size in a mortar or the like. Regarding the type of zeolite used, it is preferable to try crystallization using several types. This is because crystal precipitation is considered to depend on the size of the pores of the zeolite. For example, when molecular sieve is used as zeolite, it is preferable to attempt crystallization for 3A, 4A, 5A, 13X and the like.

  The crystallization reagent (also referred to as “crystallization agent” in the present specification) used in the present invention is a reagent in which protein crystals are confirmed to be obtained, and the components differ depending on the type of protein. It is normal. Generally, the crystallization reagent is a solution containing polyethylene glycol (PEG), glycerol, a suitable buffer (for example, a citrate buffer having a pH of 5 to 6) and the like.

  Any kind of protein may be used for the crystallization method of the present invention.

  The protein crystallization conditions in the present invention, for example, the crystallization time, temperature, pH, etc. are appropriately determined according to the protein to be crystallized, the crystallization reagent, the type and properties of the zeolite, the desired protein crystal morphology and space group. You can choose.

The following is a typical procedure for the protein crystallization method of the present invention. This is merely a typical example and is not limited. The crystallization method is not particularly limited, and any method can be used as long as molecular sieve can be added. For example, a microbatch method, a hanging drop method, or a sitting drop method may be used. The following procedure is for the microbatch method. The molecular sieve may be a commercially available product.
1. A reagent (crystallization reagent) from which crystals are obtained is prepared.
2. Crush the molecular sieve to an appropriate size (so that it can enter the container). For example, it is crushed in a mortar and processed into a size that fits into a well of a crystallization plate of about 0.1 mm to 0.8 mm.
3. Place one or two molecular sieves in the well of the crystallization plate.
4). A crystallization solution and a protein solution are added thereto, and finally covered with paraffin oil if necessary.
5. The above operation is performed with each molecular sieve (for example, 3A, 4A, 5A, 13X).

  In yet another aspect, the present invention relates to a protein crystallization kit comprising zeolite as a constituent component. The kit may include, for example, several protein crystallization reagents, as well as several molecular sieves in separate containers. Further, for example, a plurality of types obtained by mixing a molecular sieve and a crystallization agent may be prepared. The kit may further include an apparatus and instruments for protein crystallization. In general, an instruction manual is attached to the kit.

  EXAMPLES The present invention will be described more specifically and in detail below with reference to examples, but the examples should not be construed as limiting the present invention.

The usefulness of this crystallization method was confirmed using a protein sample derived from Pyrococcus horikoshii OT3. This protein sample has been confirmed to be crystallized by a crystallization agent of the following composition:
22.5% (w / w) PEG 4000, 0.1 M citrate buffer, pH 5.7.

In this experiment, crystallization was performed using the microbatch method under the crystallization conditions in which the molecular sieves 3A, 4A, 5A, and 13X were added to the crystallization reagent, and the molecular sieve was not added as a reference. These molecular sieves were crushed to a size of about 0.1 mm to 0.8 mm on a mortar and processed to fit into the well of the crystallization plate. In the case where the molecular sieve was not added, columnar protein crystals were precipitated in 1 to 2 days (FIG. 1a). On the other hand, among the four types of crystallization conditions to which the molecular sieve was added, plate-like crystals were precipitated from the surface of the molecular sieve 5A only 6 days after the crystallization only in the condition where the molecular sieve 5A was added (FIG. 1b). Crystal precipitation is thought to depend on the pore size of the molecular sieve. Next, a diffraction experiment was performed using the precipitated crystals (Table 1). The results of the diffraction experiment are shown in Table 1. When there was no molecular sieve, the space group was P3 ₁ 21, whereas when molecular sieve 5A was added, it changed to P3 ₂ 21. The resolution was 2.2 angstroms in the absence of molecular sieve, whereas the addition of molecular sieve 5A resulted in a resolution of 1.60 angstroms. This is considered to be an effect of the molecular sieve 5A.

Here, the quality of the diffraction data is greatly influenced by the cryoprotectant. Thus, crystallization and diffraction experiments similar to those in Experiment 1 were attempted using a crystallization agent having the following composition obtained by adding 30% (w / w) glycerol to the crystallization solution:
22.5% (w / w) PEG 4000, 30% (w / w) glycerol, 0.1 M citrate buffer, pH 5.7.

If a crystal is obtained under these crystallization conditions, a cryoprotectant is not required for the diffraction experiment. As a result, in the case where the molecular sieve was not added, block-like protein crystals were precipitated after 1 to 2 weeks (FIG. 1c). Of the crystallization conditions to which the molecular sieve was added, only under the conditions where the molecular sieve 5A was added, block crystals were precipitated from the surface of the molecular sieve 5A 3 to 4 weeks after crystallization (FIG. 1d). Moreover, the shape of the precipitated crystals was different. Next, a diffraction experiment was performed using these crystals (Table 1). As a result, when there was no molecular sieve, the space group was P3 ₁ 21, whereas when there was molecular sieve 5A, the space group was P3 ₂ 21. The resolution was 2.3 angstroms in the absence of molecular sieve, whereas the resolution was 1.35 angstroms in the presence of molecular sieve 5A. Compared with crystals obtained from crystallization conditions without addition of 30% glycerol, the crystals obtained in Experiment 2 have a difference in resolution, but their space group and cell length are the same. Obtained. From these experimental results, it is considered that the molecular sieve, which is a zeolite, can easily change the space group just by adding it to the protein crystallization solution, and in some cases, the resolution can be greatly improved by changing the space group. It is done.

  According to the present invention, the shape and space group of a protein crystal can be changed by a simple operation. Therefore, the present invention can be used in various fields dealing with proteins, for example, in structural analysis of proteins. .

It is a figure which shows the influence of the molecular sieve which acts on protein crystallization. (A) No molecular sieve under the conditions described in Example 1, (b) Molecular sieve 5A added under the conditions described in Example 1, (c) No molecular sieve under the conditions described in Example 2, ( d) Addition of molecular sieve 5A under the conditions described in Example 2.

Claims (4)

  A protein crystallization method comprising crystallizing a protein in the presence of zeolite.   The process according to claim 1, wherein the zeolite is a molecular sieve.   A protein crystallization kit containing zeolite as a constituent component. The kit according to claim 3, wherein the zeolite is a molecular sieve.