KR101083400B1 - Method for Crystallizing human recombinant SARAH domain of Rassf5 （Nore1） - Google Patents

Abstract

The present invention relates to a method for crystallizing a recombinant protein of human Rassf5 (Nore1) SARAH domain involved in cell division and apoptosis by using a drop mixing vapor equilibrium method. Rassf5-SARAH domain protein crystals can be obtained that are easy to analyze and excellent in crystallinity.

Rassf5, Nore1, SARAH Domain, Crystallization, Droplet Mixture Vapor Equilibrium, Nitrogen Cooling, X-ray Crystallography

Description

Method for Crystallizing human recombinant SARAH domain of Rassf5 (Nore1)}

The present invention is to clone the SARAH domain of recombinant Rassf5 (Nore1) obtained from humans in order to identify the structure of the SARAH domain of recombinant Rassf5 (Nore1) obtained from humans, which is a cell division and apoptosis-related disease protein by X-ray crystallization, Purification and crystallization methods.

The present invention uses SARAH domain protein crystals of recombinant Rassf5 (Nore1) obtained from humans by the above method, and the SARAH domain of recombinant Rassf5 (Nore1) obtained from humans using fast nitrogen cooling to identify the tertiary structure of the protein. The crystal relates to a method of protection from x-rays.

Schee, H. et al., Current biology , 13 , pp. R899-900, 2003

2 Gilliland, G.L. et al., Acta. Cryst., Pp. D 50: 408, 1994

3 Cudney, R. et al., Acta. Cryst., Pp. D 50: 414, 1994

[4] Jancarik, J et al., J. Appl. Cryst . 24 , pp. 409-411, 1991

[5] Bradford, M., Analytical Biochemistry , 72 , pp. 248-254, 1976

6 Garman EF et al., J. Appl. cryst., 30 , pp. 211-237, 1997

Recently, the mechanism of cell division and apoptosis through Mst1 protein has been newly discovered, which is mediated by the protein interacting with a cancer suppressor protein called RASSF through a new structure called SARAH domain. In other words, when apoptosis receptors such as Fas or TNF-alpha are stimulated or treated with apoptosis-producing substances, a protease called caspase-3 acts to activate Mst1, and this protein enters the nucleus, and the histone ( histone) protein phosphorylation causes apoptosis. Apoptosis and cell cycle arrest by the Ras->RASSF-> MST pathway in eukaryotic cells are caused by interactions between SARAH (for Sav / Rassf / Hpo) domains, the C-terminal part of the tumor suppressor. Adjusted. Rassf SARAH domains regulate apoptosis by mutually binding to the Mst1 SARAH domain to form a heterodimer (Schee, H. et al., Current biology , 13 , pp. R899-900, 2003).

 The importance of SARAH domains that regulate apoptosis has been suggested, and if the three-dimensional structure is revealed, the mechanism of apoptosis through protein interactions will become more apparent. Tertiary structure and function studies may provide new clues to the treatment of cell division and apoptosis-related diseases such as cancer, stroke and myocardial infarction. The three-dimensional structure of apoptosis control SARAH domain can be used to identify protein functions and secure source technologies for the discovery of structure-based new drugs.

 It is essential to identify the tertiary structure of the SARAH domain of Rassf5 (Nore1), and to determine the tertiary structure of proteins using x-ray crystallization, it is necessary to first make protein crystals with good physical properties. (Gilliland, GL et al., Acta.Cyst., pp. D 50: 408, 1994; Cudney, R. et al., Acta. Cryst., pp. D 50: 414, 1994)

X-ray crystallography is used to identify the three-dimensional structure of the protein, and various crystallization methods are performed to use this x-ray crystallization. Among these, a method of crystallizing by a hanging-drop vapor diffusion method (Jancarik, J et al., J. Appl. Cryst . 24 , pp. 409-411, 1991) is mainly used. The crystallization principle is as follows. When small droplets of mother liquor and a much larger reservoir solution coexist in an enclosed space, the movement of water or other volatiles occurs between them. There is a super saturation in the solution condition of the protein. In such thermodynamic metastable state, the protein precipitates according to the change of the precipitant, and at this time, it becomes a stable crystallization state with a slow progress. Known precipitants can reduce the solubility of concentrated protein solutions. In order to reduce the relative adsorption layer around the protein molecules, the proteins gather together and form crystals. The reservoir solution is a mixture of these precipitants, buffers, salts, surfactants, etc. in different concentrations, so that the protein solution and the reservoir solution under these various conditions are usually mixed at a ratio of 1: 1 to form droplets. do. The droplets thus obtained are obtained on a glass slide coated with silicon and placed on an inverted state to seal the prepared plate. Initially, the concentration of protein in the droplets differs from that of the reservoir solution, so that the protein does not exist in the crystalline state. When left in this sealed state, equilibrium is gradually achieved, in which crystals are formed under specific conditions according to the principles described above. In such a drop-mixed vapor equilibrium method, the type and proper concentrations of salts, buffers and surfactants, as well as the precipitants in the reservoir solution, the pH, and the experimental temperature of the solution are different depending on the type of protein, and in some cases, It is a very important factor.

In addition, if there is a lot of fluidity in the protein crystals are not generated well, it is very important to increase the probability of success of the crystallization to predict and remove the portion in advance. However, the SARAH domain of recombinant Rassf5 (Nore1) obtained from humans that can be used in the methods of the present invention does not remove any amino acid residues at the amino terminus of the human Nore-SARHA domain, and provides glutathione to facilitate purification at the amino terminus. Fusion using S-translated fusion protein was used. Protein crystals have a short lifespan when exposed to high energy of x-rays, and the strength of data is weakened, which may affect the structural analysis. This is necessary to cool the liquid nitrogen to a temperature of 100 K and then expose it continuously during the experiment to obtain the data of the tertiary structure continuously.

Therefore, the present inventors determined the SARAH domain of recombinant Rassf5 (Nore1) to identify the tertiary structure of the protein of SARAH domain of Rassf5 (Nore1), which is involved in the treatment of cell division and apoptosis-related diseases such as cancer, stroke, and myocardial infarction. Were prepared and analyzed.

In order to achieve the above object, the present invention is to determine the SARAH domain of the recombinant Rassf5 (Nore1) obtained from humans by a hanging-drop vapour diffusion method using a reservoir solution containing sodium-citric acid as a precipitant It provides a method for crystallizing the SARAH domain of recombinant Rassf5 (Nore1) obtained from humans, characterized in that it is characterized.

The SARAH domain of recombinant Rassf5 (Nore1) obtained from humans was fused to glutathione S transferase fusion protein to facilitate purification at the amino terminus without removing any amino acid residues at the amino terminus of the human Nore-SARHA domain. It is characterized by.

In a hanging-drop vapour diffusion method using a reservoir solution as defined herein, the reservoir solution comprises a precipitant and a buffer solution, and additionally 0.1 to 1 (w / w)%, preferably About 0.5 (w / w)% OG (Octyl ß-D glucopyranoside) can be added to obtain improved protein crystals.

The droplet mixing vapor equilibrium reaction time is about 1 to 1 week, preferably about 2 to 4 days at a temperature of about 15 to 25 ℃, preferably about 18 to 22 ℃, more preferably 20 ℃ Preference is given to carrying out the reaction.

As the buffer, it is preferable to use sodium-cacodylate buffer having a concentration of 0.01 to 1 M, preferably 0.05 to 0.5 M, more preferably 0.08 to 0.12 M.

As a precipitant for precipitating the protein, sodium-citric acid at a concentration of 0.5 to 1.5 M, preferably 0.8 to 1.5 M, more preferably 1.0 to 1.4 M, and the pH of the reservoir solution is 3.0 to 10.0, Preferably from 5.0 to 7.0, more preferably from 6.0 to 7.0.

Preferred embodiments of the present invention include sodium-citric acid having a concentration of 0.8 to 1.5 M, preferably 1.0 to 1.4 M as a precipitating agent of the reservoir; Buffers are sodium-cacodylate buffers having a concentration of 0.05 to 0.5 M, preferably 0.08 to 0.12 M; It is characterized by a drop-mixing vapor equilibrium method further using 0.1 to 1 (w / w)%, preferably about 0.5 (w / w)% OG (Octyl ß-D glucopyranoside).

The present invention also provides a SARAH domain of crystalline recombinant Rassf5 (Nore1) having the following crystalline form, obtained from the crystalline human produced by the above-mentioned preparation.

Crystal Form I

Space Group of Crystals: Hexagonal Space Group (P6)

Lattice unit: a = 73.041Å, b = 73.041Å, c = 66.092Å, α = 90˚, β = 90˚, γ = 120˚

Lattice Volume of Crystal: 352600 Å ³

Rsym: 6.1%

In order to analyze the structure of the SARAH domain of recombinant Rassf5 (Nore1) obtained from crystalline humans prepared by the above method, X-rays are used. In this case, crystals exposed to high energy of X-rays have a short lifetime. The strength of the data is also weakened, which affects structural analysis. To avoid this drawback, it is desirable to rapidly nitrogen cool the SARAH domain crystals of recombinant Rassf5 (Nore1) obtained from humans prior to x-ray analysis.

In order to analyze the structure of the SARAH domain of recombinant Rassf5 (Nore1) obtained from crystalline humans prepared above, the SARAH domain crystal of recombinant Rassf5 (Nore1) obtained from humans was rapidly cooled by nitrogen and subjected to high energy X before X-ray analysis. -Protects the SARAH domain crystals of recombinant Rassf5 (Nore1) obtained from humans from the line.

In addition, the present invention provides a recombinant Rassf5 obtained from humans during liquid nitrogen cooling by using a solution for high-speed cooling containing sodium-citric acid as a precipitant, sodium-cacodylate (pH 6.5) as a buffer, and a mixture of glycerol and sucrose as a cryoprotectant. (Nore1) can effectively protect SARAH domain decisions.

Accordingly, the present invention is directed to the SARAH domain of recombinant Rassf5 (Nore1) obtained from crystalline humans obtained above, by nitrogen cooling in a solution for high speed cooling containing sodium-citric acid precipitant, sodium-cacodylate buffer, glycerol, and sucrose. It provides a method for cooling the SARAH domain of recombinant Rassf5 (Nore1) characterized by the Shikim.

In the cooling method defined herein, the concentration of sodium-cacodylate buffer is 0.01 to 0.20 M, preferably 0.05 to 0.15 M, more preferably 0.08 to 0.15 M; the pH is from 3.0 to 10.0, preferably from 5.0 to 7.0, more preferably from 6.0 to 7.0; The concentration of sodium-citric acid is from 0.5 to 2.0 M, preferably from 1.0 to 1.5 M, more preferably from 1.0 to 1.20 M; 10 to 50 (w / w)%, preferably 20 to 40 (w / w)% glycerol; And a fast cooling solution containing 10 to 20 (w / w)%, preferably 12 to 15 (w / w)% sucrose, at a temperature of 80 K to 120 K, preferably 90 to 110 K, at a high temperature nitrogen cooling method. It is characterized by that.

According to the method of the present invention, it is possible to obtain SARAH domain determination of human Rassf5 (Nore1), which is easy to x-ray analysis and excellent in crystallinity. It can be used to secure the source technology for excavation, and it is expected to provide new clues for the treatment of cell division and apoptosis-related diseases such as cancer, stroke, and myocardial infarction through structural and functional studies.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are merely examples for the present invention, and the scope of the present invention is not limited thereto.

Reference Example 1. Amplification and Expression of SARAH Domain Gene of Rassf5 (Nore1) from Humans

Step 1: amplification of the gene

<1-1> Primer Preparation

Based on the genomic sequence information of human Rassf5 (Nore1), the primers of Table 1 were prepared based on the sequence of the gene site considered to be the SARAH domain.

NORE (RASFF5) primer
pGEX4T-1 (N-terminal GST + thrombin site) SEQ ID NO: 1 5'- CGCGGATCCGAGGTAGAGTGGGATGCC-3 ' SEQ ID NO: 2 5'-GGCCTCGAGTTACCCAGGTTTGCCCTG-3 '

A primer having a nucleotide sequence of SEQ ID NO: 1 includes a nucleotide sequence corresponding to a BamHI restriction enzyme recognition site (underlined), and a primer having a nucleotide sequence of SEQ ID NO: 2 is a base corresponding to a Xho I restriction enzyme recognition site (underlined). Sequence.

<1-2> PCR (Polymerase Chain Reaction)

PCR was performed to obtain the gene of the SARAH domain of human Rassf5 (Nore1). Human genomic DNA was used as a template for PCR. Primers were prepared by inserting the BamHI restriction enzyme site in front of the initiation codon in order to make use of subcloning, containing the stop codon and inserting the Xho I restriction enzyme site for purification of the expressed gene product. This primer was used by requesting synthesis to Bioneer. The composition of the reaction mixture for PCR was 100 μl of each primer, 100 ng of template DNA, 10X PCR buffer, 0.2 mmol of dNTP, and 5 units of Taq polymerase (Takara, Japan). The conditions of the PCR reaction were predenaturation at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 30 seconds, annealing at 57 ° C. for 1 minute, and 72 ° C. for 1 minute. The extension reaction was carried out for 45 cycles and finally extended at 72 ° C. for 10 minutes. The PCR resulted in the SARAH domain gene of human Rassf5 (Nore1).

Step 2: Preparation of Expression Vector Comprising SARAH Domain of Human Rassf5 (Nore1)

Subcloning of the SARAH Domain of Human Rassf5 (Nore1)

<2-1> Ligation

Genes obtained through PCR were mixed with pGEX4T-1 vector in a ratio of 3: 1, and 2X rapid ligation buffer and T4DNA ligase (Promega, USA) were added to make the final volume 10µl. Next, recombinants were made at room temperature for 1 hour.

<2-2> transformation

E. coli DH5α transformable cells stored at -70 ° C were dissolved in ice, and then a ligation reaction mixture was added thereto, left for 20 minutes on ice and then 90 ° C at 42 ° C for 10 minutes on ice. After standing, 500 μl of LB broth was added and incubated for 1 hour at 37 ° C., and then plated in LB agar medium containing antibiotic ampicillin and incubated overnight at 37 ° C. The resulting colonies were incubated in 10 ml of LB broth and then plasmid DNA having the SARAH domain gene of human Rassf5 (Nore1) was isolated using a plasmid DNA prep kit purchased from QIAGEN. The transformed plasmid DNA was isolated through restriction enzyme treatment and sequencing.

<2-3> Secondary Transformation

The E. coli BL21 transformable cells stored at 70 ° C. were dissolved in ice, and then a conjugated reaction mixture was added thereto and left for 30 minutes on ice, then left at 42 ° C. for 90 seconds and then on ice for 10 minutes, followed by LB broth. 500 μl of broth was added thereto, followed by incubation for 1 hour at 37 ° C., and then plated in LB agar medium containing the antibiotic ampicillin and incubated overnight at 37 ° C.

Step 3: Expression in Escherichia Coli of the SARAH Domain of Human Rassf5 (Nore1)

10 ml of the inoculated recombinant was added to a 2000 ml Erlenmeyer flask containing 1000 ml of LB broth, incubated at 37 ° C and 200 rpm, and cell growth was measured at 600 nm until the OD reached 0.5. Next, 0.5 mM of isoproyl-β-D-thiogalactopyranoside (IPTG) was added thereto, followed by further incubation at 25 ° C. and 200 rpm to induce overexpression.

Reference Example 2 Purification of the SARAH Domain Protein of RASSF5 (Nore1)

Step 1: Cultivation and Crushing of E. Coli Cells

E. coli cells expressing the SARAH domain variant of Rassf5 (Nore1) prepared in Examples 1 and 2 were inoculated into a 2000 ml Erlenmeyer flask containing 1000 ml of LB broth in the same manner as in Step 3 of Reference Example 1, respectively. 10 ml of one recombinant was added and incubated at 37 ° C. and 200 rpm. Cell growth was measured at 600 nm until the OD was 0.8-1.0, followed by 0.5 mM of IPTG (isoproyl-β-D-thiogalactopyranoside). After incubation at 25 ° C., 200 rpm for 6 hours, the overexpression was induced. Cells were collected by centrifugation (5,000 rpm, 20 min, 4 ° C.) from the above medium, and then 30 ml of 1 × binding buffer (binding buffer, 140 mM NaCl, 2.7 mM KCl, 10 mM NaH 2 PO 4, 1.8 mM KH 2 PO 4 pH 7.3). Resuspend. This was sonicated for 20 minutes using an ultrasonic crusher (Sonicator, Cole-Parmer Instruments, USA), the cells were completely disrupted and centrifuged (15,000 rpm, 45 min, 4 ° C) to collect the supernatant and extract the cells. (cell-free extract) was prepared.

Step 2: Purification by Column Chromatography

The supernatant obtained in step 1 was bound to a glutathione S-transferase (GST) column (Pharmacia, Sweden) previously equilibrated with the above buffer solution, and then the buffer solution was used as an eluate, and reduced glutathione. After eluting with ON buffer, fractions containing SARAH domain protein of Rassf5 (Nore1) were identified by SDS-PAGE, and used for the following procedure. The SARAH domain protein fraction of Rassf5 (Nore1) obtained in the above procedure was collected and concentrated to about 5 ml. Gel permation column (superdex 75, 16) pre-equilibrated with a buffer solution of 25 mM HEPES (7.0), 100 mM sodium chloride and 2 mM DTT. / 60, Pharmacia, Sweden), eluted with the buffer and separated by the molecular weight of the protein. Fractions containing SARAH domain variant protein of Rassf5 (Nore1) were identified and collected by SDS-PAGE.

Example 1 Conditions of Solution for Purification and Crystallization of SARAH Domain Protein of Human Rassf5 (Nore1)

The SARAH domain protein of Rassf5 (Nore1) obtained in the above reference example was obtained by genetic engineering method and then crystallized by applying the drop-mixed vapor equilibrium method described in the literature to this experiment (Jancarik, J. et. al., J. Appl. Cryst., 24 , pp. 409-411, 1991).

It crystallized by the drop mixing vapor equilibrium method under the following conditions. A protein solution was prepared by adding the SARAH domain of Rassf5 (Nore1) to a solution consisting of 25 mM HEPES (7.0), 100 mM sodium chloride, 2 mM DTT, and adjusting the protein concentration to 13.8 mg / ml. Final protein concentration was determined by the Bradford method (Bradford, M., Analytical Biochemistry , 72 , pp. 248-254, 1976).

The final protein solution and initial screen solution (from Hampton research Inc, Emerald Biosystems Inc.) were searched step by step for a determination. A reservoir consisting of 1.2 M sodium-citric acid precipitate and 0.1 M sodium-cacodylate buffer (pH 6.5) The best crystals were obtained when solution (C) was used. Specifically, 1 μl of the SARAH domain protein solution of Rassf5 (Nore1) and 1 μl of the reservoir solution (B) were brought into contact with the surface of the glass slide (A) coated with silicon, and the slide was 0.5 ml of the reservoir solution (C). Cover the plate containing the stored at 20 ℃ constant temperature (see Figure 1). After 3 days, crystals were formed and the space group represented a hexagonal space group. A photograph taken with an Olympus camera is shown in FIG. 2.

Example 2. Solution Conditions for Rapid Cooling Experiments of SARAH Domain Crystals of Rassf5 (Nore1)

In order to solve the problem derived from direct exposure of the SARAH domain crystal of Rassf5 (Nore1) obtained in Example 1 to direct x-rays, the SARAH domain crystal of Rassf5 (Nore1) was described as follows in a high-speed nitrogen cooling method (Garman EF). et al., J. Appl.cryst., 30 , pp. 211-237, 1997).

Searching for the optimum conditions of the fast nitrogen cooling method revealed that 1.2 M sodium-citric acid precipitant, 0.1 M sodium-cacodylate buffer (pH 6.5), 30 (w / w)% glycerol, and 14 (w / w)% sucrose The configured fast cooling solution was shown to best tolerate 100 K liquid nitrogen stream without harming Rassf5 (Nore1) SARAH domain determination. In addition, the ice ring (a phenomenon in which water freezes when the high-speed nitrogen cooling is incomplete) does not appear frequently during the experiment.

Example 3 X-Ray Data Collection and Analysis of SARAH Domain Determination of Rassf5 (Nore1) via Radiation Accelerator

Using SARS domain determination of Rassf5 (Nore1) obtained in Example 2, an experiment was performed to collect data from a radiation accelerator (Macromolecular Beam Line PL-4A) in Pohang University and University. The detector of the accelerator is Q210 (Area Detector Systems Co. USA), and the data limit of the crystal in Fig. 2 is 2.7 Å, the spatial group of the crystal is a hexagonal space group (P6), and the lattice unit is a = 73.041 Å, b = 73.041 Å , c = 66.092 Å, α = 90 °, β = 90 ° and γ = 120 °. As a result of analyzing and calculating the data obtained above, the lattice volume of the crystal was 352600 Å ³ , and as a result of the data processing, the Rsym was 6.1%.

1 is a schematic diagram of a drop mixed vapor equilibrium method for generating SARAH domain crystals of recombinant Rassf5 (Nore1) obtained from humans,

Figure 2 shows a crystallographic picture of the SARAH domain protein of recombinant Rassf5 (Nore1) obtained from humans.

<110> KOREA BASIC SCIENCE INC <120> Method for Crystallizing human recombinant SARAH domain of Rassf5          (Nore1) <130> DIF / 2008-12-0001 / RS <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> nore (rasff5) -1 <400> 1 cgcggatccg aggtagagtg ggatgcc 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> nore (rasff5) -2 <400> 2 ggcctcgagt tacccaggtt tgccctg 27  

Claims (16)

The SARAH domain of recombinant Rassf5 (Nore1) obtained from humans was converted to 0.5-1.5 M sodium-citrate precipitator, 0.01-1 M sodium-cacodylate buffer and 0.1-1 (w / w)% OG (Octyl ß-D glucopyranoside). ) Is pH is crystallized by performing the reaction for 1 to 1 week at a temperature of 15 to 25 ℃ by a hanging-drop vapour diffusion method using a reservoir solution of 3.0 to 10.0 SARAH domain crystallization method of recombinant Rassf5 (Nore1) obtained from human. delete delete delete delete delete delete delete SARAH domain of crystalline recombinant Rassf5 (Nore1) having the following crystalline form, obtained from a crystalline human produced by the method of claim 1: Crystal Form I Space Group of Crystals: Hexagonal Space Group (P6) Lattice unit: a = 73.041Å, b = 73.041Å, c = 66.092Å, α = 90˚, β = 90˚, γ = 120˚ Lattice Volume of Crystal: 352600 Å ³ Rsym: 6.1% The SARAH domain of recombinant Rassf5 (Nore1) obtained from crystalline humans according to claim 9 was prepared with 0.5 to 2.0 M sodium-citric acid precipitation agent, 0.01 to 0.20 M sodium-cacodylate buffer, 10 to 50 (w / w)% glycerol and Cooling method for cooling the SARAH domain of recombinant Rassf5 (Nore1) characterized by fast nitrogen cooling at a temperature of 80K to 120K in a solution for fast cooling of pH 3.0 to 10.0 containing 10 to 20 (w / w)% sucrose . delete delete delete delete delete delete

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