KR100970770B1 - A crystallized recombinant human hSpoT and the method for crystalizing the same - Google Patents

Abstract

The present invention provides a recombinant protein SpoT (hereinafter referred to as 'hSpoT') obtained from human (Homo sapiens), an enzyme that hydrolyzes a growth inhibitory gene (lacking nutrient signaling material) ppGpp (guanosine 5'-diphosphate 3'-diphosphate). This method relates to crystallization using the drop-mixed vapor equilibrium method, hSpoT according to the present invention is easy to X-ray analysis and excellent crystallinity, it is possible to identify the mechanism related to the transmission of nutrient deficiency signal in eukaryotes such as human It can be useful for the study of protein tertiary structure.

ppGpp (guanosine 5'-diphosphate 3'-diphosphate), undernourished signal transduction, hSpoT, drop mixed vapor equilibrium, crystallization

Description

A crystallized recombinant human hSpoT and the method for crystalizing the same}

The present invention relates to hSpoT, an enzyme that hydrolyzes a growth inhibitory gene (nutrition deficiency signaling material) ppGpp (guanosine 5'-diphosphate 3'-diphosphate), and a method for crystallizing hSpoT using a drop-mixed vapor equilibrium method. .

Literature 1 hanging-drop vapor diffusion method; Jancarik, J et al., J. Appl . Cryst . , 24, pp 409-411, 1991

[Reference 2] Gentry, D.R. et al., J. Bacteriol., 1993, p. 7982-7989, Lange, R. et al., J. Bacteriol., 1995, p. 4676-4680

3 Nucleic Acids Research, Givens, R.M. et al., J. Biol. Chem., 2004, 7495-7504

Kasai, K., et al., Nucleic Acids Research, 2002, p. 4985-4992, van der Biezen, EA, et al., PNAS, 2000, p. 3747-3752, Givens, RM, J Biol. Chem., 2004, p.7495-7504

[Reference 5] Garman, E., et al., J. Appl. Cryst., 30, p211-237, 1997

6, Bradford, M., Analytical Biochemistry, 72, p248-254, 1976

The present invention relates to recombinant hSpoT obtained from the human genome and a method of crystallizing it using droplet mixed vapor equilibrium.

In the absence of amino acids, the bacteria respond by reducing protein synthesis and controlling metabolism. This reaction is achieved by ppGpp acting as a secondary messenger. ppGpp is synthesized from GDP and ATP by the genetic product relA and is activated by uncharged transfer RNA attached to ribosomes. Since all transition RNAs are charged by amino acids in the nutrient rich state, ppGpp is synthesized only in the absence of amino acids. Accumulated ppGpp attaches to the beta subunits of RNA polymerases to regulate their function and specificity. ppGpp inhibits the synthesis of ribosomal RNA and ribosomal proteins by regulating RNA polymerase. It also activates a stationary sigma factor (s ^S ) to transcrib several genes needed to cope with stress (Gentry, DR et al., J. Bacteriol., 1993, p.7982-7989, Lange, R et al., J. Bacteriol., 1995, p. 4676-4680).

The synthesized ppGpp is hydrolyzed by the genetic product SpoT, and because ppGpp has a function of inhibiting growth, its abnormal accumulation is fatal to microorganisms. Indeed, E. coli without the SpoT gene dies, and mutants with reduced transcription of SpoT grow slower, even with sufficient nutrients. However, mutants without both the relA and spoT genes cannot synthesize ppGpp, so they live well in rich nutrients but do not grow in medium without amino acids. In addition, SpoT has a weak activity of synthesizing ppGpp, which is achieved by responding to osmotic shock and oxidative stress in addition to amino acid deficiency. Thus, the reactions made by ppGpp are essential for bacteria to survive in response to various stresses, including amino acid deficiencies (Nucleic Acids Research, Givens, R.M. et al., J. Biol. Chem., 2004, 7495-7504).

Although signaling of ppGpp is conserved in various prokaryotes, it has not been investigated in eukaryotes during the 20th century. Recently, however, a team found picomolar amounts of ppGpp in plants, indicating the presence of ppGpp molecules in higher eukaryotes. Indeed, homologues of RelA and SpoT have been found in various eukaryotes such as Chlamydomonas, Arabidopsis, and Nicotiana (Kasai, K., et al., Nucleic Acids Research, 2002, p. .4985-4992, van der Biezen, EA, et al., PNAS, 2000, p. 3747-3752, Givens, RM, J. Biol. Chem., 2004, p.7495-7504).

In order to elucidate the clear reaction mechanism of SpoT protein of higher eukaryotes, it is necessary to study the tertiary structure of protein by X-ray crystallization method. These studies may reveal that metabolism-related signaling mechanisms similar to those present in microorganisms exist in eukaryotes such as humans. In order to reveal the tertiary structure of a protein, it is necessary to make a protein with good physical properties.

X-ray crystallography is used to identify the three-dimensional structure of the protein, and various crystallization methods are performed as a pretreatment step for using such 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 such precipitants, buffers, salts, and detergents (additives, surfactants) in various concentrations.In general, the solution of the protein and the reservoir solution under these various conditions are mixed at a ratio of 1: 1 to form drops. Done. The droplets thus obtained are placed on a glass slide coated with silicon and inverted on a prepared plate to be sealed. Initially, the protein concentration 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. At this time, crystals are formed under specific conditions by the above-described principle.

In this drop-mixed vapor equilibrium, the type and titration of salts, buffers and additives as well as the precipitants in the reservoir solution, the pH and experimental temperature of the solution are chosen differently depending on the type of protein and in some cases the determination of the protein. It is a very important factor in its formation.

In addition, protein crystals use fast nitrogen cooling because their properties are shortened when exposed to high energy of X-rays and the strength of the data may be affected by structural analysis. It is necessary to continuously obtain the tertiary structure data by cooling and then exposing it continuously during the experiment (Garman, E., et al., J. Appl. Cryst., 30 , p211-237, 1997).

Therefore, the present inventors have completed the present invention by preparing a crystal of hSpoT that is easy to X-ray analysis and excellent crystallinity by separating and purifying recombinant hSpoT protein obtained from the human genome.

An object of the present invention is to provide a method for crystallizing hSpoT from a recombinant protein hSpoT obtained from a human genome using a hanging-drop vapour diffusion method using a reservoir solution.

The recombinant protein hSpoT includes the following SEQ ID NO: 1, characterized in that fusion of six histidines at the amino terminus to the protein.

SEQ ID NO: 1 Met Gly Ser Glu Ala Ala Gln Leu Leu Glu Ala Ala Asp Phe Ala Ala Arg Lys His Arg Gln Gln Arg Arg Lys Asp Pro Glu Gly Thr Pro Tyr Ile Asn His Pro Ile Gly Val Ala Arg Ile Leu Thr His Glu Ala Gly Ile Thr Asp Ile Val Val Leu Gln Ala Ala Leu Leu His Asp Thr Val Glu Asp Thr Asp Thr Thr Leu Asp Glu Val Glu Leu His Phe Gly Ala Gln Val Arg Arg Leu Val Glu Glu Val Thr Asp Asp Lys Thr Leu Pro Lys Leu Glu Arg Lys Arg Leu Gln Val Glu Gln Ala Pro His Ser Ser Pro Gly Ala Lys Leu Val Lys Leu Ala Asp Lys Leu Tyr Asn Leu Arg Asp Leu Asn Arg Cys Thr Pro Glu Gly Trp Ser Glu His Arg Val Gln Glu Tyr Phe Glu Trp Ala Ala Gln Val Val Lys Gly Leu Gln Gly Thr Asn Arg Gln Leu Glu Glu Ala Leu Lys His Leu Phe Lys Gln Arg Gly Leu Thr Ile.

The “drop mixed vapor equilibrium method” is known in the art (hanging-drop vapour diffusion method; Jancarik, J et al., J. Appl. Cryst. , 24, pp 409-411, 1991) A first step of forming a droplet by mixing the purified protein solution and the reservoir solution in a ratio of 1: 1; A second step of sealing the drops obtained in the first step by placing them on a glass slide coated with silicon and inverting the prepared plates; A mixture of protein and reservoir solution at a lower concentration than the reservoir solution is sealed and kept at a temperature of 15-25 ° C., preferably 18-22 ° C., for 1-10 days, preferably 3-5 days. Allowing it to slowly equilibrate to include the third step of manufacturing the hSpoT protein crystals under specific conditions.

The pH of the reservoir solution is characterized in that 2.0 to 8.0, preferably 3.0 to 6.0.

The reservoir solution as defined herein is characterized as comprising a precipitant and a salt solution.

In a preferred embodiment of the present invention, the precipitant of the reservoir solution is 10% to 40%, preferably 15% to 35% polyethylene glycol 3,350 (PEG 3,350), the salt solution is 0.1 to 1M, preferably 0.2 to 0.5 M chloride solution of citric acid.

The present invention also provides hSpoT crystalline protein contained in the subunit lattice having the following physical properties produced by the above-mentioned manufacturing method.

Crystalline form

Spatial Group of Crystals: Rhombic Space Group (C2221)

Lattice Units: a = 72.713, b = 79.796, c = 62.474

α = 90˚, β = 90˚, γ = 90˚

Number of shooting points: 218,533

Number of required grid points: 36,875

       Lattice Volume of the Crystal: 362,587.1

       Rsym: 10.9%

In order to analyze the crystalline structure of the recombinant protein hSpoT obtained from the human prepared above, the hSpoT crystals are nitrogen-cooled at high speed prior to X-ray analysis to effectively protect the hSpoT crystals from high energy X-rays.

In addition, the present invention provides a crystalline hSpoT obtained in the above concentration of 10 to 40 (w / w)%, preferably 15 to 35 (w / w)% of polyethylene glycol 3,350 (PEG 3,350), 0.1 to 1M as a salt solution, It is characterized in that the nitrogen-cooled solution containing a solution of 0.2 to 0.5M citric acid and 10 to 50 (w / w) ethylene glycol at a temperature of 80K to 120K, preferably 90K to 110K It provides a method of cooling hSpoT protein.

According to the present invention, the recombinant protein hSpoT obtained from the human genome is easy to X-ray analysis and has excellent crystallinity. It can be usefully used.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example  One. hSpoT  Gene Subcloning  And expression

Step 1: hSpoT  Gene Cloning

1-1. primer  making

The DNA sequence of the gene of hSpoT was prepared primers of Table 1 based on Gene ID: 374659 (NCBI nucleotide ID: XM_351013) (provided by Professor KAIST Jung Jong-kyung).

SEQ ID NO: 2 5'- ACT CATATG GGC TCT GAG GCG GCG CAG CTG -3 ' SEQ ID NO: 3 5'- ACT CTCGAG TCA GGT TGT CAG CCC CCG CTG CT -3 '

The primer having the nucleotide sequence of SEQ ID NO: 2 includes a nucleotide sequence corresponding to the Nde I restriction enzyme recognition site (underline), and the primer having the nucleotide sequence of SEQ ID NO: 3 corresponds to the Xho I restriction enzyme recognition site (underline) Including the nucleotide sequence, the primers were used in the synthesis by Genotech (Korea).

1-2. PCR  ( Polymerase Chain Reaction )

As template DNA for PCR, a plasmid cloned with the hSpoT gene in pGEX 4T-1 was used. 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. Extension reaction was carried out for 30 cycles and finally extended at 72 ° C. for 10 minutes. As a result of the PCR, the hSpoT gene to be inserted into the expression vector was obtained.

1-3. Restriction Enzyme Treatment

Add the restriction enzymes Nde I (10 U) and Xho I (10 U) to the hSpoT gene and pET-28a (+) expression vector (Novagen) obtained by PCR, and then add 10X buffer solution so that the final volume was 20 μl. Next, the reaction was carried out at 37 ° C. for 1 hour 30 minutes.

1-4. Gel elution elution )

DNA bands from sections obtained by electrophoresis of the gene products obtained through restriction enzyme treatment on a 1% agarose gel were cut from the gel, and the gene products were eluted according to the method provided using the QIA quick Gel Extraction Kit (QIAGEN, USA). elution).

1-5. join( ligation )

A commercially available pET-28a (+) vector (Novagen) was treated with the same restriction enzyme (Nde I / Xho I), and the DNA ratio of the hSpoT gene obtained through gel elution was mixed 1: 3, and the 10X conjugation buffer was added thereto. And T4 DNA conjugated enzyme (Takara, Japan) were added to the final volume of 20 μl and left overnight at 16 ° C. to make a recombinant expression vector into which the hSpoT gene was inserted.

1-6. Transformation ( transformation )

E. coli BL21 (DE3) transformable cells stored at -70 ° C were dissolved in ice, and then the conjugated reaction mixture was added thereto and left for 30 minutes on ice. After standing for 2 minutes, 500 μl of LB broth was added and incubated for 1 hour at 37 ° C., and then plated in LB agar medium containing antibiotic kanamycin and incubated overnight at 37 ° C. The resulting colonies were incubated in 10 ml of LB broth and then plasmid DNA having each hSpoT gene was isolated using a plasmid DNA prep kit purchased from Intron (Korea). The transformed plasmid DNA was identified by sequencing (SolGent Co., Ltd, Korea).

Step 2: hSpoT  Protein In E. coli  Expression

10 ml of the recombinant inoculated into a 2000 ml Erlenmeyer flask containing 1000 ml of LB broth was incubated at 37 ° C and 200 rpm, followed by cell growth measurement at 600 nm under UV / Vis spectrophotometer. Was raised to 0.5-0.7 and then 1 mM of IPTG (isoproyl-β-D-thiogalactopyranoside) was added thereto, followed by further incubation at 18 ° C. and 200 rpm overnight to induce overexpression.

Example  2. hSpoT  Purification and Crystallization of Proteins

2-1. hSpoT  Purification of Protein

Step a: Cultivation and disruption of E. coli cells

       Escherichia coli cells expressing hSpoT variants were overexpressed in the same manner as in Step 2 of Example 1, and the cells were collected by centrifugation (5,000 rpm, 20 min, 4 ° C.), and then bound with 1 × binding buffer (binding buffer, 5 mM imidazole, 500 mM NaCl, 25 mM sodium phosphate, pH 7.0) 20 ml was resuspended, and the cells were lysed with an ultrasonic sonicator while standing on ice. After sonication of the cells for 10 minutes using an ultrasonic sonicator, the cells were completely disrupted and centrifuged (15,000 rpm, 45 min, 4 ° C) to collect the supernatant to prepare a cell-free extract. It was.

Step b: column  Purification by Chromatography

The supernatant obtained in step a was bound to a nickel-nitrilotriacetic acid (Ni-NTA) column (Pharmacia, Sweden) previously equilibrated with the above buffer solution, and then the buffer solution was used as an eluate and the 250 mM imidazole buffer solution After elution, fractions containing hSpoT protein were identified and collected by SDS-PAGE and used in the following procedure. The imidazole in the elution solution was removed using dialysis buffer (dialysis buffer: 20 mM Tris-Cl, pH 7.0). Histidine tag was removed by treatment with thrombin, and the remaining thrombin was removed using a Benzamidine column. The fraction of hSpoT protein obtained in the above process was collected and concentrated to about 5ml, gel permeation column (GPC: gel permeation column, superdex 75, 16) pre-equilibrated with a buffer solution of 20 mM phosphate (pH7.0), 50 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 hSpoT variant protein were identified and collected by SDS-PAGE.

2-2. hSpoT  Crystallization of Proteins

After obtaining the hSpoT protein of Example 2-1 by genetic engineering method, the droplet mixing vapor equilibrium method described in the following literature was applied and crystallized in this experiment (hanging-drop diffusion method; Jancarik, J et al., J. Appl . Cryst . , 24 , pp 409-411, 1991).

A protein solution was prepared by adding hSpoT protein to a solution consisting of 20 mM phosphate (pH7.0), 50 mM sodium chloride, and 2 mM DTT, and adjusting the protein concentration to 20 mg / ml. Final protein concentrations were determined by the Bradford method described in Bradford, M., Analytical Biochemistry, 72, p248-254, 1976.

The final protein solution and initial screen solution (Hampton research Inc, Emerald Biosystems Inc) were used to search for crystallization conditions, using a Hydra II plus automatic crystallization instrument (Matrix, Co.). In order to obtain many protein crystals required for the X-ray experiment, specifically, 2 μl of purified hSpoT protein solution and 2 μl of reservoir solution (B) were brought into contact with a silicon coated glass slide (A). Was placed on a plate containing 0.5 ml of reservoir solution (C) and stored at 20 ° C. constant temperature (see FIG. 1). After 1 day, crystals were formed and the space group represented a tetragonal space group. After one week, the crystals grew to 0.1 x 0.1 x 0.4 mm and had a square pillar shape. The resulting crystals taken with an Olympus camera (C7070, Olympus) are shown in FIG.

Example  3. hSpoT  Fast Cooling Method of Protein Crystals

In order to solve the problem derived when the hSpoT crystals obtained in Example 2 are directly exposed to X-rays, hSpoT crystals were prepared by the high-speed nitrogen cooling method described in the literature (Garman, E., et al., J. Appl. Cryst., 30, p211-237, 1997). As a result of searching for the optimum condition of the high-speed nitrogen cooling method, hSpoT was found to be 15 to 35 w / w% polyethylene glycol 3,350 (PEG 3,350), 0.2 to 0.5 M chloride solution of citric acid and 10 to 50 w / w% ethylene as a salt solution. Fast cooling solutions containing glycols have been shown to best tolerate a 100 K liquid nitrogen stream without harming the hSpoT crystals. In addition, there was no ice ring phenomenon, which is a phenomenon in which water freezes when the high-speed nitrogen cooling that is frequently performed during the experiment is not complete.

Example  4. Through radiation accelerator hSpoT  X-ray data collection and analysis of protein crystals

Using the hSpoT protein crystals obtained in Example 3, 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), which has a data limit of type I crystals of 1.7, the spatial group of crystals is a tetragonal space group, and the lattice units are a = 72.713, b = 79.796, c = 62.474, α = 90˚, β = 90˚ and γ = 90˚. The total number of grid points was 218,533, of which 36,875 were required and represented about 5.93 times multiplicity.

As a result of analyzing the data obtained above, the crystal lattice volume is 362,587.1 and it is calculated that one hSpoT protein is contained in the subunit lattice. As a result of the data processing, Rsym exhibited a value of 10.9%.

1 is a diagram illustrating a drop mixed vapor equilibrium method for generating recombinant protein hSpoT crystals obtained from the human genome,

(A: glass slide, B: protein solution, reservoir solution mixed solution, C: reservoir solution)

2 is a diagram showing a crystallization picture of the recombinant protein hSpoT obtained from the human genome.

<110> Korea Basic Science Institute <120> A crystallized recombinant human hSpoT and the method for          crystalizing the same <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 179 <212> PRT <213> Artificial Sequence <220> <223> hSpoT <400> 1 Met Gly Ser Glu Ala Ala Gln Leu Leu Glu Ala Ala Asp Phe Ala Ala   1 5 10 15 Arg Lys His Arg Gln Gln Arg Arg Lys Asp Pro Glu Gly Thr Pro Tyr              20 25 30 Ile Asn His Pro Ile Gly Val Ala Arg Ile Leu Thr His Glu Ala Gly          35 40 45 Ile Thr Asp Ile Val Val Leu Gln Ala Ala Leu Leu His Asp Thr Val      50 55 60 Glu Asp Thr Asp Thr Thr Leu Asp Glu Val Glu Leu His Phe Gly Ala  65 70 75 80 Gln Val Arg Arg Leu Val Glu Glu Val Thr Asp Asp Lys Thr Leu Pro                  85 90 95 Lys Leu Glu Arg Lys Arg Leu Gln Val Glu Gln Ala Pro His Ser Ser             100 105 110 Pro Gly Ala Lys Leu Val Lys Leu Ala Asp Lys Leu Tyr Asn Leu Arg         115 120 125 Asp Leu Asn Arg Cys Thr Pro Glu Gly Trp Ser Glu His Arg Val Gln     130 135 140 Glu Tyr Phe Glu Trp Ala Ala Gln Val Val Lys Gly Leu Gln Gly Thr 145 150 155 160 Asn Arg Gln Leu Glu Glu Ala Leu Lys His Leu Phe Lys Gln Arg Gly                 165 170 175 Leu Thr Ile             <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> hSpoT <400> 2 actcatatgg gctctgaggc ggcgcagctg 30 <210> 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> hSpoT <400> 3 actctcgagt caggttgtca gcccccgctg ct 32  

Claims (7)

Human Genome reservoir containing (genome) Purification of SEQ ID NO: 1, recombinant protein hSpoT solution and precipitant chloride citric acid solution having a concentration of 10% to 40% of polyethylene glycol 3,350 (PEG 3,350), a concentration of 0.1 to 1M as a salt solution as to the Mixing the solution in a ratio of 1: 1 to form drops; A second step of sealing the drops obtained in the first step by placing them on a glass slide coated with silicon and inverting the prepared plates; In the third step, hSpoT protein crystals are formed in a condition where the mixed droplets of the protein and the reservoir solution having a lower concentration than the reservoir solution are kept in a sealed state at a temperature of 15 to 25 ° C. for 1 to 10 days to be slowly equilibrated. Crystallization method to crystallize the hSpoT protein contained in the subunit lattice with the following physical properties including the manufacturing process: Spatial Group of Crystals: Monoclinic Space Group (C2221) Lattice Units: a = 72.713, b = 79.796, c = 62.474 α = 90˚, β = 90˚, γ = 90˚ Number of shooting points: 218,533 Number of required grid points: 36,875        Lattice Volume of the Crystal: 362,587.1        Rsym: 10.9%. delete delete delete delete delete       The crystalline protein hSpoT according to claim 1 is used in a concentration of 10 to 40 (w / w)% polyethylene glycol 3,350 (PEG 3,350), 0.1 to 1 M chloride solution of citric acid and 10 to 50 (w / w)% ethylene glycol as a salt solution. Cooling method of the hSpoT protein, characterized in that the cooling in a high-speed cooling solution comprising a nitrogen at 80K to 120K.

Images