KR101398895B1 - Method of Crystallizing LsrK and Crystal Thereof - Google Patents

Abstract

The present invention provides a method and a method for crystallizing LsrK involved in the quorum sensing signal transmission of bacteria.

Description

Crystallization method of LsrK and its determination {Method of Crystallizing LsrK and Crystal Thereof}

The present invention relates to a method for producing a protein single crystal and the development of a protein activity modifier using the three-dimensional structure of the single crystal. Specifically, the present invention relates to the preparation of a single crystal of LsrK protein and its activity regulator, which are important components of bacterial quorum recognition.

Quorum sensing is a signaling process in which bacteria communicate with each other in the same species or heterogeneity to measure the bacterial density around them and thereby regulate the expression of a wide range of genes. Quorum recognition is a signaling pathway behind a variety of phenomena such as bacterial motility, surface attachment, biofilm formation, and pathogenicity. Therefore, development of means to control bacterial quorum recognition can be useful in various fields such as the development of new antibiotics, the control of resistance to existing antibiotics, the regulation of human intestinal flora, and the prevention of biofilms on the surfaces of sewage pipes and in vivo implants. Can be used to.

In quorum recognition, a small molecule called autoinducer is used as a signaling molecule. Among them, AI-2 is a universal quorum-sensing signal transduction agent that can act on various bacteria to cause changes in gene expression depending on the number of bacteria (Xue, T. et al., Cell Research (2009): 1258-1268). The specific form of AI-2 differs slightly from that of bacterial species, but the evolutionary phase of Lux-S, which is a highly conserved synthetic enzyme in many species of bacteria, is responsible for the synthesis of 4,5-dihydroxypentane- , And 4,5-dihydroxy-2,3-pentanedione (DPD) are generated in the process. The structure of 4,5-dihydroxypentane-2,3-dione is shown in Formula (1).

The final product, AI-2, is a linear or cyclic form of furanone, a group of substances produced by spontaneous conversion from DPD, the intermediate. Since this spontaneous mutual conversion is possible in aqueous solution, several types of AI-2 including DPD can coexist in the intracellular environment of bacteria having LuxS (Miller ST. Et al., Mol Cell 15: 677-687) (See Xavier KB, Bassler BL (2005) Nature 437: 750-753) among bacteria producing different forms of AI-2.

Quorum recognition by autoinducer-2 is common to many bacteria, but is slightly different from each other, so it has both universality and selectivity. And it was confirmed that mutants lacking the genes of the individual components involved in the signal transduction pathway could survive. Therefore, it is highly likely that the activity modifier of AI-2 quorum recognition does not induce resistance to bacterial resistance. In this respect, AI-2 quorum recognition is an attractive subject.

FIG. 1 shows a schematic diagram of a quorum recognition process by the self-induction agent-2. As an example of E. coli, an autoinducer-2 (black pentagon in FIG. 1) present in the surrounding environment enters the germ through the LsrABCD receptor complex and lsr Transcription of six operon dominated genes ( lsrACDBFG ) and lsrK and lsrR occurs. When no autologous inducer-2 is present, these genes are transcriptionally blocked by an LsrR transcription inhibitor that attaches to the promoter of the operon. The autoinducer-2 is phosphorylated by kinase, LsrK. When the phosphorylated autoinducer-2 binds to LsrR, the LsrR bound to the promoter dissociates from the promoter, resulting in full-scale transcription of the above-mentioned genes. However, LsrR does not dissociate in the promoter when only non-phosphorylated autoinducer-2 is present (Xue, T. et al., Cell Research (2009): 1258-1268). Transcriptional regulation of bacteria by LsrR also affects the expression of genes other than those described above.

5-phospho-4-hydroxypentane-2,3-dione (P-DPD), a form of phosphorylation of autoinducer-2, is a natural substrate of LsrR ) Or a ligand. LsrK is a kinase that transfers the phosphate group of ATP to the hydroxyl group of the 5th carbon, which is the terminal carbon, based on DPD, and phosphorylates AI-2 family substances including DPD. LsrK is also not very rigorous in substrate selectivity, such as phosphorylating ribose-5-phosphate, which is similar in structure to phosphorylated autoinducer-2, and ribose-5-phosphate However, it binds to LsrR.

In suppression of lsr operon by LsrR transcription inhibitor, LsrK plays a key role in the control circuit of quorum recognition. It is known that if LsrK is not expressed, induction of transcription by AI-2 does not occur and that the effect of transcription alone is not obtained by AI-2 that is not phosphorylated. Therefore, the means for enhancing or inhibiting the activity of LsrK is expected to be a useful means of controlling the quorum recognition of bacteria. However, information on the high-resolution three-dimensional structure of LsrK is essential for the development of active modulators of LsrK and for a deeper understanding of quorum recognition. However, to date there have been few studies on single crystal of LsrK protein and its preparation method.

One of the technical problems of the present invention is to provide a method of manufacturing LsrK single crystal capable of providing high-resolution three-dimensional structure information of LsrK and a substrate bonded thereto.

According to an aspect of the present invention, there is provided a method of forming a hexagonal space group P3 comprising asymmetric units including LsrK protein and HPr protein, wherein the lattice unit is a = 100 Å, b = 100 Å, c = 353 A, the triaxial angle gives a crystal of LsrK with α = β = 90 ° and γ = 120 °.

In another aspect of the present invention, there is provided a hexagonal space group P21212, wherein the asymmetric unit includes LsrK protein and HPr protein, wherein the lattice unit is a = 100 Å, b = 173 Å, c = 352 Å, = gamma = 90 [deg.].

In one embodiment of the present invention, the LsrK protein constituting the LsrK crystal is a full length sequence derived from Escherichia coli. In another embodiment of the present invention, the LsrK protein of the crystal is an LsrK protein which is 80% to 99.5% homologous to the E. coli-derived sequence.

In another embodiment of the present invention, the above-mentioned LsrK crystal may be a substrate such as 4,5-dihydroxypentane-2,3-dione (DPD) or a substrate containing a substrate analogue such as AMP- It is a crystal (cocrystal).

In another aspect of the present invention, there is provided a method for producing the aforementioned LsrK crystal. The LsrK crystallization method of the present invention comprises the steps of preparing an LsrK storage liquid in which LsrK is dispersed at a concentration of 5 to 20 mg / mL, mixing the LsrK storage solution and the crystallization buffer solution at a volume ratio of 1: 1 And placing the mixed droplets in an enclosed space containing the container such that vapor diffusion equilibrium is established between the mixed droplets and the container containing the crystallization buffer solution. The composition of the crystallization buffer solution is described later in this specification.

In a specific embodiment of the crystallization method of the present invention, the expression process of LsrK further comprises an osmotic shock stage in which 250 to 500 mM of sodium chloride is added to a culture medium of a bacterium encoding LsrK, a temperature of the culture medium A heat shock step of giving a heat shock to the bacteria encoding the LsrK at a higher temperature and then lowering the temperature of the medium rapidly; inducing LsrK gene transcription in the bacteria encoding the LsrK; Followed by quenching at a temperature of 0 ° C to 4 ° C, followed by quenching at a temperature below room temperature, for example, at 15 to 25 ° C.

The LsrK crystal of the present invention and its crystallization method are crucial for understanding the three-dimensional structure of LsrK, which plays a key role in the quorum sensing signal transmission of bacteria. This includes new antibiotic discovery, biofilm control, Vibrio cholerae , Salmonella typhimurium and Pseudomonas can be used as a tool to study the quorum detection of bacteria directly linked to the inhibition and prevention of major infectious bacterial diseases including aerunginosa .

Brief Description of the Drawings Fig. 1 is a schematic diagram showing the main components of the quorum detection of bacteria by autoinducer-2.
2 shows a crystal photograph of LsrK derived from Escherichia coli obtained in Example 1 of the present invention. FIG. 2A is a co-crystal of LsrK and HPr and 4,5-dihydroxypentane-2,3-dione obtained according to the condition (a) of Example 1, And the co-crystals of LsrK and HPr and 4,5-dihydroxypentane-2,3-dione obtained.

Hereinafter, the present invention will be described in more detail. In interpreting the terms or words used in the present specification and claims, it is to be understood that the interpretation of terms or words used herein is necessarily conventional, based on the principle that the inventor can properly define the concept of the term to describe its invention in the best way It is to be understood that the invention is not to be interpreted as being limited only by the precautionary sense, but should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention as described in the present specification.

In the present application, when two protein sequences are homologous or homologous, the two sequences to be compared are sorted according to appropriate criteria, and then the identity between the amino acids is scaled according to the standard Percentage means that the scale exceeds the threshold. Alignment of the sequences can be accomplished using known computer algorithms. For example, Needleman, SB and Wunsch, CD (1970) A General Method Applicable to the Search for Similar Proteins in the Amino Acid Sequence of Two Proteins. J. Mol . Biol . 48: 442-453, and currently known methods such as FASTA, Smith-Waterman and BLASTP use this algorithm. The value of sequence matching can also be obtained using an appropriate scoring function. In order to obtain the percentage homology, a well-known scoring matrix such as BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM 70, BLOSUM 75, BLOSUM 80, BLOSUM 85 and BLOSUM 90 ). ≪ / RTI > Specifically, percentage sequence homology can be obtained by the following known methods. For example, using a program available in the Wisconsin Sequence Analysis Package, 9.1 edition (Devreux J. et al., Nucleic Acid Res . , 12: 387-39, 1984), for example BESTFIT and GAP, (%) And similarity (%) can be measured. BESTFTT uses a "partial homology" of Smith and Waterman ( J. Mol . Biol. , 147, 195-197, 1981) and reveals a single domain of maximum similarity between both sequences. BESTPTT, a program whose short sequence is assumed to represent part of a long sequence, is more suitable for comparing two polynucleotide sequences of different lengths. The GAP program reveals the "maximum similarity" according to the Needleman and Wunsch algorithm described above. GAP is more suitable for comparing sequences that are expected to be aligned over the entire length and about the same length. Preferably, the parameters "Gap Weight" and "Length Weight" used in each program are 12 and 4 for the polypeptide sequence. Preferably, when the two sequences to be compared are optimally aligned, agreement (%) and similarity (%) are measured. Other programs that measure the degree of similarity and / or similarity between sequences are well known in the art and include, for example, the programs of the BLAST series described above (Altschul SF, Nucleic acid Res . , 25: 3389 to 3402, 1997) can be used. BLAST is available from the National Biology Information Center (NCBI) website.

In one aspect of the present invention, a method of crystallizing LsrK protein is provided. The crystallization method of the present invention is a crystallization method based on a vapor diffusion method. This crystallization method

(1) a step of preparing an LsrK storage liquid in which the LsrK is dispersed at a concentration of 5 to 20 mg / mL, and a step of mixing the LsrK storage solution and the crystallization buffer solution to obtain a mixed solution

(2) placing the mixed droplets in an enclosed space containing the container such that vapor diffusion equilibrium is established between the mixed droplets and the container containing the crystallization buffer solution.

The crystallization method of the present invention can be applied to various kinds of LsrK.

In one specific embodiment of the crystallization method of the present invention, LsrK to be dispersed in the LsrK stock solution of step (1) is a polypeptide of the entire length (1 to 530 amino acid residues) derived from Escherichia coli described in SEQ ID NO: 1. In another embodiment of the crystallization method of the present invention, the crystallization method of the present invention is applied to a heterologous sequence of LsrK, for example, a polypeptide of another organism-derived or artificial LsrK-like sequence. That is, a heterologous LsrK that is not an E. coli-derived sequence is sufficiently similar to the above sequence, the crystallization method of the present invention can be carried out. As an example of a heterologous sequence, the crystallization method of the present invention can be applied to a partial length LsrK in which a part of a sequence at a terminal site, particularly a sequence at a N-terminal site, is deleted, which is often used in the art for protein crystallization. For example, the stability of the single crystal and the solubility of LsrK can be increased by using the partial length LsrK, which eliminates the part expected to exist in a disordered structure without forming the secondary structure of LsrK.

In one embodiment of the present invention, LsrK to be dispersed in the above-mentioned LsrK stock solution has a sufficient level of homology as compared with the Escherichia coli-derived sequence as a polypeptide in which a part of the sequence is substituted, deleted or inserted in the above-mentioned Escherichia coli-derived sequence . Using the amino acid sequence alignment and homology comparison algorithm, homology can be measured for the number of amino acid residues or for substituted LsrK-like sequences. In the natural structure of the above-mentioned natural structure, in the native structure of the partial length LsrK in which a part of the sequence is deleted, a polypeptide in which some amino acid residues at the N-terminus are deleted can calculate homology with this principle, and the amino acid residues deleted, inserted or substituted in the corresponding heterologous sequence Can be easily detected using the algorithm described above and a computer program published to the public.

Furthermore, since the three-dimensional structure of the LsrK protein is conserved among different bacterial species, the LsrK protein of another bacterium or the LsrK heterologous sequence having sufficient homology is substantially identical It takes a three-dimensional structure and exhibits similar behavior in crystal formation. More specifically, a sequence having such a sufficient level of homology includes, for example, at least preferably about 70% or more homologous, such as 75% or more homologous, such as 80% or more homologous to the sequence of SEQ ID NO: 1, Such as greater than or equal to 95% homology, such as greater than or equal to 96% homology, such as greater than or equal to 97% homology, such as greater than or equal to 85% homology, such as greater than or equal to 90% homology, But is not limited to, sequences that are 98% or more homologous, such as 99% or more, for example, 99.5% or more homologous.

The LsrK protein to be crystallized can be prepared using methods that are conventional in the art. For example, a method of overexpressing and purifying recombinant LsrK using molecular cloning and genetic engineering methods can be used. Since such cloning and genetic engineering overexpression are well known in the art, a general description of the purification and expression of recombinant LsrK is herein omitted.

In an embodiment of the present invention for overexpression, osmotic shock and heat shock can be performed in parallel, and the amount of expression can be increased by low-temperature treatment. For example, through such osmotic shock, heat shock, and low-temperature treatment, the amount of soluble LsrK expressed in Escherichia coli can be increased about 5 to 10 times as compared with the conventional expression method.

The conditions of this osmotic shock, heat shock and low temperature treatment can be made, for example, as follows.

(i) an osmotic shock phase in which 250 to 500 mM of sodium chloride is added to a culture medium of a bacterium encoding LsrK,

(ii) a heat shock step of subjecting the bacteria encoding the LsrK to heat shock at a high temperature higher than the temperature of the culture medium, and then rapidly lowering the temperature of the medium,

(iii) transferring bacteria that encode the LsrK under the conditions of 0 ° C to 4 ° C and quenching

(iv) inducing LsrK gene transcription at 15 ° C to 25 ° C in a bacterium encoding said LsrK to increase the amount of protein expressed in soluble form.

In one specific embodiment, the osmotic shock step may further increase 0.2-3.0% glucose in a conventional LB (Luria-Bertani) medium with the above-described amount of sodium chloride to increase recombinant E. coli growth at low temperatures.

Specifically, in order to increase the amount of the LsrK protein expressed in a soluble form, when the absorbance of the strain expressing the recombinant LsrK becomes about 0.5 or more, it is placed in a 42 ° C shaker incubator for 60 minutes to give a heat shock, Allow to cool in ice for a sufficient time, for example 0.5-1.0 hours.

Subsequent low temperature treatment leads to transcription induction and low temperature treatment of the LsrK gene. For example, a substance inducing transcription of IPTG (isopropyl beta-D-1-thiogalactopyranoside) or other genes is added to induce LsrK protein expression. After induction of transcription, the strain expressing LsrK is transferred again to a low temperature, for example, 0 ° C ice or 4 ° C refrigerator. Followed by quenching followed by a low temperature (eg overnight at 15-25 ° C.) to induce low temperature expression.

In one embodiment of the present invention, LsrK is first expressed in the form of a fusion protein in order to maintain the stability of LsrK during expression and purification and to prevent precipitation formation, and then, after purification, an unwanted portion is removed from the fusion protein, . Isolation using a fusion protein is well known in the art and is not particularly limited. For example, in a more specific embodiment of the present invention, glutathione-S-transferase is used as such a fusion protein.

In the crystallization method of the present invention, the LsrK storage solution of step (1) may be of a composition similar to that used in the buffer solution used for the expression and purification of LsrK. That is, the LsrK storage solution may be a buffer solution that can prevent precipitation or denaturation of LsrK, and may be dispersed at a desired concentration, for example, at a concentration of 5 to 20 mg / mL to maintain its activity. In the crystallization method of the present invention, the LsrK stock solution may further comprise a natural substrate or ligand of LsrK and a precursor or derivative thereof together with HPr protein. In one embodiment of the present invention, the LsrK stock solution comprises a pH of 7 to 8, an ammonium ion at a concentration of 50 mM or more, an HPr protein at 1 to 3 times the molar concentration of LsrK, and a selective component, AMP-PNP, 0.5 to 2.0 mM 4,5-dihydroxypentane-2,3-dione. Two more specific examples of such a storage solution include: (a) 10 mM Tris (HCl) chloride, pH 200, NaCl 200 mM, and DTT 1 mM or? -Mercaptoethanol 5 mM as a reducing component (B) 10 mM of tris hydrogen chloride, 50 mM of NH ₄ Cl, 150 mM of NaCl and 1 mM of DTT or 5 mM of? -Mercaptoethanol as a reducing component. In particular, when ammonium ions of 50 mM or more are contained in the buffer solution, the solution stability of the protein is increased. The exemplary storage solution may further comprise additional components such as glycerol, buffer substances other than tris, and the concentrations of sodium chloride and dithiothreitol may be added or subtracted by the average skilled artisan in the art.

The HPr protein is an important component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS), a small protein in the cytoplasm of bacteria that plays an important role in the carbohydrate delivery system of bacteria. PTS manages the phosphorylation of sugars, a key regulator of bacterial migration through the cell membrane. The source of the phosphate group that phosphorylates the sugar is phosphoenolpyruvic acid, and HPr is a phosphocarrier protein that plays a role in relaying the phosphate group. Since HPr is an important component of the means by which bacterial cells sense the presence of a nutrient sugar outside the cell, it is a tool for recognizing the environment in which the bacterial cells are located.

In one embodiment of the present invention for expressing and purifying LsrK in Escherichia coli, when LsrK is purified from affinity chromatography and size exclusion chromatography from cell extracts, LsrK and HPr are almost always isolated, and LsrK and HPr ≪ / RTI > stable co-crystals of < RTI ID = 0.0 > While not intending to be bound by any particular theory of the working principle of the present invention, this finding can be interpreted as a close crosstalk between the PTS pathway that recognizes the bacterial food environment and the quorum recognition pathway. Dihydroxypentane-2,3-dione (DPD) and AMP-PNP are some examples of such natural substrates or ligands or precursors or derivatives that may be included in the LsrK stock solution. AMP-PNP is in vivo environment adenosyl -P (= O) present in the form of ^{(O -) -OP (= O} ) (O -) -NH-OP (= O) (O - -) (O) It is an ATP analog that is not hydrolyzed. In one specific embodiment of the present invention, DPD and AMP-PNP may be contained in the LsrK stock solution at a concentration of about 1-5 mM. When these natural substrates or the like are further included in the LsrK storage solution, co-crystals of LsrK with the corresponding natural substrates can be obtained.

In the crystallization method of the present invention, the vapor diffusion equilibrium of step (2) may use a mixed drop method. In the droplet mixing method, droplets mixed with a small amount of the LsrK storage solution and a small amount of the crystallization buffer solution are placed in a closed space, which is a large amount of the same crystallization buffer solution and vapor diffusion equilibrium in a physically- . The volatile components such as water in the mixed droplets are changed in the process of reaching the equilibrium. As a result, the solution composition of the mixed droplets becomes the same as the target composition, that is, the composition of the crystallization buffer solution. The vapor diffusion equilibrium method can use a commonly used suspended drop or a sitting drop. As a modification of this method, a vapor diffusion equilibrium method using a glass capillary can also be used for crystallization.

The method of making the mixed droplets is not particularly limited, and usually, for convenient convenience, it is common to mix the desired protein storage solution and the crystallization buffer solution at a ratio of 1: 1. The amount of the crystallization buffer solution in the reservoir may be sufficient for the mixed droplets. For example, 1 mL of the crystallization buffer solution may be used as a reservoir for mixed droplets mixed with 1 μL of the LsrK stock solution and the crystallization buffer solution.

In the crystallization method of the present invention, as the crystallization buffer solution, an aqueous solution having the following composition (a) to (e) can be used:

(A) polyethylene glycol, 0.05 to 0.2 M citric acid salt at pH 4.5 to 5.5 or 0.02 to 0.1 M ammonium dihydrogen phosphate (KH ₂ PO ₄ )

(B) ammonium dihydrogen phosphate 0.1 to 1.0 M,

(C) 1.0 to 1.2 M ammonium dihydrogen phosphate and 0.05 to 0.5 M Bis-Tris having a pH of 6.0 to 7.5,

(D) 0.85 to 1.5 M of magnesium sulfate hydrate, 0.05 to 0.2 M of citrate of pH 4.5 to 5.5,

(E) 0.05 to 0.1 M guanidine hydrochloride, 0.05 to 0.1 M lithium chloride, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) as an additive to the aqueous solution of the composition exemplified in (a) HCl, tris (2-carboxyethyl) phosphine hydrochloride) 0.01 to 0.02 M.

The polyethylene glycol in the above composition (a) is 20 to 30% (w / v) of PEG-300. In another specific example, polyethylene glycol having a higher molecular weight than PEG-300 may be used at a lower content, or polyethylene glycol having a lower molecular weight may be used at a higher content. For example, a polyethylene glycol having a molecular weight of 10 to 25% (w / v) is used when the molecular weight is larger than that of PEG-300, and a polyethylene glycol having a molecular weight of 25 to 40% (w / v) Can be used.

In one specific embodiment of the invention, the salt of citric acid is used in the form of a sodium salt.

In one embodiment of the present invention, a high-speed nitrogen cooling method (for example, Garman EF et al . , J. Appl . Cryst . , 30 , 211-237, 1997), and the X-ray diffraction is carried out by cooling the crystal. As the freezing buffer solution to be used in the high-speed nitrogen cooling method, the crystallization buffer solution itself may be used. In other cases, (1) the concentration of polyethylene glycol in the crystallization solution is increased or (2) a crystallization buffer solution is added with a cryo-protectant such as PEG-200, PET-400, glycerol or ethylene glycol It is acceptable to use. The concentration of the used cryoprotectant is raised until the solution is able to freeze transparently during cooling.

For example, as the freezing buffer solution of LsrK crystals, those shown in the following compositions (a) to (d) can be used:

(A) polyethylene glycol, 0.05-0.2 M citric acid salt with pH 4.5-5.5 or 0.02-0.1 M ammonium dihydrogenphosphate (KH ₂ PO ₄ ) and 25-30% (v / v) glycerol,

(B) Ammonium hydrogen phosphate 0.1-0.4 M and glycerol 25-30% (v / v),

(C) 0.05 to 0.5 M Bis-Tris with pH 1.0 to 1.2 M ammonium dihydrogen phosphate, 25 to 30% (v / v) glycerol,

(D) 0.85 to 1.5 M of magnesium sulfate hydrate, 0.05 to 0.2 M of citric acid salt having a pH of 4.5 to 5.5 and 25 to 30% (v / v) of glycerol,

The polyethylene glycol in the composition (a) is 20 to 30% (w / v) of PEG-300. In another specific example, polyethylene glycol having a higher molecular weight than PEG-300 may be used at a lower content, or polyethylene glycol having a lower molecular weight may be used at a higher content. For example, a polyethylene glycol having a molecular weight of 10 to 25% (w / v) is used when the molecular weight is larger than that of PEG-300, and a polyethylene glycol having a molecular weight of 25 to 40% (w / v) Can be used.

In another aspect of the present invention, there is provided a crystal of LsrK obtained by the above-mentioned production method. The LsrK crystal of the present invention includes both LsrK and HPr. In addition, the LsrK crystal of the present invention encompasses a co-crystal in which a substrate or a substrate derivative is bound to an active site, and contains an amino acid sequence having a full length LsrK and a partial length LsrK and sufficient homology to these polypeptides.

In one embodiment of the present invention, a single crystal of the entire length LsrK derived from Escherichia coli is a space group P3 of hexagonal system, and provides crystals of LsrK including an LsrK protein, an HPr protein and a substrate (analog) of LsrK in an asymmetric unit. In a more specific embodiment of the present invention, the lattice unit of this single crystal is a = 100 Å, b = 100 Å, c = 353 Å, and the angle of the triaxial is α = β = 90 ° and γ = 120 °. This crystal has a resolution of 3.2 A after refinement of the structure.

In one embodiment of the present invention, a single crystal of the entire length LsrK derived from Escherichia coli is a space group P21212 of a hexagonal system, and provides crystals of LsrK including an LsrK protein, an HPr protein and a substrate (analog) of LsrK in an asymmetric unit. In a more specific embodiment of the present invention, the lattice unit of this single crystal is a = 100 Å, b = 100 Å, c = 353 Å, and the angle of the triaxial is α = β = 90 ° and γ = 120 °. This crystal has a resolution of 3.6 A after refinement of the structure.

[ Example ]

Hereinafter, the present invention will be described in more detail with reference to Production Examples and Experimental Examples. The following examples are intended to illustrate the invention in detail and are not intended to limit the scope of the invention in any way.

≪ Preparation Example 1 > LsrK gene cloning

Based on the LsrK sequence information of the Escherichia coli genome, primers for cloning the full length LsrK gene of amino acids 1 to 530 were prepared and the length and partial length of Escherichia coli LsrK gene were cloned using a polymerase chain reaction (PCR) Respectively. For convenience of subcloning these primers were equipped with restriction enzyme recognition sites. The pGEX-4T-1 vector, which is a glutathione-S-transferase (GST) fusion protein expression vector of GE Life Sciences, USA, was digested with restriction enzyme digestion and ligated with the above-described full-length or partial-length LsrK base sequence Were subcloned. This recombinant is expressed as a fusion protein of LsrK and GST (LsrK-GST) to which the glutathione-sulfur transferase is linked at the N-terminus of the corresponding LsrK. This recombinant expression vector was used to transform DH5α and BL21 transformable E. coli strains.

≪ Preparation Example 2 > Expression of LsrK in E. coli

The E. coli BL21 transformable cells stored at -80 ° C were dissolved in ice, and the ligation reaction mixture was added thereto. The reaction mixture was allowed to stand in ice for 30 minutes, again left at 42 ° C for 90 seconds, on ice for 2 minutes, and LB agar containing antibiotic ampicillin And cultured overnight at 37 ° C. About 5 colonies of average size colonies were inoculated into 15 mL of LB culture medium and cultured in a shaker incubator at 37 ° C for 160 ~ 200 rpm overnight. 5 ~ 10 mL of culture E. coli was inoculated into 1 L of LB culture medium Respectively. The 1 L LB broth was prepared in a 2.8-L wrinkled Erlenmeyer flask, and 250-500 mM NaCl and 0.4% glucose for osmotic shock were added to increase soluble protein expression. After culturing at 37 ° C until the absorbance of E. coli reached 0.5 (600 nm), the medium was transferred to a previously prepared shaker incubator for heat shock at 42 ° C and cultured for one hour. The culture was transferred to ice and the medium was allowed to cool for 0.5-1.0 hours. Then, 0.5-1.0 mM of IPTG (isopropyl-beta-D-thiogalactopyranoside) was added to the culture to induce LsrK transcription, and then 0.5 to 1.0 mM IPTG was added to the culture at a low temperature of about 20 ° C Was added and left overnight (about 18 to 20 hours) to induce overexpression of LsrK.

PREPARATION EXAMPLE 3 Purification of Expressed LsrK

Step 1: Culture and disruption of E. coli cells

E. coli cells overexpressing LsrK of Production Example 2 were collected by centrifugation (4,500 rpm, 25 minutes, 4 ° C) and then washed with 1 × binding buffer (pH 7.0, 50 mM Tris.HCl, 500 mM NaCl, mM [beta] -mercaptoethanol). Proteinase inhibitors were added to the suspension, the cells were disrupted using an ultrasonic disrupter and centrifuged (18,000 rpm, 50 minutes, 4 ° C). The supernatant obtained by centrifugation was collected to obtain a cell-free extract.

Step 2: column  Purification by chromatography

The supernatant obtained in Step 1 was added to a glutathione affinity chromatography column (GSTrap) preliminarily equilibrated with the buffer solution described above. The unbound protein was washed out in a column, and elution buffer solution (pH 8.0, 10 mM Tris.HCl, 200 mM NaCl, 10 mM glutathione), the LsrK-GST fusion protein was eluted and the fractions were collected. Whether the fraction of affinity chromatography contained LsrK-GST was confirmed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Step 3: GST Removal of

To the eluate obtained in step 2, glycerol was added at 5% (v / v) and the concentration of the GST-LsrK fusion protein was adjusted to an appropriate concentration of 5 mg / mL or less. The concentrate was left overnight at 4 ° C with thrombin, and 1.0 mM of fluorinated 4-2-aminoethylbenzenesulfonyl (ABESF) and 1 mM of benzamidine were added to stop thrombin digestion.

Step 4: Thrombin  Size Exclusion Chromatography of Digestion Mixture

The digested mixture was then injected into a size exclusion chromatography column (Superdex 200 and Hitrap-GST, GE Life Sciences, USA) pre-equilibrated with a buffer of pH 8.0, 10 mM Tris.HCl, 200 mM NaCl, Respectively. The Hitrap-GST column was connected in series behind the Superdex 200 to remove the non-cleaved cleaved glutathione sulfur transferase from the Superdex 200 and the untrimmed GST-LsrK protein. By doing so, the total length LsrK or partial length LsrK was separated and the corresponding fractions were collected. Whether the fraction of size exclusion chromatography contained LsrK was confirmed by SDS-polyacrylamide gel electrophoresis.

total length LsrK Crystal formation

In order to form protein crystals of only the entire length LsrK obtained in the above-mentioned production example or LsrK and its substrate, 4,5-dihydroxypentan-2,3-dione (DPD) or AMP- Crystallization conditions were searched based on the vapor diffusion method, which is a known method (for example, Jancarik, J. et al . , J. Appl . Cryst. , 24 , 409-411, 1991). The retrieval method is briefly described as follows.

The purified LsrK protein was dispersed in an LsrK storage solution (basic composition) composed of 10 mM Tris-HCl, 50 mM NH ₄ Cl, 150 mM NaCl and 5 mM? -mercaptoethanol at pH 7.5 and the concentration was adjusted to 9.2 mg / mL To 16 mg / mL to prepare the final protein solution of LsrK to be used for the crystallization condition search. In some cases, HPr, DPD and / or AMP-PNP were added to this base composition and the concentration of this final protein solution was calculated by measuring the absorbance at 280 nm using a spectrophotometer.

The final protein solution and initial screen solution (Hampton Research Inc, Emerald Biosystems Inc) were used to search for the determination conditions step by step. Specifically, a mixed solution of 1 μL of the final protein solution of LsrK and a crystallization buffer solution to be searched for crystal formation ability was prepared by dropping on a silicon-coated slide surface, and the slide was placed on a plate containing 1.0 mL of crystallization buffer And stored at a constant temperature of 20 ° C. When crystals were formed, pictures were taken with an Olympus camera. Successfully, about three days later, crystals of LsrK were obtained. When the crystals were left in the crystallization buffer solution for a long time, the crystals were corroded. Therefore, the crystals aged in the crystallization buffer solution for 3 to 4 days were analyzed by X-ray diffraction . The crystallization conditions (a) to (e) including the crystallization buffer solution capable of obtaining the diffraction signal of the total length LsrK thus obtained are summarized below.

(a) LsrK storage solution: LsrK 16 mg / mL. Basic composition

Crystallization buffer: 0.1 M sodium citrate and 20 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HCl) at pH 5.5 to 20 to 30% (w / v) PEG-300 0.01 to 0.02 M;

(b) LsrK storage solution: LsrK 10 mg / mL. The basic composition further contains 3 times wild type HPr and 2 mM DPD of LsrK molar concentration.

Crystallization buffer: ammonium dihydrogen phosphate 0.1-0.4 M;

(c) LsrK storage solution: LsrK 11 mg / mL. The basic composition further contains 3 times wild type HPr and 2 mM DPD of LsrK molar concentration.

Crystallization Buffer: Magnesium sulfate hydrate 0.85 to 1.5 M and sodium citrate 0.1 M at pH 5.5;

(d) LsrK storage solution: LsrK 10 mg / mL. Basic composition

Crystallization buffer: an additive selected from 0.1 to 0.4 M ammonium dihydrogen phosphate 0.1 M guanidine hydrochloride and 0.05-0.1 M lithium chloride; And

(e) LsrK storage solution: LsrK 9.2 mg / mL. Contains more than 1 mM DPD or AMP-PNP in the base composition.

Crystallization Buffer: 0.1 M Bis-Tris with pH 1.0 to 1.2 M ammonium dihydrogen phosphate and 0.1 M lithium chloride;

2A shows a photograph of the co-crystallization of the entire length LsrK-HPr-DPD derived from E. coli crystallized under the condition of (a) above. The single crystals were hexagonal or hexagonal horns with the same point on both sides, belonging to the P3 hexagonal space group, and the accuracy of the X-ray diffraction signal was 3.2 Å.

4B shows a crystal photograph of the co-crystallized whole length LsrK-HPr-DPD derived from E. coli crystallized under the condition of (b) above. This single crystal had the same hexagonal horn or semi-hexagonal horn shape on both sides, which was the same as that of the P3 hexagonal crystal, but belonged to the P21212 hexagonal space group and the X-ray diffraction signal had an accuracy of 3.6 Å.

High-speed nitrogen cooling

(Garman EF et al . , J. Appl . Cryst . , 30 , 211-237, 1997) described in the literature was applied to solve the problem of being induced when the LsrK crystal was directly exposed to X- Lt; / RTI > As a result of searching the optimal condition of the high-speed nitrogen cooling method, it was found that the high-speed cooling solution having the composition obtained by adding ethylene glycol to the composition of the crystallization buffer solution described above did not adversely affect the LsrK crystal, Respectively. In addition, there was no ice ring (phenomenon in which water freezes when high-speed nitrogen cooling is not completed) frequently occurred during the experiment.

The compositions (a ') to (e') of the solution for rapid cooling corresponding to the LsrK crystallization conditions (a) to (e)

(a ') 0.1 M of sodium citrate at pH 5.5 to 20 to 30% (w / v) of PEG-300, 0.01 to 0.02 M of tris (2-carboxyethyl) phosphine hydrochloride (TCEP.HCl) (v / v).

(b ') Ammonium dihydrogen phosphate 0.1-0.4 M, ammonium dihydrogen phosphate 0.3 M, sodium chloride 0.2 M, glycerol 3-30% (v / v) gradient.

(c ') 0.1 M magnesium sulfate hydrate, 0.1 M sodium citrate, pH 5.5, 0.01 M tris (HCl) chloride, 0.2 M sodium chloride, and 30% glycerol (v / v).

(d ') 30% (v / v) glycerol additive selected from 0.1 M guanidine hydrochloride 0.1 M and 0.1 M lithium chloride to 0.1-0.4 M ammonium dihydrogen phosphate.

(e ') Bis-Tris 0.1 M, ammonium chloride 0.1 M, glycerol 32.5% (v / v) 1.0-2.2 M ammonium dihydrogen phosphate, pH 6.5.

Experiments were conducted to collect data from the Pohang Accelerator Laboratory and the Japan Photon Factory for LsrK crystals cooled at high speed in Example 2.

The data collected by X-ray diffraction of the LsrK single crystal according to the conditions (a) and (b) of Example 1 and the structure refinement results are summarized in Table 1, respectively.

The crystals obtained according to the conditions (c) to (e) of Example 1 had a low resolving power of the diffraction signal of about 3.5 to 4.0 Å and could not obtain a more detailed three-dimensional structure as in (a) or (b) .

In Table 1, R _sym = Σ | I _hkl - <I _hkl > | / Σ <I _hkl >, I _hkl is the intensity of diffraction at the hkl reflection plane, and <I _hkl > is the weighted average of hkl diffraction.

From the data in Table 1, it can be confirmed that the crystallization method of the present invention can obtain crystals of LsrK, which can provide a three-dimensional structure with excellent resolution, in a co-crystal form with HPr.

While the present invention has been particularly shown and described with reference to certain exemplary embodiments thereof, it should be understood that the same is by way of illustration and example only and is not to be taken by way of limitation. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. .

Accordingly, all equivalents of the claims and their equivalents, as well as the embodiments described hereinabove and the appended claims, are also within the scope of the inventive concept.

Claims (7)

A hexagonal space group P3, an asymmetric unit containing an LsrK protein and an HPr protein of SEQ ID NO: 1 and having a lattice unit of a = 100 Å, b = 100 Å, c = 353 Å, &Lt; / RTI &gt; and &lt; RTI ID = 0.0 &gt; y = 120. A hexagonal space group P21212, an asymmetric unit containing an LsrK protein and an HPr protein of SEQ ID NO: 1 and having a lattice unit of a = 100 Å, b = 173 Å, c = 352 Å, = 90 [deg.]. delete 3. The crystal of LsrK according to claim 1 or 2, characterized in that the crystal further contains AMP-PNP or 4,5-dihydroxypentane-2,3-dione in the asymmetric unit. As a crystallization method of LsrK,
Preparing an LsrK stock solution dispersed at a concentration of 5 to 20 mg / mL;
Mixing the LsrK storage solution and the crystallization buffer solution to obtain a mixed droplet; And
Placing the mixed droplet in an enclosed space containing the vessel such that vapor diffusion equilibrium is established between the mixed droplet and the vessel containing the crystallization buffer solution,
Wherein the crystallization buffer solution is an aqueous solution of LsrK having a composition containing any of the following materials (a) to (c):
(A) 0.05 to 0.2 M citrate of pH 4.5 to 5.5,
(B) ammonium dihydrogen phosphate 0.1 to 1.0 M,
(C) Tris (2-carboxyethyl) phosphine hydrochloride (TCEP · HCl, tris (2-carboxyethyl) phosphine hydrochloride) as an additive in an aqueous solution of the composition exemplified in (a) or (b) Aqueous solution,
However, the polyethylene glycol is 20-30% (w / v) PEG-300. 6. The method according to claim 5, wherein the LsrK stock solution comprises a pH of 7 to 8, an ammonium ion at a concentration of 50 mM or more, an HPr protein having 1 to 3 times the molar concentration of LsrK and a selective component, , 5-dihydroxypentane-2,3-dione. 6. The method according to claim 5, wherein the crystallization method of LsrK further comprises an expression step,
The expression process may include an osmotic shock phase in which 250 to 500 mM of sodium chloride is added to a culture medium of a bacterium encoding LsrK;
Subjecting the bacteria encoding the LsrK to heat shock at a high temperature higher than the temperature of the culture medium, and then rapidly lowering the temperature of the medium;
Transferring bacteria that encode the LsrK under the conditions of 0 占 폚 to 4 占 폚 and quenching;
And inducing LsrK gene transcription at 15 ° C to 25 ° C in the bacterium encoding LsrK to increase the amount of protein expressed in a soluble form.

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