KR20060003494A - High-throughput assay of lethal factor using immobilized full-length substrate with a defined orientation based on immuno-fluorescence method - Google Patents

Abstract

The present invention relates to a high-throughput activity assay method of anthrax lethality using immunofluorescence, and more particularly, the action at the cleavage site and the C-terminal site involved in the specific reaction between anthrax lethality and the substrate. To produce the fusion protein of GST (glutathione-S-transferase) -native form substrate using subcloning to cover the entire mechanism and to easily observe the cleavage site, GST of the fusion protein Glutathione, which binds specifically to the moiety, is used as a capture ligand to directionally fix the fusion protein to a solid support, resulting in high sensitivity and specificity when assaying anthrax lethal activity The small amount of enzyme allows not only the activity assay for a large number of samples in a short time, but also the reaction solution and cleavage after the reaction. Of, anthrax lethal factor using an immunofluorescence improved to be easily removed with water it is possible to reduce the experimental error caused by chemicals used for screening high-throughput relates to activity assay method.

Anthrax lethality, fusion protein of GST-structure substrate, immunofluorescence, high-throughput

Description

High-throughput assay of lethal factor using immobilized full-length substrate with a defined orientation based on immuno-fluorescence method             

1 is a glutathione (GSH) as a capture ligand in accordance with the present invention after the reaction by putting the lethal element in the microplate to fix the GST-substrate fusion protein in a specific direction, to remove the cleavage product and the lethal element And an assay for activity of anthrax lethality, by measuring an amount of an uncleaved substrate using an antibody that specifically binds to a cleavage site,

Figure 2 shows the change of substrate by lethal element (E), ie the presence of cleavage of very short amino terminus by preparing a fusion protein of GST (glutathione-S-transferase) -native form (S). Showing the activity assay proteolytic reaction of the present invention,

Figure 3 shows the appearance of the substrate cleaved by the lethal element of (1) and (2) and (3) when the GST-substrate fusion protein is fixed to the microplate in a certain direction according to the present invention ,

Figure 4 shows the changes in optical density and microplate surface according to the treatment of glutathione on the microplates, blocking solution treatment, fixation of GST-MEK fusion protein and treatment of antibodies,

Figure 5 shows the appearance of the graph and the plate surface measured the optical density while changing the concentration of GST-MEK fusion protein according to the present invention,

6 is a graph illustrating reactivity over time by putting 20 nM of lethal element in a microplate to which a GST-MEK fusion protein is bound,

Figure 7 considers that the lethal element is a metal dependent proteolytic enzyme by varying the metal ions in various, comparing the reactivity according to the present invention and the SDS-PAGE method.

The present invention relates to a high-throughput activity assay method of anthrax lethality using immunofluorescence, and more particularly, the action at the cleavage site and the C-terminal site involved in the specific reaction between anthrax lethality and the substrate. To produce the fusion protein of GST (glutathione-S-transferase) -native form substrate using subcloning to cover the entire mechanism and to easily observe the cleavage site, GST of the fusion protein Glutathione, which binds specifically to the moiety, is used as a capture ligand to directionally fix the fusion protein to a solid support, thereby providing high sensitivity and specificity when assaying the activity of anthrax lethality. This allows you to perform activity assays on multiple samples in a short time with a small amount of enzyme, as well as reaction solution and cleavage The present invention relates to a high-throughput activity assay method for anthrax lethality using immunofluorescence, which can be easily removed to reduce experimental errors caused by chemicals used for screening.

Anthrax is an inherited common infectious disease that is primarily infected by livestock but also by humans by the Gram-positive bacillus Bacillus anthracis. In Korea, this is classified as a statutory epidemic, and it has been confirmed worldwide in Africa, the Middle East, China, Russia, the United States, and Europe. Anthrax generated in livestock can be transmitted to humans by eating meat from lung meat or through skin, and if infected, it can be life threatening. Therefore, the need for control of anthrax has emerged.

It is known that the pathogenicity of anthrax is determined by toxin production ability and capsular formation. Proteins affecting the ability of anthrax toxin to produce are protective antigen, edema factor, and lethal factor (LF), each of which is not toxic, Combined to form edema toxins, defense antigens and lethal elements combine to form lethal toxins (lethal toxin).

Therefore, in order to control anthrax, a series of biochemical pathways related to proteins affecting the toxin-producing ability of the anthracnose are studied, and genes or proteins related to resistance and sensitivity according to host cell reactions are identified. The process of analyzing the characteristics is essential.

Conventionally, high-throughput assays using Fluorescence resonance energy transfer (FRET) have been used for activity assays for lethal elements [ Proc. Natl. Acad. Sci. USA, M. Mock and BR Roques (2002) 99: 6527-6529; Proc. Natl. Acad. Sci. USA , RT Cummings et al. (2002) 99: 6603-6606. More specifically, this method uses 14 to 16 amino acids using the amino acid sequence of the cleavage site of MEK2 belonging to the group of Mitogen-activated protein kinase kinase (MAPKK), known as the substrate of the lethal element. It is a method of forming an active peptide using a fluorescent resonance energy transfer (FRET) according to whether the peptide is cleaved by forming different synthetic peptides and connecting different phosphors at both ends of the peptides. .

This activity assay has the advantage of being able to process many samples in a short time with high sensitivity by using phosphors, but has the following problems.

First, this method is based on the premise that enzyme-substrate binding and catalysis occur only between the synthetic peptide and the lethal element of the cleavage site of the substrate, but the specific binding of the carboxy terminal and the enzyme of the substrate away from the cleavage site And the carboxy terminus plays an important role in the lethal factor cleavage reaction [ Anthrax lethal factor proteolysis and inactivation of MAPK kinase 2003, J. Biol. Chem. 278: 9402-9406. Therefore, when developing inhibitors for lethal elements, full-length native substrates of lethal element substrates are used rather than using synthetic peptides with only the protein corresponding to the cleavage region to ensure a variety of inhibitor collections. Inhibitor screening is preferred.

Secondly, this method is a method of assaying by changing the energy amount of fluorescent substance labeled on the synthetic peptide, and there is no method to separate the reaction solution and the cleavage product separately. Since the fluorescence was measured in this state, an error could occur, and the noise signal must be tested.

Recently, the second problem was solved, using an improved streptavidin-coupled fluorescence peptide to separate the synthetic peptide after the reaction [ An enzymatic electrochemiluminescence assay for the lethal factor of anthrax , 2003. Anal. Biochem. 321: 125-130] and chemical screening by mass spectrometry to identify inhibitos of anthrax lethal factor , 2004, for binding non-fluorescent labeled peptides to gold plated plates and assaying their cleavage sites. Nature Biotechnol. May 16. In press]. However, both methods still had a first problem as a method of using synthetic peptides based on substrate cleavage sites, and thus should cover the mechanism of action of various proteins other than substrate cleavage proteins in inhibitor screening. There was no limit.

Accordingly, the present inventors have made efforts to solve the above-mentioned conventional problems. As a result, the separate substrate portion other than the cleavage site is an important factor for determining the cleavage efficiency due to the specificity between the lethal element and the substrate and the lethal element. Based on the fact, the fusion of the overexpressed GST-wide structure substrate by subcloning the full-length cDNA of the lethal element substrate instead of the 14-16 synthetic peptides prepared using only the amino acid sequence of the cleavage site as the substrate. The use of the protein allows screening of anthrax inhibitors of various mechanisms of action, and the binding of the fusion protein to the solid support in a specific direction using glutathione (GSH), which specifically binds to the GST portion of the fusion protein, results in cleavage of the substrate. Not only is it easy to determine the chemistry of reaction solutions, cleavage products, enzymes, etc. It is aware of the fact that reducing the error due to the high quality of the present invention have completed the throughput test methods.

Accordingly, the present invention provides a fusion protein of GST (glutathione-S-transferase) -total structure substrate by subcloning the full-length cDNA of anthrax lethal substrate and fixing it in a specific direction to the solid support. It is an object of the present invention to provide a method for assaying activity of anthrax lethal element, characterized in that

The present invention provides a method for preparing a fusion protein of a GST-wide structure substrate by subcloning the full-length cDNA of anthrax urea substrate, and coating a capture ligand that specifically binds the GST to a solid support. Characterized in that the activity assay method of anthrax lethal element comprising a.

The present invention will be described in more detail as follows.

The present invention encompasses the mechanism of action at the C-terminal site and the cleavage site of the substrate involved in the specific reaction between the anthrax lethal element and the substrate, and uses GST (glutachchi) using subcloning to easily observe the cleavage site. The fusion protein is prepared by preparing a fusion protein of on-S-transferase-native form substrate and using glutathione (GSH), which specifically binds to the GST portion of the fusion protein, as a capture ligand. By directional fixation to the support, it has high sensitivity and specificity when assaying the activity of anthrax lethality, and can perform activity assays for a large number of samples in a short time with a small amount of enzyme. Reaction solution and cleavage products can be easily removed after the reaction to reduce the experimental error caused by the chemicals used in the screening. The high of the anthrax lethal factor using an immunofluorescence relates to the traffic activity assay method.

The reaction of the lethal element with the fusion protein follows the simple enzyme kinetics of Scheme 1 below, called the Michels-Manton Kinetics.

Simple enzyme kinetics is the theory that under a constant enzyme concentration, the enzyme reaction rate is proportional to the substrate concentration when the substrate concentration is low, and the reaction rate does not increase above a certain concentration even when the substrate concentration is high. The chemical reaction takes place in the enzyme-substrate complex (ES ^* ) to which (S) is bound, resulting in a reaction product (P). Thus, according to simple enzyme kinetics, the development of a substance that inhibits the activity of lethal elements involves two steps: the binding step between the enzyme and the substrate (E + S) and the catalysis step of the activated enzyme-substrate complex (ES ^* ). Can be adjusted.

However, in the past, since the peptide prepared using the cleavage site of the substrate protein molecule was used as a substrate, only the catalytic step of the enzyme-substrate complex could be observed, and therefore, there were limitations in screening anthrax inhibitors of various mechanisms of action. In the present invention, the entire structural protein (native form) is used as a substrate to observe and reflect both of the above steps through which lethal elements react with the substrate.

The solid support to which the GST-substrate fusion protein used in the present invention is immobilized is prepared as follows.

The GST-substrate fusion protein of the present invention is the full-length of proteins (eg MEK1, MEK2, etc.) belonging to the group of Mitogen-Activiated Protein Kinase Kinases known as substrates of lethal elements. cDNA is prepared by subcloning.

Detailed description of the preparation and purification of the GST-substrate fusion protein is as follows.

In order to prepare a fusion protein of the entire structure, an amplification system such as polymerase chain reaction (PCR) is used for MEK cDNA. To this end, rat liver is isolated, total RNA is extracted, and cDNA is obtained through reverse transcription using a random hexamer. Based on the obtained cDNA, the MEK-specific oligonucleotides (primers) are used to amplify and purify the whole MEK gene. The obtained MEK gene is restriction enzyme treated and inserted into a bacterial overexpression vector. The expression vector is expressed in a form in which about 26 kDa GST is fused to the amino terminus (N-terminus) of the protein to be expressed. Linker genes may be inserted to position five glycines between the GST and MEK genes to reduce folding and functional interference of the target protein that may result from fusion protein expression. The substrate overexpression vector (pGEX-MEK) thus secured is inserted into an expression host, cultured in a medium, and collected by centrifugation. It is suspended using a buffer and the cells are crushed to separate GST-MEK fusion proteins.

GST, which is fused with a protein belonging to the MAPKK group, is a glutathione-S-transferase, which is relatively large in size and shifted to the amino terminus (Pro8-Ile9), making it difficult to determine whether it is cleaved. The problem can be solved and it is advantageous to observe the enzyme cleavage site of the fusion protein.

GST fused to the amino terminus of the fusion protein serves to fix the GST-substrate fusion protein to a solid support (microplate, etc.) in a fixed direction. The solid support may be the same as that used for the ELISA, and is generally made of polystyrene and preferably having a plurality of wells. The surface of the solid support should be able to be easily bound through hydrophobic interaction with the protein.

A well of the solid support is coated with a capture ligand capable of specifically binding to the GST portion of the fusion protein of the GST-structure substrate to immobilize the fusion protein. As the capture ligand, any substance capable of specifically binding to GST can be used, but glutathione is preferable. In addition, a blocking solution can be used if necessary to suppress nonspecific binding, and a representative solution of blocking solution is 1% BSA. Reacting the coated solid support with the previously prepared GST-substrate fusion protein fixes the GST moiety toward the bottom of the plate and the substrate moiety on the opposite side so that the substrate and the lethal element can easily react (FIG. 3).

This defined orientation fixation facilitates the observation of cuts of the lethal element and can also improve the sensitivity of the assay method. That is, when the fusion protein of the GST-structure substrate is fixed by the capture ligand as shown in (1) of FIG. 3, when the GST portion is directed toward the bottom of the plate and the substrate portion is oriented on the opposite side, the reaction is completed. The C-terminal part is removed by removing the solution from the well, but if it is not immobilized with the capture ligand, it can be fixed to the plate such as (2) or (3), making it difficult to remove the cleavage product after the cleavage reaction occurs. The sensitivity to the reaction is greatly reduced. In the case of (2), the C-terminus, which plays an important role in binding, is fixed to the well plate, which may affect the reaction with lethal elements. In case of (3), it is difficult to observe the change of the substrate after the reaction. it's difficult.

The coating of capture ligands on the well surface, the immobilization of the fusion proteins of the GST-structure substrate, and the substrate specificity of the signals obtained from the assay system are not the primary antibodies that specifically recognize the C-terminal region of the substrate. Each non-negative control (negative control) is put, and confirmed through the color reaction of the secondary antibody (secondary antibody conjugated to HRP) to the TMB solution (Fig. 4).

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to Examples.

Example: Activity test of anthrax lethal element using immunofluorescence

First, a fusion protein was prepared and purified through the following procedure.

Rat liver was isolated, total RNA was extracted, and cDNA was obtained by reverse transcription using a random hexamer. Based on the obtained cDNA, MEK1 specific oligonucleotides (primers) were used to amplify and purify the whole MEK1 gene and then purified. The obtained MEK1 gene was treated with restriction enzyme (EcoRI) and inserted into a bacterial overexpression vector (pGEX-2T, Amersham Pharmacia) to express the GST of about 26 kDa at the amino terminus (N-terminus) of the protein. In this example, five glycines were inserted into the linker gene between the GST and MEK1 genes to reduce folding and functional interference of the fusion protein.

The pGEX-MEK thus obtained was inserted into BL21 (E. coli) and then cultured in LBA (LB medium containing 50 mg / ml ampicillin) until OD ₆₀₀ = 0.7 to 0.8. IPTG (final 1 mM) was added thereto and incubated at 30 ° C. for 5 hours. The cultured cells were collected by centrifugation followed by PBST buffer (150 mM NaCl, 16 mM Na ₂ HPO ₄ , 4 mM NaH ₂ PO ₄ , pH 7.4, 2 mM EDTA, 1% Triton X-100, 0.1% BME ) And suspended cells via sonication. Only cell extracts were obtained through centrifugation, and the GST-MEK fusion proteins were separated through a glutathione-sepharose column. The multiwell plate was coated with glutathione that specifically binds to the GST moiety, and the GST-substrate fusion protein 280 nM was fixed in a specific direction according to the present invention and then reacted with 20 nM of lethal element.

This result is shown in FIG. 6, and it can be observed that the reaction is almost terminated after about 30 minutes. This was observed by the researchers through SDS-PAGE [ Implication of pH I the catalytic properties of anthrax lethal factor Biochem. Biophys. Res. Commun. 2004, 313: 217-222, it can be seen that the desired result can be obtained by using a small amount of lethality of 20 nM. Therefore, this assay method can satisfy both the rapid response time and the low cost of enzyme, which are essential for high-throughput assays.

Experimental Example 1 Confirmation Experiment of Assay System

The microplates confirmed the ability of substrate specific detection of the treatment of blocking ligands, blocking, immobilization of GST-substrate fusion proteins, and assay methods (FIG. 4). This is a process to check the specificity of the high-throughput analysis method and to verify that each step has been completed during system construction. Plates are made of polystyrene and can be bound to proteins via hydrophobic interaction, so if no treatment is applied to the surface of the plate, all of them will bind to the plate and display a high signal when the antibody, antibody, is inserted. . Treatment of the capture ligand (GSH) and blocking solution (1% BSA) on the surface of the plate gradually reduces the untreated plate surface, and the addition of substrate-specific antibodies reduces the signal. This can be confirmed as a signal that decreases as each process proceeds to the surface of the plate, as shown in Figure 4, after the substrate (GST-MEK) is put on the plate treated with the capture ligand (GSH) and blocking solution When detected using a substrate-specific antibody, the signal can be seen to increase rapidly, but when detected using a substrate-specific antibody, it can be observed that there is almost no increase in signal.

Experimental Example 2 Assay Range According to Substrate Concentration

In order to bind the GST-substrate fusion protein to a specific orientation, it was examined whether the various concentrations of substrates were placed in one plate and measured by the assay method according to the present invention. As can be seen from the experimental results shown in FIG. 5, an increase in the signal can be observed in a linear form in the range of about 2 nM to 200 nM, and it can be seen that it has a considerably wide measurement range. This is a measure of how precisely and accurately the amount of substrate remaining after the reaction can be assayed.

Experimental Example 3 Comparison of the Invention and SDS-PAGE

Conventionally, in order to examine the reaction between the anthrax lethal element and the substrate, the amount of change in the substrate was measured by using an electrophoresis (SDS-polyacrylamide gel electrophoresis), which separates the protein by the size of the enzyme through the gel. However, since the substrates of the lethal elements were cleaved at the amino terminus (Pro9-Ile9), it was difficult to judge the protein size after the cleavage reaction, and until now, the western blot had to determine the reaction. However, in the case of the present invention, 26-28 kDa sized GST is bound to the amino terminal region of the anthrax substrate, resulting in a marked difference in protein size after cleavage reaction. GST-MEK was 72 kDa and MEK after cleavage was 44 kDa [ Production and proteolytic assay of lethal factor from Bacillus anthracis , Protein Exp. Purif. 2003, 30: 293-300.

The SDS-PAGE photograph of FIG. 7 is a photograph subjected to electrophoresis after reacting under the same conditions as the present invention, and refers to a substrate in which a cleavage reaction does not occur in a portion indicated by GST-MEK. Intensity of the protein band in the SDS-PAGE photograph means the amount of the substrate, which means that the more cleavage reaction occurs as the band intensity decreases. Considering that the lethal element is a metal dependent proteolytic enzyme, the experiments on the metal ions required for the lethal element by combining metal ions of various conditions were carried out to include various metal ions such as Zn ²⁺ . The activity of urea was observed to be in perfect agreement with the results obtained through SDS-PAGE. It was also possible to quantitate the difference in their activities.

As described above, the method of activity assay of anthrax urea using the immunofluorescence method of the present invention is performed by subcloning the full-length cDNA of the anthrax urea substrate to obtain GST (glutathione-S-transferase) -total structure. It has a high sensitivity and specificity when assaying the activity of anthrax lethality, including preparing a fusion protein of a substrate and coating a glutathione that specifically binds GST to a solid support. As a result, it is possible to perform the activity assay on a large number of samples in a short time with a small amount of enzyme, and to easily remove the reaction solution and the cleavage product after the reaction, thereby reducing the experimental error caused by the chemical used in the screening. . In addition, many potential chemical inhibitors for the development of various inhibitors of anthrax urea are expected to be searched in a short time, which may be widely applied in the pharmaceutical and pesticide fields.

Claims (2)

Subcloning the full-length cDNA of anthrax toxin substrate to produce a fusion protein of GST (glutathione-S-transferase) -total structure substrate, and specifically to the GST on a solid support A method for assaying activity of anthrax lethality, comprising coating a binding glutathione. The method of claim 1, wherein said anthrax lethal substrate is MEK1.

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