KR100241264B1 - Antagonist detecting method of binding to sh2 - Google Patents

Abstract

The present invention relates to a method of searching for an antagonist acting on an SH2 site by using a protein comprising an SH2 site that binds to alkaline phosphatase (AP). Specifically, the present invention is a fusion protein (hereinafter, abbreviated as "Ap-SrcSH2 fusion protein") that combines a protein containing the SH2 site involved in cell signaling process with alkaline phosphatase, which can be produced in large quantities in E. coli The present invention relates to a method for producing an expression vector, an AP-SH2 fusion protein using the same, and a method for searching for an antagonist acting on an SH2 site using alkaline phosphatase, and the method for searching according to the present invention is simple to operate without using radioactivity. Modulators and the like acting on cell signaling can be effectively selected.

Description

Search for an antagonist that binds to a protein comprising a SH2 site involved in cell signaling.

The present invention relates to a method of searching for an antagonist acting on an SH2 site by using a protein comprising an SH2 site that binds to alkaline phosphatase (AP).

More specifically, the present invention is a fusion protein (Ap-SrcSH2 fusion protein) that combines a protein containing an SH2 site involved in the cell signaling process with alkaline phosphatase, an expression vector capable of producing a large amount in E. coli, The present invention relates to a method for producing an AP-SH2 fusion protein and a method for searching for an antagonist that acts on an SH2 site using alkaline lane phosphatase, and the method for searching according to the present invention is easy to operate without using radioactivity and thus cell signal transduction. Regulators that act on the process can be effectively selected.

Cancer is caused by genetic damage of normal cells by carcinogens such as viruses, chemicals, or radiation, resulting in abnormal cell signaling processes involved in cell growth and proliferation. In this intracellular signaling process, various protein kinases act. These kinases are activated to phosphorylate their substrates, thereby inducing various physiological responses. Specifically, in order for a cell to function normally, various organs must interact with each other. In this process, extracellular signaling agents such as growth factors or cytokines are applied to receptors present in the cell membrane through information of their amino acid primary sequence. It transmits extracellular information to induce a cellular response through which various proteins are produced intracellularly.

These intracellular actions are transmitted externally by intracellular signaling by receptor tyrosine kinase, binding receptors for G protein, cytokine receptors, ion channels, serine-threonine kinase receptors and chemokine receptors. Triggered. In particular, these receptors, when activated primarily as receptor tyrosine kinase (RTK), transfer external signals into cells by phosphorylating tyrosine residues of various substrates in the cell, including itself (Cohen, Nature, 276: 409-410, 1978). ). For example, the src product, a cancer gene of Ras' sarcoma virus, has been reported to be tyrosine kinase, and it has been confirmed that the cancer gene product that continuously proliferates cells is tyrosine kinase and cell growth is regulated by activation of these enzymes. It has also been reported that when some normal growth factor receptors bind to signals from outside, the site inside the cell membrane of the receptor protein acts as a protein tyrosine kinase. In addition, when the receptors are overexpressed or their gain of function mutations are induced, diseases such as insulin-resistant diabetes and pibaldism may occur, and this phosphorylation process may be caused by protein tyrosine phosphatase (PTK). Is controlled by

As a result, specific genes are expressed in the cell nucleus through the intracellular signal transduction process (Schlessinger, J., Neuron, 9: 383-391, 1992), and various proteins produced in the process are used for cell growth and metabolism. And control the differentiation process directly or indirectly. For example, in fibroblast cells, platelet-derived growth factor (PDGF) regulates cell proliferation and motility, NGF (nerve growth factor) causes neuronal differentiation, and insulin (Insulin) is a target. Activates metabolic mechanisms of cells

Protein tyrosine kinase (PTK), which acts on cellular signaling, can be classified into receptor tyrosine kinase (RTK) and nonreceptor tyrosine kinase (RTTK). epidermal growth factor receptor (PDGFR), platelet-derived growth factor receptor (PDGFR), nerve growth factor receptor (NGFR), fibroblast growth factor receptor (FGFR), EPH, AXL, TIE, hepatocyte growth factor receptor (HGFR), and InsulinR. Non-receptor tyrosine kinase enzymes include Src, Abl, Fes / Fer, Syk, Jak, Tec, Fak, Ack, Csk and the like. The protein tyrosine kinase (PTK) has been reported to be closely related to various diseases including cancer. Tyrosine phosphorylated receptors or substrates bind to target proteins present in the cell to phosphorylate tyrosine residues or structurally bind proteins. Conformational change or alteration of cellular location of the protein. At this time, the phosphorylated proteins specifically bind and interact with the signaling protein, which is composed of an amino acid residue immediately adjacent to the phosphorylated tyrosine residue and an SH2 region (Src homology 2 domain) consisting of about 100 amino acids of the signaling protein. It can be explained by the reaction of the liver.

These SH2 sites exhibit unique specificities that are structurally or functionally different for each signaling protein (Koch, C. A., Science, 252, 668-674, 1992). Specifically, the SH2 site recognizes a short amino acid sequence including phosphorylated tyrosine residues. The types and number of SH2 sites included in each signaling protein are different. It plays an important role in the proliferation and differentiation of cells because it acts as a molecular switch that recognizes specific peptides in target proteins and induces interactions between signaling proteins. Thus, signaling proteins containing phosphorylated tyrosine and SH2 sites that act early in the signaling system in the treatment of cancer, where genetic information related to cell growth and proliferation is impaired, resulting in abnormalities in signaling pathways related to cell growth. Many studies have been attempted to develop inhibitors that inhibit the binding of livers.

As shown above, the specificity of the binding process of the specific protein phosphorylated to the SH2 site depends on the amino acid sequence of several (about 4-5) amino acids around the phosphorylated tyrosine and the amino acid sequence of the SH2 site. These bonds have very high affinity at least a few nM (Songyang, Cell, 1993, 72: 767-778). For example, the SH2 region of the Src protein is a peptide consisting of four amino acids that are phosphorylated. It has affinity of about 4nM with pYEEI monomer, SH2 site of p85 protein has affinity of about 0.3-3nM with peptide having amino acid sequence such as pY (M / V) (D / E / P) M, and dissociation The dissociation rate is also very high (Felder, S. et al., Mol. Cell. Biol., 1993, 13: 1449). This suggests that short linear peptides consisting of about five amino acids can specifically regulate the activity of signaling proteins and further develop compound drugs that act on this process. Specifically, positive results have already been realized and reported (Beattie, J., Cell Signal, 1996, 8: 75-86; Masamitsu, TM, Mol. Cell. Biol., 1995, 15: 6829-6837 ).

As shown above, the SH2 site plays an important role in regulating the interaction of proteins involved in early signaling in cell signaling, so developing an effective antagonist that acts on the SH2 site leads to failure of early signaling. Or as a therapeutic agent for various diseases including cancer caused by abnormal problems.

However, in the case of proteins containing most SH2 sites including SH2 sites without enzymatic activity, their affinity, dissociation and specificity in the course of reaction with antagonists, peptides and agonists that can bind to them, etc. There are many difficulties in evaluating the back. Thus, in order to select an appropriate antagonist that binds to a protein comprising the SH2 site, an assay method is required to measure the activity of the antagonist and to evaluate the results effectively.

In order to effectively search for antagonists acting on the SH2 site, the present inventors have identified a fusion protein which combines alkaline phosphatase with a portion of the most well known oncogenic Src protein among tyrosine kinase as a protein including the SH2 site. Ap-SrcSH2 fusion protein) was prepared, and the present invention was completed by developing a method of analyzing an antagonist that binds to the SH2 region of the fusion protein by analyzing the activity of alkaline phosphatase.

An object of the present invention is to provide a screening method for selecting an antagonist binding to SH2 site by enzymatic reaction using a fusion protein in which part or all of the protein including the SH2 site is combined with alkaline phosphatase (AP). .

In this case, the fusion protein may include all kinds of proteins having the SH2 region, and may include only the SH2 region or the entire protein thereof.

Specifically, the present invention reacts the fusion protein with a compound immobilized on a solid support, and then selects an antagonist that binds to the SH2 site by measuring alkaline phosphatase activity in the fusion protein bound to the compound.

The present invention relates to a fusion protein (Ap-) which combines a portion of the Src protein, which is a tyrosine kinase (hereinafter, abbreviated as "Src SH2 site") or whole and alkaline phosphatase (AP), as a protein including an SH2 site. SrcSH2 fusion protein) and its genes.

In addition, an object of the present invention is to provide an expression vector capable of producing the Ap-SrcSH2 fusion protein in E. coli. Specifically, the present invention provides an expression vector pRGT7 Ap-SrcSH2 comprising a gene having a nucleotide sequence of SEQ ID NO: 5 obtained by combining an AP gene obtained from E. coli and an SH2 region gene of Src protein, and an amino acid sequence of SEQ ID NO: 6 inferred therefrom. To provide.

In addition, the present invention deposited Escherichia coli transformants obtained by transforming the expression vector pRGT7 Ap-SrcSH2 into Escherichia coli to the Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute, attached on February 3, 1997 (Accession No .: KCTC 0313BP). ).

In addition, an object of the present invention is to provide a method for producing the Ap-SrcSH2 fusion protein in the crude cell eluate after inducing the expression of the protein by culturing the E. coli transformant.

Figure 1 shows the gene sequence of the fusion protein (Ap-SrcSH2 protein) that combines the SH2 site and alkaline phosphatase of the Src protein of the present invention and the amino acid sequence inferred therefrom. At this time, the base number 1420 to 1820 corresponds to the SH2 region gene.

Figure 2 shows the overall cloning process of the expression vector pRGT7 Ap-SrcSH2 of the present invention.

Figure 3 shows the results confirmed by SDS-polyacrylamide gel electrophoresis by expressing the Ap-SrcSH2 fusion protein of the present invention in E. coli.

Lane C: control; Lane M: standard molecular weight labeled protein

Lanes 1, 2 and 3: expressed Ap-SrcSH2 fusion proteins

Figure 4 shows the analysis method for the SH2 site of the present invention as a schematic (prototype, prototype).

Figure 5 shows the comparison of alkaline phosphatase activity in the cell lysate with the Ap-SrcSH2 fusion protein of the present invention and the cell lysate used as a control.

Lane 1: control group; Lane 2: Cell expressing Ap-SrcSH2 fusion protein

Figure 6 shows the results of measuring the interaction of Ap-SrcSH2 fusion protein and biotin-peptide using alkaline phosphatase.

Lane 1: in a cell to which the peptide is attached, a cell lysate of the control group is added;

Lane 2: in the cell lysate in which the Ap-SrcSH2 fusion protein was expressed;

Lane 3: attaching phosphotyrosine antibody followed by attaching alkaline phosphate bound secondary antibody

The present invention provides a screening method for screening an antagonist that binds to a SH2 site by an enzymatic reaction using a fusion protein that binds a protein comprising an SH2 site with an alkaline phosphatase (AP).

Specifically, the present invention provides a method for preparing a fusion protein including 1) an SH2 site and an AP enzyme active site in a multi-well, 2) reacting a compound immobilized on a solid support, and 3) binding the compound. The present invention provides a method for screening an antagonist that binds to an SH2 site by measuring alkaline phosphatase activity in a fusion protein, 4) recovering a compound that exhibits appropriate AP enzyme activity, and confirming its mass, structure, purity, and concentration. .

In this case, the fusion protein may include all kinds of proteins having the SH2 region, and may include only the SH2 region or the entire protein thereof.

The search method of the present invention can be performed by a model (protoytpe assay) as shown in Figure 4 using multi-well. Therefore, the method of the present invention can easily analyze the affinity, dissociation, and specificity of each compound as well as the interaction (binding or binding) with the SH2 site of each compound, so that many kinds of compounds such as HTS (High Throughput Screening) It can be useful to analyze them together.

The present invention, in order to develop a screening method for screening an antagonist or the like acting on the SH2 site, as part of the protein containing the SH2 or a portion of the entirety of the Src protein tyrosine kinase and alkaline phosphatase (alkaline phosphatase, AP) Bound fusion protein (Ap-SrcSH2 fusion protein) and genes thereof are provided (see FIG. 1).

Specifically, the present invention performs a polymerase chain reaction using the primers of SEQ ID NO: 1 and SEQ ID NO: 2 using a human placenta cDNA library as a template to obtain the SH2 region gene of the most well known Src protein among tyrosine kinase. In this case, the primer having the nucleotide sequence of SEQ ID NO: 1 has a restriction enzyme Xba I recognition site and the nucleotide sequence corresponding to the amino terminus of the Src SH2 site, and the primer having the nucleotide sequence of SEQ ID NO: 2 has a restriction enzyme Xho I recognition site From the 14th base has a complementary base sequence corresponding to the carboxy terminus of the Src SH2 site. In addition, the AP gene is obtained by performing a polymerase chain reaction (PCR) using the primers of SEQ ID NO: 3 and SEQ ID NO: 4 using the chromosomal gene of E. coli as a template. In this case, the primer having the nucleotide sequence of SEQ ID NO: 3 has a nucleotide sequence corresponding to the amino terminus of the secreted AP and the restriction enzyme Nde I recognition site, and the primer having the nucleotide sequence of SEQ ID NO: 4 has a restriction enzyme Xba I recognition site 12 From the first base has a complementary base sequence corresponding to the carboxy terminus of the AP.

The present invention also provides an expression vector capable of producing the Ap-SrcSH2 fusion protein in E. coli. Specifically, the present invention provides an expression vector pRGT7 Ap-SrcSH2 comprising a nucleotide sequence of SEQ ID NO: 5 obtained by combining an AP gene obtained from Escherichia coli and the SH2 region gene of Src protein, and an amino acid sequence of SEQ ID NO: 6 inferred therefrom. (See Figure 2).

In addition, the present invention provides a method for producing a large amount of Ap-SrcSH2 fusion protein in crude cell eluate after inducing the expression of the protein by culturing E. coli transformants obtained by transforming E. coli with the expression vector pRGT7 Ap-SrcSH2. (See FIG. 3).

In order to measure alkaline phosphatase activity present in the Ap-SrcSH2 fusion protein of the present invention, E. coli cells expressing the protein are suspended in AP buffer and p-nitrophenyl phosphate disodium salt (p-nitrophenyl phosphate) as a substrate solution. , disodium salt, pNPP) and the like (see FIG. 5).

In addition, in order to investigate the interaction between the SH2 site of the Ap-SrcSH2 fusion protein and the peptide binding thereto, the interaction of the Ap-SrcSH2 fusion protein with the biotin-peptide (B-S003) was measured using alkaline phosphatase. The biotin-peptides present in the wells are identified by reaction with a phosphotyrosine monoclonal antibody and a secondary antibody to which the AP is bound (see FIG. 6).

Hereinafter, the present invention will be described in detail by way of examples.

The following examples illustrate the invention and are not intended to limit the scope of the invention.

Example 1 Isolation and Cloning of SH2 Gene and Alkaline Phosphate Gene

Alkaline phosphatase (AP) genes were cloned by purifying the chromosomal genes of E. coli HB101 (ATCC 33694) cells and performing a polymerase chain reaction (PCR) using the template as a template. In addition, by performing a polymerase chain reaction using a human placenta cDNA library as a template, a SH2 region (Src SH2 region) of the src gene was obtained and finally a gene (Ap-SrcSH2 gene) was combined with the AP gene. . The specific process is as described below.

(Step 1) Design and synthesis of primers

To obtain the Src SH2 gene by polymerase chain reaction using a human placenta cDNA library (Clontech company, Cat # HL4025AH) as a template, two oligonucleotides were synthesized using a nucleic acid synthesizer (DNA Synthesizer, Applied Biosystems INC., USA). Used as primer. In this case, the primer having the nucleotide sequence of SEQ ID NO: 1 has a restriction enzyme Xba I recognition site and the nucleotide sequence corresponding to the amino terminus of the Src SH2 site, and the primer having the nucleotide sequence of SEQ ID NO: 2 has a restriction enzyme Xho I recognition site From the 14th base has a complementary base sequence corresponding to the carboxy terminus of the Src SH2 site.

In addition, two oligonucleotides were synthesized and used as primers to obtain an AP gene for binding to the amino terminus of the Src SH2 region by polymerase chain reaction using the entire gene of E. coli as a template. In this case, the primer having the nucleotide sequence of SEQ ID NO: 3 has a nucleotide sequence corresponding to the amino terminus of the secreted AP and the restriction enzyme Nde I recognition site, and the primer having the nucleotide sequence of SEQ ID NO: 4 has a restriction enzyme Xba I recognition site 12 From the first base has a complementary base sequence corresponding to the carboxy terminus of the AP.

(Step 2) Gene Amplification

Polymerase chain reaction was performed to 1) amplify the SrcSH2 gene from the cDNA library and 2) amplify the AP gene from all of E. coli. Specifically, each gene, that is, the human placenta cDNA library or the whole gene of E. coli, 2 μl of template, 10 μl of 10X concentrated reaction solution, 6 μl of 25 mM magnesium chloride, 2 μl of 10 mM dNTP (dATP, dGTP, dCTP, dTTP) 3 μl, 0.75 μg of primer 1 of the sequence 1, 3 μl of 0.75 ug of primer 2, and 0.4 μl of Taq polymerase (5 U / μl, Promega, USA) Reaction liquid I was made. In addition, 3 μl of a 0.75ug sequence 3 primer and 3μl of a 0.75ug sequence 4 primer were placed in a tube containing only the above-described primers, thereby preparing Reaction Solution II. 1 minute 30 seconds at 94 ℃ to the reaction solution; 1 minute at 50 ° C .; As a result of repeating the reaction of 1 minute 30 seconds at 72 ° C. for 30 times, the Src SH2 gene of 399 base pairs in the reaction solution I and the AP gene of 1416 base pairs in the reaction solution II were amplified. Next, DNA was extracted using phenol / chloroform (phenol / Chloroform) and 100% ethanol, and then dissolved in 20 μl of TE buffer and used in the next step.

(Step 3) Preparation of Expression Vector pRGT7 Ap-SrcSH2

Take 16 μl of each of the reaction solutions I and II, and add 1 μl of restriction enzymes Xba I and Xho I to the reaction solution I, and add 1 μl of the restriction enzymes Nde I and Xba I to the reaction solution II, and then add 10 × restriction enzyme. 2 µl of the reaction solution was added again, and a DNA fragment having a desired restriction enzyme recognition site was obtained by a conventional method of reacting at 37 ° C. for 1 hour. In addition, the plasmid vector pRGT7 (E. coli vector with T7 agonist, Hyun-ho Chung, Sciece, 259, 806, 1993) was also completely cleaved with restriction enzymes Nde I and Xho I and isolated on 1% agarose gel. It was.

In order to prepare the expression vector of the present invention by connecting two DNA fragments to the plasmid vector, the plasmid vector pRGT7, which was digested with 0.1 ug of SrcSH2 and AP gene fragments and 0.1 ug of restriction enzymes Nde I and Xho I, Add 2 μl of 10X ligation solution (500mM Tris-HCl, pH 7.8, 10mM Magnesium Chloride, 100mM DTT, 10mM ATP), 10U of T4 DNA Ligase, add distilled water to make 20µl of total volume, and then add 12 ° C at 16 ℃. The reaction was carried out for a time. The reaction solution was added to E. coli HB101 (ATCC # 33694) competent cells to transform E. coli, and then, E. coli transformants were selected by plating on LB-Epicillin (LB medium containing 50 ug / ml empicillin) plate. . Cloning of the expression vector pRGT7-Ap-SrcSH2 is shown in FIG.

Example 2 Expression of Ap-SrcSH2 Fusion Proteins

In order to express the Ap-SrcSH2 fusion protein of the present invention in Escherichia coli, the expression vector pRGT7 Ap-SrcSH2 was extracted in a large amount and added to E. coli BL21 competent cells to provide a single method (J. Mol. Biol., 116, 557). , 1983) and E. coli transformants were selected by plating on the LB plate. The transformed E. coli strains were inoculated in 3 ml of LB-empicillin medium and further incubated for 3 hours at 37 ° C., followed by further incubation for 3 hours by adding IPTG (isopropylthiogalactoside) at a concentration of 0.4 mM.

Centrifuge the cell culture solution at 12,000 g for 5 minutes to discard the culture supernatant, add 100 μl of TE buffer solution to the precipitate, shake well, and then 100 μl Ramley buffer solution (Laemli, et. Al., Nature, 227, 680, 1970) and mixed well to perform 12% SDS-polyacrylamide gel electrophoresis and staining the protein with Coomassie blue solution was confirmed that the expression of Ap-SrcSH2 fusion protein of about 66kDa size (see Fig. 3) ).

The transformed Escherichia coli BL21 / pRGT7 Ap-SrcSH2 was deposited on February 3, 1997, to the Gene Bank of Biotechnology Research Institute, affiliated with the Korea Institute of Science and Technology (Accession Number: KCTC 0313BP).

Example 3 Determination of Enzyme Activity of Ap-SrcSH2 Fusion Proteins

In order to measure the enzymatic activity of the Ap-SrcSH2 protein of the present invention, 1 L of E. coli BL21 strain transformed with the expression vector pRGT7 Ap-SrcSH2 was shaken at 37 ° C. When the absorbance at 600 nm reached a value between 0.3 and 0.6, add IPTG to a concentration of 1 mM, incubate for another 12 hours, and then use a JA-10 (Beckmann) rotor for 10 minutes at 5,000 rpm. Centrifuged. The culture supernatant was then discarded and the cell mass suspended in 50 ml AP buffer [1M diethanolamine, 0.5 mM magnesium chloride, pH 9.8].

The cell suspension was crushed at a pressure of 13,000 psi using French pressure and centrifuged at 10,000 rpm for 20 minutes using a JA-14 rotor (Beckmann), and then the supernatant thereof was taken out. 10 μl (10 μg) of the cell lysate thus obtained was put in 96-well plates, respectively, and then 6.2 ml AP buffer was added to the substrate solution [1 mM p-nitrophenyl phosphate, disodium salt, pNPP). Dissolved in solution] was further added thereto and reacted at 37 ° C. for 1 hour. After completion of the reaction, 25 μl of 1N sodium hydroxide was added, and the result was read by an enzyme-linked immunosorbent assay (ELISA) reader at 405 nm.

At this time, E. coli BL21 strain transformed with the expression vector pRGT7 TGF1 as a control was prepared in the same manner as above and used to analyze the enzyme activity. FIG. 5 is a graphical representation of measuring the activity of alkaline phosphatase in the cell lysate of the control group (E. coli BL21 pRGT7 TGF1) and the cell lysate expressing the Ap-SrcSH2 fusion protein as described above.

Example 4 Biotinylation of Peptides Interacting with Ap-SrcSH2 Protein and Purification thereof

A substance capable of binding a peptide binding to Src SH2 protein (peptide S003; amino acid sequence is amino terminus-EPQpYEEIPIYL-carboxy terminus, pY is phosphotyrosine; product of Eppendorf Co.) with avidin or streptoavidin (Steptavidin) The peptide was biotinylated using already activated NHS-biotin (NHS-biotin, Boehringer Mannheim Co.). The biotin-combined biotin-peptide (B-S003) complex was then purified by HPLC (Waters Co.), lyophilized, and the powder was used in the next step.

Example 5 Measurement of Ap-SrcSH2 Protein and Biotin-Peptide Interaction Using Alkaline Phosphate

(Step 1) Measurement of reaction of SH2 protein with biotin-peptide (B-S003)

In a 96-well plate coated with avidin (coating), biotin-peptide (100 ng) was dissolved per well (dissolved in 100 μl of phosphate buffer solution) and reacted at 37 ° C. for 1 hour. Next, wash each well more than 10 times with PBST buffer solution (20mM sodium phosphate, pH 7.5, 150mM sodium chloride, Triton X-100 1%) and add 100µl (100ug) of cell lysate containing Ap-SrcSH2 protein. The reaction was carried out at 1 ° C. for 1 hour. As a negative control, the control cell lysate was added in the same amount as above to react together. Then wash each well more than 10 times with PBST buffer solution, add 75µl of substrate solution, react for 1 hour at 37 ℃, stop the reaction with 25µl of 1N sodium hydroxide, and measure the result at 405nm. Read by.

(Step 2) Identification of biotin-peptides present in the wells using phosphotyrosine monoclonal antibodies

In order to confirm that the biotin-peptide is attached to the wells examined above, the wells obtained by reacting the biotin-peptide (B-S003) with 100ng at 37 ° C were washed with PBST buffer and the phosphotyrosine monoclonal antibody was 37 ° C. After diluting at 1000-fold for 1 hour, the wells were washed again with PBST buffer, and alkaline phosphate-bound secondary antibody (AP conjugated 2nd Antibody) was diluted 3000-fold and attached at 37 ° C for 1 hour. At this time, a negative standard was used that did not react the biotin-peptide to the well. Next, 75 μl of substrate solution was added to react at 37 ° C. for 1 hour, and 25 μl sodium hydroxide was added thereto to terminate the reaction. The result was read at 405 nm.

FIG. 6 shows biotin-peptide attached to an avidin-coated 96-well and reacted with cell disruption solution expressing Ap-SrcSH2 protein to measure its AP enzyme activity (lane 2). To determine if there is a phosphotyrosine monoclonal antibody attached and then reacted with alkaline phosphate-conjugated secondary antibody to measure AP activity (lane 3).

In the present invention, the Ap-SrcSH2 fusion protein is selected using alkaline phosphatase activity, so that not only whether various compounds or peptides interact with the SH2 site can be determined, but also their affinity, dissociation degree, etc. can be confirmed. This can be very useful when analyzing various compounds such as high throughput screening (HTS). Therefore, the screening method of the present invention is easy to operate without using radioactivity, it is expected to select an effective regulator involved in the cell signaling process and is expected to contribute greatly to the prevention and treatment of cancer.

(Sequence list)

SEQ ID NO: 1

Sequence length: 30

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 1]

(Sequence list)

SEQ ID NO: 31

Sequence length: 30

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 2]

(Sequence list)

SEQ ID NO: 3

Sequence length: 53

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 3]

(Sequence list)

SEQ ID NO: 4

Sequence length: 35

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[SEQ ID NO 4]

(Sequence list)

SEQ ID NO: 5

Sequence length: 1821

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: DNA

[SEQ ID NO: 5a]

[SEQ ID NO: 5b]

[SEQ ID NO: 5c]

(Sequence list)

SEQ ID NO: 6

Sequence length: 606

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[SEQ ID NO: 6a]

[SEQ ID NO: 6b]

[SEQ ID NO: 6c]

Claims (10)

A method for screening an antagonist that binds to a SH2 site by AP enzyme reaction using a fusion protein that combines a portion or all of a protein including an SH2 site with an alkaline phosphatase (AP). The method of claim 1, wherein the fusion protein is reacted with a compound immobilized on a solid support. The method of claim 1, wherein the protein comprising the SH2 site is an Src protein. A fusion protein comprising the amino acid sequence of SEQ ID NO: 6 in which the SH2 site of Src protein and alkaline phosphatase are combined. The fusion protein gene of claim 4, comprising the nucleotide sequence of SEQ ID NO: 5. An expression vector capable of producing the fusion protein of claim 4. The expression vector pRGT7 Ap-SrcSH2. Escherichia coli transformed with the expression vector of claim 6. The Escherichia coli transformant BL21 / pRGT7 Ap-SrcSH2 (Accession No .: KCTC 0313BP). A method for producing Ap-SrcSH2 fusion protein comprising the step of culturing E. coli under the conditions suitable for expression of Ap-SrcSH2 fusion protein and separating and purifying Ap-SrcSH2 fusion protein.

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