KR100426648B1 - Method for preparing mitogen-activated protein kinase kinase-1(mapkk-1 or mek-1) with high purity - Google Patents

Abstract

PURPOSE: A method for preparing mitogen-activated protein kinase kinase-1(MAPKK-1 or MEK-1) with high purity is provided, which protein MEK-1 is useful for analysis of mutual relation between protooncogene such as ras and cancer, so that it can be useful for prevention and treatment of cancer. CONSTITUTION: The method for preparing mitogen-activated protein kinase kinase-1(MAPKK-1 or MEK-1) with high purity comprises the steps of: preparing MEK-1 gene in which the nucleotide sequence encoding amino acids at amino acid position 33 to 52 are deleted, the nucleotide sequence encoding an amino acid at amino acid position 222, serine is substituted by aspartic acid, the nucleotide sequence encoding histidine set forth in SEQ ID NO:1 consisting of 5'-ATG CAT CAT CAC CAC CAT CAC GGA TCC CAT ATG CCC AAG AAG AAG CCG-3' is present in the 5'-terminal of the nucleotide sequence; constructing an expression vector pBacHis MEK-1 containing the MEK-1 gene; transforming Baculovirus with the expression vector pBacHis MEK-1 to produce Baculovirus AcMNPV MEK-1(KCTC 0287BP); and culturing Baculovirus AcMNPV MEK-1(KCTC 0287BP) and purifying protein MEK-1 from the cultured Baculovirus cells.

Description

Method for preparing high purity activated mitogen activating protein kinase kinase-1

The present invention provides a high purity of representative mitogen-activated protein kinase kinase-1 (MAPKK-1 or MEK-1; hereinafter MEK-1) present in higher animal cells. The present invention relates to a method of preparing a high-purity activated MEK-1 protein, specifically, by expressing activated MEK-1 in an insect cell, and then disrupting the cell, nickel affinity chromatography, dialysis, and gel filtration chromatography. It is about how to.

Cell signaling systems regulate the differentiation of cells, which are fundamentally necessary for linkage with various diseases and maintenance of life phenomena, and thus research on them has become one of the most prominent fields in recent years. In particular, the mitogen-activated protein kinase (MAPK) signaling pathway works when cells are stimulated by various mitogens such as growth factors, cytokines, and hormones. Is delivered internally in the nucleus, and its mechanism of action is well preserved in a variety of organisms, from yeast to mammals (Davis, RJ, et al., TIBS, 19, 470 (1994)).

Mammalian cells have various forms of MAPK pathways (Neiman, A., TIGS, 9, 390 (1993); Marshall, CJ, Cell, 80, 179 (1995)). The Ras-MAPK signaling system, in which Ras acts as one of the important signaling materials, is the most well known mammalian MAPK signaling system, and many of the proteins involved in this signaling are protooncogene products. It is known that abnormalities caused by mutations of these genes, ie, abnormal binding between proteins, overproduction of proteins by gene amplification, and changes in activity of component proteins, all cause cancer.

MEK protein is a protein that phosphorylates extracellular signal-regulated protein kinase-1 (ERK-1) and ERK-2 proteins that act on the ends of MAPK signaling system in the cytoplasm. It acts as an important mediator to deliver external signals into the nucleus and modulates MAPK activity changes that induce a variety of physiological changes, including cell growth differentiation. The MEK protein activates the protein by phosphorylating threonine (Thr), the 183th amino acid, and tyrosine (Tyr), the 185th amino acid, in signaling (Marshall, CJ, et al., Cell, 80, 179 (1995). )), The activated ERK protein phosphorylates in vitro the threonine residues of myelin basic protein (MBP). Since phosphorylation of threonine and tyrosine residues plays a critical role in MAPK activation, it has been reported that enzymes are not activated when these amino acid residues are substituted with other amino acid residues (Robbins, DJ, et al., J. Biol. Chem. 268, 5097 (1993).

Another kind of MAPK, JNK (Jun kinase), was first discovered as a protein that phosphorylates the transcription factor c-Jun when cells are exposed to ultraviolet light (Hibi, M., et al., Genes Dev). , 7, 2135 (1993). Amino acid sequencing revealed that JNK was very similar to ERK and activated through phosphorylation of the same threonine and tyrosine residues, but the phosphorylation site in JNK was reported to be different from ERK as the threonine-proline (Pro) -tyrosine site. JNK is also known to be activated by tumor necrosis factor (TNF), interleukin-1 (IL-1), etc. in addition to UV (Hibi, M., et al., Genes Dev., 7, 2135 (1993)).

p38, the most recently known phosphorylated protein of the MAPK family, was first identified by phosphorylation of tyrosine (Tyr) residues when exposed to Gram-negative endotoxin (Han, J., et al., Science, 265). , 808 (1994)). Like JNK, p38 is activated by TNF, IL-1, lipopolysaccharide (LPS), or osmotic stress. The relationship between substrate specificity for the p38 protein and the ERK and JNK signaling pathways is not well known, but p38 has the amino acid sequence most similar to the yeast high-osmolarity glycerol response 1 (HOG1) and the double phosphorylation site of Thr-Gly-Tyr. It has a dual phosphorylation motif and is known to replace HOG1 when expressed in yeast (Han, J., et al., Science, 265, 808 (1994)).

MEK activating ERK does not activate MAPKs such as JNK or p38 (Derijard, B., et al., Science, 267, 682 (1995)), and 12-o-tetradecanoylphorball-13-acetate (12 -o-tetradecanoylphorbol-13-acetate (TPA) strongly activates ERK but does not activate JNK and p38, indicating high specificity between them. In addition, actinomycin, okadaic acid and UV irradiation activate JNK strongly but do not significantly affect ERK. These results confirm that MAPK acts specifically. The relationship between JNK and p38 is not well known, but is known to use the same mitogens.

On the other hand, abnormalities of primary cancer genes such as ras, src, raf, and mos are known to cause cancer by sustaining the active state of several levels of signaling substances in the MAPK signaling system (Gu, Z., et al., J. Virol., 69, 8051 (1995); Pelech, SL, et al., TIBS, 17, 233 (1992). Therefore, knowing the correlation between cancer caused by mutations in the primary cancer genes such as ras and MAPK activity can develop a treatment method for cancer by regulating MAPK activity, and if a drug that modulates the activity of MAPK is developed, It may be an important anticancer agent that works effectively on various types of cancer caused by defects in upstream components of various signaling systems such as raf. Compared to this importance, the research on the development of a substance that can regulate the activity of MAPK is far less than the development of new drugs aimed at other higher stages, and it is hardly in practical use stage. In addition, the MEK-1 protein, which is used in the process of drug discovery and the interrelationship of the signaling system, is mostly in the form of a GST (glutathione-S-Transferase) fusion protein having a molecular weight of 25 kDa. Or inadequate to study the structure of proteins and new compounds (David, T. Dudley, et al., Proc. Natl. Acad. Sci. USA, 92, 7686-7689 (1995)).

Accordingly, the present inventors have succeeded in mass-purifying the MEK-1 gene in a form having activity in insect cells and purifying it with high purity as a basic step for developing a therapeutic agent for controlling the activity of MEK-1. It is possible to provide tertiary structural analysis by resonance spectroscopy and X-ray crystallography and to provide an activated MEK-1 protein suitable for the development of inhibitors that can inhibit cancer development.

It is an object of the present invention to prepare high purity activated mitogen active protein kinase kinase-1.

Figure 1 shows the nucleotide sequence and amino acid sequence of the activated mitogen active protein kinase kinase-1 (MEK-1).

Figure 2 shows the process of cloning the activated MEK-1 gene with the expression vector pBacHis MEK-1.

Figure 3 is the result of electrophoresis of the fractions obtained in the nickel affinity chromatography step of purification of activated MEK-1 protein.

Figure 4 is an elution graph of the gel filtration chromatography step of purification of activated MEK-1 protein.

Figure 5 is the result of electrophoresis of the fractions obtained in the gel filtration chromatography step of purification of the activated MEK-1 protein.

6 is a result of measuring the phosphorylation activity of the activated MEK-1 protein purified according to the method of the present invention.

Figure 7 is the result of measuring the phosphorylation activity of ERK-1 and ERK-2 protein activated by activated MEK-1 protein purified according to the method of the present invention.

8 shows the results of measuring activity inhibition by the kinase activity inhibitor of the activated MEK-1 protein purified according to the method of the present invention.

According to the above object, in the present invention, amino acids 33 to 52 of amino acid sequence of mitogen-activated protein kinase kinase-1 (MAPKK-1 or MEK-1) are removed. , 222nd amino acid serine is transformed into aspartic acid, the MEK-1 gene comprising a nucleotide sequence encoding histidine in the 5'-terminal sequence, the expression vector comprising the gene, transformed with the expression vector A method of preparing activated mitogen-activated protein kinase kinase-1 comprising culturing and purifying a cell line and the transformed cells under conditions suitable for expression of mitogen-activated protein kinase kinase-1 to provide.

The base sequence encoding the histidine at the 5′-end of the MEK-1 gene has the following SEQ ID NO: 1.

[SEQ ID NO: 1]

High purity activation through crushing recombinant insect cells expressing activated MEK-1 protein of the present invention, removing insoluble precipitates to recover soluble proteins, and performing nickel affinity chromatography, dialysis and gel filtration chromatography Obtained MEK-1 protein.

Hereinafter, the present invention will be described in more detail.

First, in order to amplify the activated MEK-1 gene using polymerase chain reaction, the plasmid pBK-RSV MEK (George F., Vande Woude Lab., Science, 265, 966) was used as a template. Primers for polymerase chain reaction (hereinafter, referred to as PCR) corresponding to the '-and 3'-ends are designed and synthesized. The 5'-terminal primer has a restriction enzyme recognition site and six histidines (His) to facilitate cloning with a baculovirus expression vector, and the corresponding 3'-terminal primer has a termination codon and a restriction. An enzyme recognition site is inserted so that the protein is synthesized from the start codon and the protein synthesis is completed at the artificially inserted end codon.

Plasmid pBK-RSV MEK was used as a template and PCR was performed using the primers to amplify about 1120 base pairs of activated MEK-1 gene, and MEK- was prepared using conventional phenol / chloroform and 100% ethanol. 1 Extract the DNA. It was inserted into a baculovirus expression vector such as plasmid pBacPAK8 (cat # 6145-1, Clontech).

The prepared expression vector is infected with an insect cell line to prepare a recombinant baculovirus, and then, the insect cell line is then infected with the insect cell line and cultured under conditions suitable for expression of the present MEK-1 protein. The resulting protein can be isolated and purified from the cultured cell line and the culture medium through cell disruption, nickel affinity chromatography, dialysis and gel filtration chromatography.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Amplification and Expression of MEK-1 Gene

(Step 1) Synthesis and Amplification of MEK-1 Gene

In order to amplify the MEK-1 gene activated from the plasmid pBK-RSV MEK (George F., Vande Woude Lab., Science, 265, 966) using polymerase chain reaction, two oligonucleotide primers were synthesized using a DNA synthesizer (DNA Synthesizer). , AppliedBiosystems INC. USA). The primer MEK Nde of SEQ ID NO: 2 has a recognition site of restriction enzyme Bgl II and six His codons, and then has a recognition site of restriction enzymes Bam HI and Nde I, and encodes a nucleotide sequence encoding the amino terminal of MEK-1. The primer MEK Xho of SEQ ID NO: 3 has a recognition site of restriction enzyme Xho I, and has a base sequence complementary to the carboxy terminus of MEK-1 from the 13th base sequence, and was designed to have a termination codon.

[SEQ ID NO 2]

[SEQ ID NO 3]

The polymerase chain reaction was carried out using the plasmid pBK-RSV MEK as a template. Into the reaction tube, 3 μl of the primer MEK Nde 0.75 μg and 3 μl of the primer MEK Xho 0.75 μg were added to the reaction tube. 2 µl of the plasmid pBK-RSV MEK, 10-fold concentrated reaction solution (500 mM KCl, 100 mM Tris-Cl (pH 9.0), 10 µl of 1% Triton X-100), 6 µl of 25 mM MgCl ₂ , 10 mM dNTP mixture (10 mM each) 2 μl of dGTP, dATP, dTTP and dCTP), 0.4 μl of Taq polymerase (5 U / μl, Promega, USA) and 75 μl of distilled water were added, followed by 1 min 30 sec (denatured) at 94 ° C., 50 ° C. PCR was performed 30 times under conditions of 1 minute (annealing step) and 72 ° C for 1 minute 30 seconds (polymerization step) to amplify the MEK-1 gene of about 1120 base pairs, and 100 with phenol / chloroform, a conventional method. MEK-1 DNA was extracted using% ethanol and dissolved in 20 µl of TE solution.

(Step 2) Preparation of Baculovirus Expression Vector of MEK-1 Gene

1 μl of restriction enzymes B gl II and Xho I were added to 16 μl of the reaction solution obtained in step 1, respectively, and a 10-fold restriction enzyme reaction solution (100 mM NaCl, 50 mM Tris-Cl, 10 mM MgCl ₂ , 1 mM DTT (pH 7.9)) was added. ) 2 μl was added and reacted at 37 ° C. for 1 hour to obtain a DNA fragment of MEK-1 having a restriction enzyme recognition site by a conventional method.

On the other hand, the plasmid pBacPAK8 (cat # 6145-1, Clontech Co., Ltd.) was completely cleaved with restriction enzymes Bam HI and Xho I and separated by 1% agarose gel electrophoresis to connect the obtained MEK-1 DNA fragment as follows.

That is, 0.1 μg of the DNA fragment of MEK-1 obtained above and 0.1 μg of the cleaved vector fragment were put into a conjugation reaction tube, and then 10-fold ligation reaction solution (500 mM Tris-HCl (pH 7.8), 100 mM MgCl ₂ , 100 mM DTT). , 10 mM ATP) 2 μl, 10 units of T4 DNA ligase and distilled water were added to make the total volume 20 μl, and the reaction was carried out at 16 ° C. for 12 hours. After the reaction, the strain was added to E. coli HB101 (ATCC # 33694) competent cells, transformed, and plated on an LB ampicillin plate (LB medium containing 50 μg / ml of ampicillin) to obtain the MEK-1 gene of the present invention. E. coli transformants were selected. The expression vector pBacHis MEK-1 was obtained from the selected clone.

Figure 2 shows the process of cloning the activated MEK-1 gene with the expression vector pBacHis MEK-1.

(Step 3) Generation of Recombinant Baculovirus

To prepare a recombinant baculovirus capable of expressing MEK-1, 2 ml of TNM-FH medium (450 ml of Grace's medium; Gibco-BRL. #) Was placed in a 35 mm diameter cell culture dish. 440-1300) + 25 ml of FBS (Gibco-BRL. # 16000-044) + 10 ml of yeastolate (Gibco-BRL. # L8190-017) + 10 ml of lactoalbumin hydrosate (Gibco-BRL # 18080-010) and 1.0 X 10 ⁶ insect cell IPLB-Sf21 cells (Clontech # K1601-E; hereinafter referred to as Sf21) were added and allowed to stand at room temperature for 1 hour to allow the cells to bottom out of the cell culture dish. The cells were then washed twice with 2 ml of Grace medium without FBS, 1 ml of FBS-free Grace medium was added, and then Lipofectin (45 μl of distilled water) was added thereto. 5 μl of lipopeptin (Gibco-BRL # 18292-011) and a DNA mixture (BacPAK6 virus DNA digested with restriction enzyme Bsu 36I). Clontech # 6411-1) 100 ng, distilled water was added to 500 ng of pBacHis MEK-1 plasmid DNA prepared in Step 2 so that the total volume was 50 μl), and the mixture was left at room temperature for 15 minutes. After 5 hours the cultures were removed and 2 ml of TNM-FH medium (450 ml of Grace media + 25 ml of FBS (Gibco-BRL # 16000-044) + 10 ml of yeastolate (Gibco-BRL #) 8190-017) + 10 ml of lactoalbumin hydrosate (Gibco-BRL # 18080-010) were added and incubated for 4 days.

Subsequently, the virus-containing medium was continuously diluted 10-fold from 10-fold to 10- ^6- fold, and 100 µl of each of the 35 mm-diameter culture plates to which 1.5 X 10 ⁶ Sf21 cells were attached was added to the culture dish of 35 mm-diameter, respectively, followed by 27 ° C. Infected for 30 minutes. Here, TNM-FH medium prepared at twice the concentration and 2% low-melting agarose (seaplaque agarose; FMC BioProducts # 50102), which were sterilized at a high temperature, were mixed at a 1: 1 ratio, and then cooled to 37 ° C. in each culture dish. Carefully dropped 1.5 ml each to solidify at room temperature, and then incubated at 27 ° C. for 4 days. 1 ml of dye solution (0.03% Neutral Red dissolved in distilled water, 0.2㎛ filter, Sigma # N7005) was added to each culture dish and dyed for 2 hours, and then the dye solution was removed. At this time, the virus-infected cells form a transparent plaque. Virus-infected plaques were then isolated and incubated with 1.0 × 10 ⁶ Sf21 cells in a 35 mm diameter dish for 3 days. The resulting cultures containing the virus were stored aseptically and virus infected cells were analyzed on 12% SDS-PAGE according to the method of Ramley (Leammli, Nature, 227, 680 (1970)). Among them, baculoviruses expressing 43kDa MEK-1 (Baculovirus AcMNPV MEK-1; hereinafter MEK-1 virus) were selected. 100 μl of the purely isolated MEK-1 virus was reinfected with 1.5 × 10 ⁷ Sf21 cells in a culture dish of 150 mm in diameter, 25 ml of the culture solution was incubated for 3 days, and a culture solution containing a high virus concentration of 1.0 × ^{10 8} pfu / ml or more was obtained. Got it.

The transformed insect cell line Baculovirus AcMNPV MEK-1 was deposited on Dec. 13, 1996, with the accession number KCTC 0287BP to the Gene Bank of Korea Institute of Science and Technology.

(Step 4) Mass expression of activated MEK-1

2 × ^{10 8} Sf21 insect cells were put in a cell culture roller bottle (Falcon # 3035) having an area of 1750 cm 2 in 300 ml TNM-FH (5% FBS) and incubated while rotating at 0.2 rpm for 24 hours. Subsequently, the culture medium was removed, and 3 × ^{10 9} pfu of MEK-1 virus was added and infected by rotating at 0.2 rpm for 30 minutes. 400 ml of TNM-FH (5% FBS) was added thereto and incubated for 48 hours. The cultured cells were centrifuged at 1,500 rpm for 5 minutes to obtain cell precipitates, and then washed once with 50 ml of phosphate saline (pH 7.4), followed by centrifugation at 1,500 rpm for 5 minutes to store the sunk cells at 70 ° C. It was.

EXAMPLE 2 Purification of Activated MEK-1 Protein

(Step 1) Cell disruption and removal of insoluble protein

Insect cell precipitate expressing the recombinant MEK-1 protein was suspended by adding about 100 ml of buffer solution 1 (5 mM imidazole, 1 M NaCl, 20 mM Tris, pH 8.0), followed by ultrasonic crusher (Ultrasonic homogenizer 4710) in ice bath. Cole-Parmer, USA) was sonicated for about 2 minutes at 50% power and 50% duty-cycle to disrupt cells. The cell homogenate thus obtained was centrifuged at 10,000 rpm for 30 minutes with a high-speed centrifuge (Beckman J2-21M, Rotor JA-14) to obtain a supernatant soluble protein.

(Step 2) Nickel Affinity Chromatography

The same volume of buffer solution 1 was added to the solution of step 1 and placed in a nickel agarose column (His Bind metal chelation resin, Novagen, USA) equilibrated with buffer solution 1 and passed at a rate of 2 ml per minute. Subsequently, 50 ml of buffer 1 was flowed through the column to remove proteins that were not adsorbed on the resin. Next, 400 ml of buffer 1 containing imidazole having a linear concentration of 0 to 1.0 M was added thereto, and the proteins adsorbed on the resin were eluted at a rate of 2 ml per minute to collect 10 ml of each fraction. The collected fractions were collected by collecting 15% SDS-polyacrylamide gel electrophoresis (PAGE) to collect only specific fractions containing MEK-1 protein separately.

Figure 3 is the result of electrophoresis of the fractions obtained in the nickel affinity chromatography step of purification of activated MEK-1 protein. Here, the first column represents a standard molecular weight protein sample, the second column represents a protein solution introduced into the resin, the third column represents a protein exiting without being adsorbed to the resin, and the fourth column is eluted. The collected fractions were electrophoresed.

(Step 3) Dialysis

The protein solution collected in step 2 was placed in a dialysis membrane (Spectrum Co, U.S.A., MW Cut-off: 12000), and then dialyzed in Buffer 2 (25 mM Tris, 0.2 M NaCl, 1 mM EDTA, pH 8.0).

(Step 4) Gel Filtration Chromatography

The sample collected in step 3 was concentrated in an ultrafilter (Amicon Co, U.S.A., MW Cut-off: 10000) so that the concentration of protein was 2 mg / ml or more. Concentrated protein samples were collected by gel filtration at a rate of 0.5 ml / min on a Superdex-200 FPLC column (Superdex-200, FPLC, Pharmacia, Sweden) and then fractionated per minute, followed by SDS-PAGE to obtain high-purity MEK- 1 protein was collected.

Figure 4 is an elution graph of the gel filtration chromatography step of purification of the activated MEK-1 protein, Figure 5 is the result of electrophoresis of the fractions obtained in this step. Here, the first column shows the standard molecular weight protein sample, the second column shows the protein introduced into the column, and the third column shows the eluted protein.

Confirmation Example 1: Phosphorylation

The activity of the MEK-1 protein prepared in the present invention was observed through phosphorylation of MAPK protein. 100 ng of ERK-1 and ERK-2 proteins were added to 20 ng of purified MEK-1 protein according to the above example, and 2 μl of ATP (Amersham, USA) containing radioisotope γ- ³² P was added thereto. Reaction buffer (30mM HEPES, pH 8.0, 1mM DTT, 1mM benzamidine, 10mM MgCl ₂ ) was added to make the total volume 30 μl. The reaction was allowed to stand at 37 ° C. for 30 minutes and then placed in an ice bowl to terminate the reaction. The same amount of electrophoretic sample solution was added to each sample, 12% acrylamide gel electrophoresis was carried out, and the gel was dried in a drier and photosensitive on a radiation film.

6 is a result of measuring the phosphorylation activity of the purified MEK-1 according to the method of the present invention. Where column 1 represents a sample of standard molecular weight protein, columns 2 to 5 represent ERK-1 protein without MEK-1, and columns 6 to 9 do not contain MEK-1. Shows the ERK-2 protein, column 10 to 13 shows the ERK-1 protein with MEK-1, column 14 to 17 shows the ERK-2 protein with MEK-1 It can be seen that MEK-1 purified according to the method of the present invention has intrinsic phosphorylation properties.

Confirmation Example 2: Confirmation of ERK-1 and ERK-2 activation by MEK-1

The activity of the purified MEK-1 protein was observed through the phosphorylation properties of MAPK proteins activated by MEK-1, ie, ERK-1 and ERK-2 proteins. To 20 ng of purified MEK-1 protein according to the above example, 100 ng of ERK-1 and ERK-2 proteins were added, and 2 μg of Myelin Basic Protein (MBP), a substrate of ERK protein, and radioisotope γ- After adding 2 μl of ATP containing ³² P (Amersham, USA), the reaction buffer solution (30 mM HEPES, pH 8.0, 1 mM DTT, 1 mM benzamidine, 10 mM MgCl ₂ ) was added so that the total volume was 30 μl. . The reaction was allowed to stand at 37 ° C. for 30 minutes and then placed in an ice bowl to terminate the reaction. The same amount of electrophoretic sample solution was added to each sample, 12% acrylamide gel electrophoresis was carried out, and the gel was dried in a drier and photosensitive on a radiation film.

Figure 7 is the result of measuring the phosphorylation activity of ERK-1 and ERK-2 protein activated by activated MEK-1 protein purified according to the method of the present invention. Here, the first column shows a sample of the standard molecular weight protein, the second column shows the phosphorylation degree of MBP of ERK-1 in the absence of MEK-1, and the third column shows the degree of phosphorylation of MBP in the absence of MEK-1. ERK-2 shows the degree of phosphorylation of MBP, column 4 shows the degree of phosphorylation of MBP of ERK-1 activated by MEK-1, and column 5 shows ERK- activated by MEK-1. By showing the degree of phosphorylation of MBP possessed by 2, it was confirmed that the MEK-1 protein expressed and purified by the present invention activates ERK-1 and ERK-2 proteins, and these activated proteins phosphorylate MBP. In other words, it can be seen that MEK-1 has intrinsic phosphorylation characteristics.

Confirmation Example 3: Confirmation of Inhibition of MEK-1 Activity by Kinase Inhibitor

It was confirmed that the activity of the MEK-1 protein prepared by the present invention was inhibited by using strourosporine (straurosporine, Boehringer Mannheim, Germany, Cat No 1055682), which has a common inhibitory effect on kinase.

Into the reaction tube of 10 nM and 100 nM of stolosporin, respectively, to 20 ng of purified MEK-1 protein according to the above embodiment, 100 ng of ERK-1 protein was added to each tube, and the radioisotope γ- ³² P was included. After adding 2 μl of ATP (Amersham, USA), a reaction buffer (30 mM HEPES, pH 8.0, 1 mM DTT, 1 mM benzamidine, 10 mM MgCl ₂ ) was added so that the total volume was 30 μl. The reaction was allowed to stand at 37 ° C. for 30 minutes and then placed in an ice bowl to terminate the reaction. The same amount of electrophoretic sample solution was added to each sample, 12% acrylamide gel electrophoresis was carried out, and the gel was dried in a drier and photosensitive on a radiation film.

8 is a result of measuring the degree of activity inhibition by the kinase activity inhibitor of MEK-1 purified according to the method of the present invention. Here, the first column shows a sample of standard molecular weight protein, the second column shows a sample without strosporin, and the third column contains 1% dimethyl sulfoide (DMSO) which is a solvent of strosporin. The fourth column shows a sample containing 100 nM of storosporin, and the fifth column shows a sample containing 10 nM of stolosporin, which is determined by storosporin, a universal kinase activity inhibitor. It can be seen that the activity of MEK-1 is inhibited.

By using the MEK-1 protein prepared according to the present invention, it is possible to know the correlation with cancer caused by mutations in the primary cancer genes such as ras, and from this, it can be used to develop a method for treating cancer by regulating MAPK activity. In addition, it can be used to develop therapeutics that regulate the activity of MAPK.

Claims (8)

In the amino acid sequence of mitogen-activated protein kinase kinase-1 (MAPKK-1 or MEK-1), amino acids 33 to 52 are removed, and 222 amino acid serine is aspart. MEK-1 gene modified with an acid and comprising a nucleotide sequence encoding histidine at the 5'-terminal sequence. The method of claim 2, MEK-1 gene, characterized in that the base sequence encoding the histidine 5'-end of the MEK-1 gene has the following sequence 1. [SEQ ID NO: 1]

An expression vector comprising the gene of claim 1. The method of claim 3, wherein Baculovirus expression vector pBacHis MEK-1. Cell line transformed with the expression vector of claim 3. The method of claim 5, wherein Insect cell line baculovirus AcMNPV MEK-1 (KCTC 0287BP). A method for preparing mitogen-activated protein kinase kinase-1, comprising culturing and purifying the cell line of claim 5 under conditions suitable for expression of mitogen-activated protein kinase kinase-1. The method of claim 7, wherein The purification step is to disrupt the recombinant insect cells expressing the mitogen active protein kinase Kinase-1, remove the insoluble precipitate to recover the soluble protein, nickel affinity chromatography, dialysis and gel filtration chromatography Method for preparing phosphorus mitogen active protein kinase kinase-1.

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