KR101297667B1 - Peptides targeting the bcl-2 protein and uses thereof - Google Patents

Abstract

The present invention relates to a peptide for targeting bcl-2 protein, a cancer induction marker, a cancer diagnostic kit and drug delivery composition using the same. It can be used as a small molecule diagnostic probe for diagnosing the expression of cancer cells according to the present invention and a drug delivery material for the treatment and prevention of cancer.

bcl-2 protein, apoptosis, probe, cancer diagnosis, drug delivery

Description

ｂｃｌ-2 protein target peptide and its use {PEPTIDES TARGETING THE BCL-2 PROTEIN AND USES THEREOF}

The present invention relates to a peptide for bcl-2 protein targeting, a cancer diagnostic kit and drug delivery composition using the same. More specifically, the present invention relates to a small molecule peptide for a diagnostic probe that specifically binds to a bcl-2 protein, which is a cancer induction marker, a cancer diagnostic kit and a drug delivery material using the same.

The smallest unit of the human body, the cell, normally divides, grows and dies by intracellular control and maintains cell balance. If a cell is damaged for some reason, it is treated and recovered to act as a normal cell, but if it is not recovered, it dies on its own. However, cancer is defined as a condition in which abnormal cells, which are not controlled for such proliferation and suppression, proliferate excessively, invade surrounding tissues and organs, and cause mass formation and destruction of normal tissues for various reasons. Cancer is such an uncontrolled proliferation of cells, which destroys the structure and function of normal cells and organs.

Apoptosis, called “apoptosis,” is distinguished from necrosis, in which cells die by necrosis or morbidity in a way that cells are controlled by genes and die. Apoptosis is responsible for the body's composition during development, and after maturation, it removes unnecessary parts and maintains homeostasis. That is, the natural process of removing unwanted cells due to physiologically scheduled death of cells. However, if apoptosis does not occur at the place where it should occur or excessive cell death occurs, it leads to various diseases. Currently, studies on the relationship between apoptosis in the development and progression of tumors are being actively conducted.

One protein involved in this apoptosis is the bcl-2 protein. When the bcl-2 protein has excessive activity, apoptosis is suppressed and the mutated cells (tumors) proliferate and eventually develop into cancer cells. In the early stages of cancer, there are many cases where there are no special symptoms, and since the symptoms are nonspecific, it is difficult to distinguish them from other diseases.

For these reasons, studies of proteins that regulate apoptosis pathways are needed. The bcl-2 family, a protein that regulates apoptosis pathways, is a protein that acts on the intrinsic apoptosis pathway by mitochondria. The bcl-2 family causes apoptosis and anti-apoptosis depending on the type of protein.

More specifically, apoptosis is mainly regulated by bcl-2 family proteins, which significantly inhibit apoptosis (bcl2, bclxl, bclw, Mcl1, A1) and induce apoptosis. Proteins (bad, PUMA, H, bcl-G, Noxa, bim, bcl-rambo, bax, bik, bid, bmf) can be divided. These bcl-2 family members combine with each other to form a complex regulatory system as homodimers and heterodimers, and the relative proportion of bcl-2 family proteins is an important factor in determining the degree of response of apoptosis. They mainly work in the mitochondria, where the pro-apoptosis regulator is involved in the outflow of cytochrome C, causing apoptosis. Pro-survival, on the other hand, acts to prevent apoptosis by blocking outflow. In other words, it plays the opposite role depending on which protein acts by which signal.

There is a great deal of evidence suggesting that abnormalities in substances involved in the apoptosis mechanism play an important role in carcinogenesis. Abnormalities of the bcl-2 family proteins are also known to play an important role in this process.

As a result, apoptosis is completed by a series of activities and reactions of various regulators in the cell. Among them, bcl-2 protein, which inhibits apoptosis, is present in endoplamic reticulum, mitochondria and nuclear membrane, and together with cytokines. Inhibits apoptosis and plays an important role in the replacement of normal cells. The bcl-2 family protein is normally associated with the BH3 subfamily bid / bim and bax and bak are present in the mitochondrial outer membrane with nothing bound. When apoptosis signal comes in, bik or bad binds to bcl-2, and bid / bim released from bcl-2 binds to bak and Bax to induce their conformational change, causing apoptosis. Let's do it. That is, bcl-2 prevents apoptosis by acting to block apoptosis mechanisms of the bax and BH3 subfamily. Inhibition of apoptosis leads to tumor expansion, with increased chances of clonal expansion, secondary genetic changes, and malignant transformation.

The relationship between cancer prognosis and apoptosis has been studied in several types of cancers. Abnormal expression of bcl-2 protein, which inhibits apoptosis in breast cancer and lung cancer, has been reported to increase resistance to anticancer drugs, and in many cases, patients have a short survival time and tumor progression. Although there is no known large structural abnormality of the bcl-2 gene, the expression of bcl-2 protein is increased in solid cancers, and studies on the correlation between bcl-2 protein expression and carcinoma are ongoing.

In other words, the cancer cells are overexpressed of the bcl-2 protein to inhibit cell death, thereby overproliferating. Increased bcl-2 protein expression in many solid cancers is known to protect cancer cells from apoptosis-inducing mechanisms such as anticancer drugs in tumor cells, and more research is needed to support this. Therefore, this bcl-2 protein corresponds to a cancer-induced labeled biomarker, and it is necessary to discover a peptide as a diagnostic probe that effectively recognizes it. Such peptides can be used to develop diagnostic kits for early cancer diagnosis and can be used as prognostic factors for independent cancers. In addition, if the peptide is used to develop a drug that actually blocks cancer cells, a new concept of a broad range of anticancer drugs is expected.

The combination of target-aware probes with new imaging techniques and anticancer agents offers the opportunity to improve the treatment of early detection of cancer and develop potential diagnostic methods. In order to develop a probe for the early detection of cancer, it is necessary to find a probe with high affinity and selectively attach it to early cancer cells and use it for early diagnosis of cancer.

There is also a lot of research going on in the field of genes. Based on the results of this genetic analysis, genetic modification studies are underway to prevent the function of cancer genes to be cut off or expressed.

According to the prior art, there is a method using polymerase chain reaction (PCR) as the most common method for diagnosing a diagnosis subject. This method analyzes the unique genes of a diagnostic subject and then in vitro In vitro ), a small amount of a diagnostic subject is analyzed by amplification. In addition, the method uses a unique nucleotide sequence of the gene to analyze the unique nucleotide sequence of the diagnosis subject only. Such PCR method can be extended by securing extensive genetic information database through recent human genome projects. In addition, the PCR method uses SSCP (Single-strand conformation polymorphism), RFLP (Restriction fragment length polymorphism) and AFLP (Amplified fragment length polymorphism) methods to check the genetic variation of the amplified genes with various probes. It provides useful information in conjunction.

However, the diagnostic method using PCR has the advantage of having a high sensitivity in obtaining a small amount of the unique nucleotide sequence that only the diagnostic subject has, but has a problem such as difficulty in pretreatment and in situ adaptation of the sample to the diagnostic system. There is a limit to use.

Cancer marker markers that can detect cancer and detect it early are being progressed through studies of antibodies that can identify early development of tumors. Blood is the most representative body fluid and is a kind of 'in vivo barometer' whose components change sensitively during disease development and progression. Plasma proteins have an estimated 50,000 or more components, and the concentration of each protein component is also very dynamic (1-10 ^-12 mg / mL), with a detection limit of about 10 ^-6 -10 ^-9 mg / mL. Antigen-antibody reactions make detection and quantitation of biomarker proteins present in low and medium concentrations very difficult. However, diagnostic kits with high sensitivity, rapidity and field adaptability, and diagnostic probes with high physicochemical stability, sensitivity, specificity, and applicability have not yet been proposed.

Since antibodies have a high specificity for cancer, many studies are being conducted for cancer detection and drug delivery, but there may be a problem of generating immune reactivity in vivo. In addition, antibodies may be denatured by heat or chemical treatment.

Recently, Enzyme-Linked Immunosorbent Assay (ELISA) is a method for determining the increase and change in the expression level of a specific protein caused by disease. In order to recognize the specific protein, an antibody that specifically binds to the specific protein is used as a diagnostic probe to isolate the diagnostic subject from a biological sample such as blood or urine, and an enzyme-antibody reaction (ECL) is used for this purpose. However, nanomolar (10 ^-9 M) level of sensitivity has a disadvantage in that it has a low sensitivity compared to PCR diagnostics, despite the advantage that it can target the protein in vivo.

As a part of related research that can overcome the above disadvantages, surface enhanced Raman spectroscopy (SERS) and bio-barcode technology combined with nanotechnology (NT) based on current diagnostic methods such as PCR and ELISA methods As developed, analysis of diagnostic subjects at femtomol ( ^10-15 M) levels has proven experimentally feasible. Both of these methods are based on the signal characteristics of Raman spectroscopy (narrow Raman bands; low sensitivity to moisture, oxygen and soakers; and low photobleaching) and high sensitivity corresponding to fluorescence analysis. The amplification was performed to recognize femtomol level of a diagnosis subject, and an approach for prostate specific antigen (PSA) using easy sample such as blood was proposed. However, these methods are based on systems using antibodies that recognize biomarkers (diagnostic subjects), all of which are limited in number of bindings due to size limitations due to the physical and chemical structures of the antibodies, as well as various chemical reactions. There is a significant disadvantage of showing instability due to structural denaturation.

Therefore, there is a need for the development of new low molecular weight diagnostic probes differentiated from the above-mentioned antibody diagnostic system technology. The low molecular peptides can be used in such a low molecular diagnostic system.

Accordingly, the present inventors have developed a novel peptide as a new diagnostic substance that can replace the antibody currently used for cancer diagnosis.

It is an object of the present invention to provide novel peptides that recognize and specifically bind cancer-derived proteins for early diagnosis, prevention and treatment of cancer.

Another object of the present invention is to provide a bcl-2 protein targeting peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 7 and a polynucleotide encoding the amino acid sequence.

Another object of the present invention is to provide a recombinant expression vector comprising the polynucleotide, a transformant transformed with the expression vector.

Another object of the present invention is to provide a cancer diagnostic kit comprising the peptide.

Another object of the present invention to provide a composition for drug delivery comprising the peptide.

The present invention provides a peptide for bcl-2 protein targeting consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-7.

The bcl-2 protein is a protein well known in the art. Specifically, it may be a protein encoded from a gene having a nucleotide sequence of SEQ ID NO: 8, but is not necessarily limited thereto.

In the present invention, in order to overcome the disadvantages of the above-mentioned antibody, a small molecule peptide is used as a target substance. These small molecule peptides have the advantage of being small in size and stabilized in three dimensions, easily passing through mucous membranes, and recognizing molecular targets deep in tissues. In addition, the small molecule peptide according to the present invention has the advantage of early diagnosis of cancer because stability can be secured through local injection and minimize the immune response. In addition, the low molecular weight peptides according to the present invention are relatively simple in mass production and weak in toxicity.

In addition, the low-molecular peptides according to the present invention have an advantage of having a higher binding strength to the target material than the antibodies, and do not cause denaturation even during thermal / chemical treatment. In addition, the small size of the molecule can be used as a fusion protein by attaching to other proteins. Specifically, since it can be used by attaching to a polymer protein chain, it is used as a diagnostic kit and drug delivery material.

The bcl-2 target peptide is characterized by binding to a specific site of the bcl-2 protein, consisting of 12 amino acids. The bcl-2 target peptide is a peptide having a high affinity for the bcl-2 protein, and includes a peptide having the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 7.

Peptides of the invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry.

In addition, the peptide of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, such as using automated DNA synthesizers (such as those sold by Biosearch or AppliedBiosystems). The constructed DNA sequence is a vector comprising one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to this DNA sequence to regulate expression of the DNA sequence. The host cell is transformed with the recombinant expression vector formed therefrom. The resulting transformants are incubated under appropriate media and conditions so that the DNA sequences are expressed to recover substantially pure peptides encoded by the DNA sequences from the culture. The recovery can be carried out using methods known in the art (eg chromatography). The term "substantially pure peptide" as used herein means that the peptide according to the present invention is substantially free of any other protein derived from the host. For genetic engineering methods for peptide synthesis of the present invention, reference may be made to Maniatis et. al . , Molecular Cloning; A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif, 1991; And Hitzeman et al . , J. Biol. Chem . , 255: 12073-12080, 1990.

In order to easily identify, detect, and quantify whether the peptide of the present invention has bound to cancer cell tissue, the peptide of the present invention may be provided in a labeled state. In other words, the detectable label may be provided in a link (eg, covalently bonded or crosslinked). The detectable label may be a colorase (e.g. peroxidase, alkaline phosphatase), radioisotope (e.g. ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ^{99 m} Tc, ³² P, ³⁵ S) , Chromophores, luminescent or fluorescent materials (e.g. FITC, RITC, rhodamine, Texas red, fluorescein, phycoerythrin, quantum dot dots)).

Similarly, the detectable label can be an antibody epitope, substrate, cofactor, inhibitor or affinity ligand. Such labeling may be carried out in the course of synthesizing the peptide of the present invention, or may be performed in addition to the peptide already synthesized.

If fluorescent material is used as a detectable label, cancer can be diagnosed by fluorescence mediated tomography (FMT). For example, the peptide of the present invention labeled with a fluorescent material can be circulated into the blood and fluorescence by the peptide can be observed by fluorescence tomography. If fluorescence is observed, it is diagnosed as cancer.

On the other hand, the present invention provides a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 7, a recombinant expression vector comprising the polynucleotide and a transformant transformed with the expression vector.

The polynucleotides may be obtained as described above, and may preferably have base sequences of SEQ ID NOs: 9 to 15, respectively.

Expression vectors refer to plasmids, viral vectors or other mediators known in the art capable of expressing inserted nucleic acids in a host cell, encoding the peptides of the invention in conventional expression vectors known in the art. The polynucleotides may be operably linked. The expression vector is generally operatively linked with a replication origin capable of proliferating in a host cell, one or more expression control sequences (eg, promoters, enhancers, etc.), selective markers, and expression control sequences that control expression. Polynucleotides encoding the peptides of the invention.

The transformant may be transformed by the expression vector. Preferably the transformant is a method known in the art, including, but not limited to, expression vectors comprising polynucleotides encoding peptides of the invention, such as transient transfection, microinjection, Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection It can be obtained by introducing into host cells by polybrene-mediated transfection, electroporation, gene guns and other known methods for introducing nucleic acids into cells (Wu et al. , J . Bio Chem, 267: 963-967, 1992; Wu and Wu, J. Bio Chem, 263:.... 14621-14624, 1988).

Host cells that can be used in the present invention may be selected from the group consisting of E. coli, yeast, heart cells, lymphocytes, liver cells, vascular endothelial cells, spleen cells, cancer cells, animal cell lines such as CHO, Cos7, NIH3T3, insect cells, Not limited to this, all possible cells can be used as host cells.

The present invention also provides an antibody specific for the bcl-2 protein targeting peptide. In the present invention, "antibody" refers to a protein molecule specific to the antigenic site. For the purposes of the present invention, an antibody refers to an antibody that specifically recognizes the bcl-2 protein targeting peptide, and includes both polyclonal and monoclonal antibodies. As described above, peptides that specifically bind to the bcl-2 protein have been identified by the present invention, so that antibodies can be easily generated using the same techniques well known in the art (Sambrook et al. al . , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition).

Monoclonal antibodies are known in the art by fusion methods (Kohler and Milstein (1976) European Jounral of Immunology , 6: 511-519), recombinant DNA methods or phage antibody libraries (Clackson et al ., Nature , 352: 624-628, 1991; Marks et al ., J. Mol . Biol . , 222: 58, 1-597, 1991).

Polyclonal antibodies can be produced by methods well known in the art for injecting a bcl-2 protein targeting peptide having the amino acid sequence of SEQ ID NOS: 1 to 7 into an animal and collecting blood from the animal to obtain a serum comprising the antibody. have. For example, the peptide may be prepared by injecting a goat or rabbit with CFA (Complete Freund's Adjuvant) by subcutaneous injection, and by booster injection with CFA subcutaneously or intraperitoneally. Such polyclonal antibodies can be prepared from any animal species host, such as sheep, monkeys, horses, pigs, bovine dogs, in addition to goat rabbits.

The antibody is preferably labeled with a marker, such as a radioactive or fluorescent marker. Antibodies can be indirectly labeled by binding to anti-goat or anti-rabbit antibodies covalently bound to the marker compound.

The present invention also provides a kit for diagnosing cancer comprising the peptide of the present invention. The peptide included in the kit may have a peptide of SEQ ID NO: 1 to SEQ ID NO: 7 or a polynucleotide of SEQ ID NO: 9 to 15 encoding the same, and may be obtained by chemical or genetic engineering methods as described above. . The diagnostic kit of the present invention may further include a buffer or a reaction solution that stably maintains the structure or physiological activity of the peptide. In addition, to maintain stability, it may be provided in a powder state or dissolved in a suitable buffer or maintained at 4 ° C.

At this time, "diagnosis" in the present invention means confirming the presence or characteristics of the pathological state. For the purposes of the present invention, the diagnosis is to identify the presence or characteristics of cancer.

In addition, in order to facilitate identification, detection and quantification of the peptide of the present invention bound to cancer cell tissue, the peptide of the present invention may be provided in a labeled state as described above.

 On the other hand, when the peptide of the present invention is provided unlabeled, the kit for diagnosing cancer of the present invention may be used to detect whether or not to bind to a cancer-causing site, particularly cancer cells, in vitro or in vivo of the peptide of the present invention. It may further comprise a component. Said component is a known compound for the labeling of the peptide of the invention, or an antibody against a peptide of the invention or a specific receptor which binds to the peptide of the invention or a secondary antibody against them for detection through an antigen-antibody reaction and It may be a reagent for its detection.

In addition, the peptide of the present invention may be provided in a form coated on the surface of the plate. In this case, after inoculating the target cells directly on the plate and reacted under appropriate conditions, cancer can be diagnosed by observing the binding of the peptide to the cancer cells on the surface of the plate.

Experimental procedures, reagents and reaction conditions that can be used in the above methods can utilize those conventionally known in the art, which is obvious to those skilled in the art.

The present invention also provides a drug delivery composition comprising the peptide. The peptide of the present invention can be used as an intelligent drug carrier for selectively delivering drugs such as anticancer agents to cancer tissues. If the peptide of the present invention is used in cancer treatment in connection with a conventional anticancer agent, since the anticancer agent is selectively delivered only to cancer cells by the peptide of the present invention, the effect of the drug can be increased and at the same time, the side effect of the anticancer agent on the normal tissue is significantly reduced. Can be.

Anticancer agents that can be linked to the peptides of the present invention can be used without limitation as long as they are used in the treatment of conventional cancers. For example, cisplain, 5-fluouracil, adriamycin, methotrexate, vinblastine, busulfan, chlorambucil, cyclo Phosphamide, melphalan, nitrogen mustard, nitrosourea, and the like.

Linkage of the anticancer agent with the peptide of the present invention can be carried out through methods known in the art, such as covalent bonds, crosslinking and the like. To this end, the peptide of the present invention may be chemically modified if necessary so long as its activity is not lost.

The amount of the peptide of the present invention included in the composition of the present invention may vary depending on the type and amount of the anticancer agent to be bound. Preferably, the amount of the anticancer agent administered for treatment may be an amount sufficient to deliver the cancer tissue. However, the effective dosage of the drug is determined by taking into consideration not only the route of administration and the number of treatments but also various factors such as the age, weight, health condition, sex, severity of the disease, diet, and excretion rate. Therefore, in view of this, one of ordinary skill in the art will be able to determine an appropriate effective amount of a peptide of the present invention to treat the cancer by combining the peptide with an anticancer agent.

The composition of the present invention may include a suitable buffer solution for maintaining and / or preserving the stability of the peptide. The composition for drug delivery containing the peptide according to the present invention is not particularly limited to its formulation, route of administration and method of administration as long as the effect of the present invention is exhibited. For example, the peptides of the present invention can be administered orally or parenterally. In the case of oral administration, in order to prevent degradation by digestive enzymes in the gastrointestinal tract, it is preferable to use the peptide of the present invention composed of L-type amino acids. The parenteral administration includes subcutaneous injection, intramuscular injection, intravenous injection, urethral injection, and the like.

In addition, the composition of the present invention may further include a pharmaceutically acceptable carrier that is commonly added to the general pharmaceutical composition. The term "pharmaceutically acceptable" refers to a physiologically acceptable and does not normally cause an allergic reaction, such as gastrointestinal disorders, dizziness, or the like, when administered to a human or animal. Pharmaceutically acceptable carriers can be used, for example, in the case of injections, as buffers, preservatives, analgesics, solubilizers, isotonic agents and stabilizers.

As such, the formulation of the composition comprising the peptide of the present invention may be prepared in various ways as described above. For example, injectables can be prepared in the form of unit dose ampoules or multiple dose inclusions. Drug delivery compositions comprising the peptides of the invention can be administered by conventional drug administration methods known in the art.

Since the peptide of the present invention specifically binds to the bcl-2 protein, cancer can be diagnosed by confirming the excessive activity of the bcl-2 protein using the peptide of the present invention as a diagnostic probe. In addition, the peptide of the present invention can be used as a drug delivery composition as a low molecular weight material. When used in the treatment of cancer by connecting drugs such as peptides and anticancer drugs of the present invention, the anticancer drugs are selectively delivered to cancer tissues to increase the effect of the drug and there are almost no side effects such as immune response or denaturation reaction.

Hereinafter, preferred examples are provided to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications can be made within the scope and spirit of the present invention. It is obvious to the skilled person, and it is natural that such variations and modifications fall within the scope of the appended claims.

Example  One: bcl -2 Mass Production and Isolation of Proteins

The bcl-2 gene (SEQ ID NO: 8) was amplified by PCR as follows: 5'-CTGGTGGACAACATCGCTCTG-3 'was used for Bcl-2 sense primer and 5'-GGTCTGCTGACCTCACTTGTG-3' was used for Bcl-2 antisense primer. . In a reaction solution containing 5% DMSO containing the synthetic primer and template DNA, 94 min for 2 minutes of pre-denaturation, denaturation 95 ° C. 45 sec, annealing 55 ° C. 1 The template DNA obtained after 28 cycles of PCR reaction was confirmed through the gene sequencing with 1 cycle of min and extension of 68 ° C for 3 min. The amplified gene was inserted into the bacterial expression vector pGEX-KG (Promega, USA) and cloned according to the manufacturer's manual (see FIG. 1), and then the expression vector was transformed into E. coli BL21 (DE3) for expression of the host protein. After conversion, production of bcl-2 protein was induced for 5 hours under conditions of 0.5 mM IPTG, 37 ° C. The transformation method is briefly described as follows: First, 5 μl of DNA was added to 100 μl of competitive cells, mixed well, and left on ice for 30 minutes. Then heat shock was applied at 42 ° C. for 90 seconds. In order to prevent contamination, the alcohol lamp was first turned on, and then 800 µl of LB medium was added and incubated at 37 ° C for 45 minutes. Finally it was applied to the LBA plate and incubated overnight in a 37 ° C. incubator.

The bacterial cells were harvested by centrifugation to examine the solubility of the proteins. As a result, most of the proteins were dissolved. Therefore, a 20 mM reduced form of glutathione (GSH) linear gradient was obtained after obtaining a water-soluble protein for isolation and purification of the GST fusion protein, bcl-2, and passing it through a GSH-Sepharose column to bind the bcl-2 protein. eluted under a gradient).

The obtained protein was removed by using a desalting column (Amersham Biosciences, USA), and the purified degree of the obtained protein was confirmed through SDS / PAGE (FIG. 2). Next, since the molecular weight of the GST-tag (26 KDa) is similar to the size of Bcl-2 (25 KDa), the large GST-tag should be removed to proceed with the screening process to find peptides that bind only to the bcl-2 protein. Peptides that specifically bind to the bcl-2 protein can be identified. To this end, GST-tags were cut using thrombin (5 units of thrombin / mg of protein). When thrombin treated, the GST fusion bcl-2 protein is divided into two fragments: GST-tag (26KDa) and bcl-2 (25KDa). Thus, the bcl-2 protein isolated and purified by the above method was treated with thrombin. The same result was confirmed. Thrombin treatment resulted in two protein fragments, GST-tag and bcl-2. 1 mM benzamidine was added to terminate the reaction. GST-free protein was obtained with a 0.5 M NaCl linear gradient via anion exchange chromatography (Mono Q HR 5/5 column) to remove the GST-tag and obtain only pure GST-free protein (F5 in FIG. 3). Due to the similar size of GST-tag and bcl-2, accurate identification on gels is difficult. Thus the reaction product was dissolved in 15% SDS / PAGE to confirm that it was bcl-2 protein and Western blot method was used.

Specifically, 15% SDS / PAGE gels were made. And each sample was hanged on the gel. Proteins were transferred to the NC membrane. After completion of the transfer, the protein band transferred to the NC membrane was visually stained with a staining solution (Ponceaus S) and marked with a marker. Then, the membrane was washed with water and blocked with shaking at 50 rpm at room temperature for 1 hour with 2% BSA. The primary antibody (Anti-bcl-2, Rabbit polyclonal Ab (1: 2000 dilution; Santacruz, USA)) was attached at room temperature for 1 hour. The membrane was then washed several times at room temperature for 1 hour with 1 × TBST buffer. A secondary antibody (Anti-Rb IgG HRP conjugate (1: 4000 dilution; Santacruz, USA) was attached at room temperature for 1 hour. Once again the membrane was washed several times at room temperature for 1 hour with X TBST buffer. (Chemiluminescence) solution was sprayed sufficiently on the membrane and reacted with the secondary antibody to give light, and finally, the film was printed to see if a band of the desired size was produced. Only two protein fragments could be seen (FIG. 4).

Example  2: M13  scrap paper Peptides  Library Screening-Phage Display

In order to fix the protein obtained in Example 1 to a 96-well plate, a polystyrene plate (SPR) having a polystyrene surface was used. By using a hydrophilic interaction between the protein and the plate surface to fix the protein to the bottom of the plate surface, a random peptide library (designed to have about 2.7 billion different amino acid sequences through a random sequence of 12 amino acids) Peptide Library-Ph.D ^™ phage display peptide library kit (New England Biolabs (NEB) purchased and used) screening process was designed to enable higher specificity and binding analysis. The bond is shown in FIG. 5.

Specifically, in Figure 5, in order to screen peptides that bind to the cancer-causing marker bcl-2, after binding the bcl-2 to the plate (Qiagen) surface, M13 phage peptide library (about 2.7 billion different amino acid sequence Peptide expression consisting of a peptide consisting of 12 amino acids with a fused to the M13 phage gp3 minor coat protein) (binding method of M13 phage and peptide library), and then binding peptides with high affinity through various binding times and washing conditions Phage were selected. Screening schemes and reaction conditions in this regard are shown in FIGS. 5 and 6, respectively.

Specifically, FIG. 5 is a schematic diagram of M13 phage screening for peptide screening with bcl-2, wherein (1) bcl-2 protein is immobilized on the surface of a 96-well plate, and (2) 12 is on the surface of M13 phage. Phage libraries expressing peptide libraries consisting of dog amino acids were combined with bcl-2 protein, followed by (3) washing them under various conditions, and (4) eluting finally bound phages to obtain phages.

The phage obtained as described above is infected with E. coli and amplified, and then bound to the bcl-2 protein, and then repeated under conditions such as a higher washing condition and a shorter reaction time, thereby finding a phage having a stronger binding force. Was repeated (biopanning).

In addition, FIG. 6 is a table of five-step biopanning conditions for detecting phage expressing peptides having high specificity and binding ability for the bcl-2 protein. In each step, the reaction conditions were variously changed with strong washing conditions and short reaction times. Accordingly, five biopannings were performed to secure phages having peptides that bind to the bcl-2 protein.

The phage obtained above was infected with E. coli ER2738 cells, which are host cells, and amplified in LB medium. Then, 50 phage plaques were selected to isolate and purify M13 phage genomic DNA (single stranded circular DNA). The amino acid sequence of the peptide portion expressed on the phage surface protein (gp3 minor coat protein) and manufactured to bind bcl-2 was identified. The results are shown in Table 1 below. Through this Clustal X sequence analysis program, a similarity diagram based on mutual homology was prepared, and all seven groups were arranged and the results are summarized in FIG. 7.

[Table 1]

SEQ ID NO: Peptide sequence One AFTHEAPKRRDS 2 SFPGPASARAGA 3 QIEESFVRGHTT 4 SLYKLRNVAAGR 5 CRWSTPTTSQCP 6 IHWSWTTVERPH 7 WPANIILSHGPKH

In addition, as a result of amino acid homology analysis of the peptides of SEQ ID NOS: 1 to 7, as shown in Figure 8 showing the sequencing of the classified seven groups, high similarity can be observed in the sequences of the peptides belonging to the same group, The colored amino acids were found to be conserved in all peptides in the same group. The results suggest that these amino acids are residues that play an important role in binding the bcl-2 protein to the peptide.

Example  3: bcl Combined with -2 Of peptide  Binding force analysis

For avidity analysis of the bcl-2 target specific peptides obtained by screening, amplify each phage expressing phage and determine the concentration of the phage solution through titering, and then by concentration on the bcl-2 bound plate The binding capacity of the peptide was inferred by measuring the amount of phage bound to each other.

Phages were placed on bcl-2 bound plates and washed to carry out ELISA (Biotrak multiwell plate reader, Amersham Bioscience, USA) using antibodies that recognize phage surface proteins. The ELISA signal intensity according to the concentration was indicated and the apparent Kd value was determined according to the saturation curve.

Kd values were obtained using known methods (Kim JM et . Al , (2006) Journal of Microbiolgy and Biotechnology 16, 1784-1790.). Specifically, bcl-2 protein (0.5 g / well) was fixed on a polystyrene plate (SPL), and then amplified phage and fixed for 1 hour. After washing 5 times with 1 X TBST buffer, anti-M13 antibody (mouse monoclonal antibody, diluted 1: 5000 in TBST, Amersham Bioscience) was attached for 1 hour. The antibody-labeled protein was then washed 10 times and the secondary antibody bound (anti-mouse IgG-HRP, diluted 1: 4000 in TBST, Santascruz) and then TMB substrate solution (3,3 '5,5). After 15 minutes with '-tetramethylbenzidine (TMB) / H ₂ O ₂ , Chemicon), 1M sulfuric acid was added to terminate the reaction, and the saturation curve was determined using the measured value at 450 nm and the Kd value was determined.

As a result, as shown in FIG. 9 and FIG. 10, six peptides among the seven peptide groups showed a binding force in the range of picomolar concentration (pM). In addition, some peptides showed excellent binding strength of 10 pM or less, thereby confirming the binding force corresponding to that of the existing antibody-antigen reaction.

Figure 1 shows a recombinant expression vector map of pGEX-KG according to an embodiment of the present invention.

Figure 2 shows the results of production, induction, isolation and purification of the bcl-2 protein cloned in a recombinant expression vector according to an embodiment of the present invention: M, molecular weight marker; BI, before induction; AI, after induction; S1, soluble protein; S2, cells cultured and ground into sonication; FT, Frowthrough; W1, meticulous with PBST buffer; Washed with W2, PBS buffer; Fractions comprising F1-8, bcl-2 protein.

Figure 3 is an electrophoresis result indicating that GST-free protein was obtained before phage display: M, molecular weight marker; P, pellets; Thr, no protein (1 U / ml).

Figure 4 is an experimental result of Western blot after thrombin treatment to confirm the separation of GST-tag and bcl-2: the membrane was labeled with anti-bcl-2 and anti-GST antibody and the delivered product is indicated by the arrow.

5 is a schematic of M13 phage screening for screening peptides that bind bcl-2.

FIG. 6 is a table summarizing the five-step biopanning conditions for detecting phage expressing peptides having high specificity and avidity for bcl-2.

7 is amplification of the phage binding to bcl-2 obtained through the five-step bio-panning of Figure 6 to perform a sequencing after separation and purification of DNA to compare the amino acid sequence of the peptide expressed on the surface of the phage and homology Based on this, the result of creating a similar schematic is shown.

8 is a view showing the conserved region in each peptide as a result of the amino acid sequencing of the seven peptides of the present invention classified as a result of homology analysis.

Figure 9 is a graph of amplifying the seven peptide expression phage of the present invention and then measured the concentration to analyze the bcl-2 and binding force.

Figure 10 summarizes the binding peptide sequence and the binding force of the peptide to bind bcl-2 obtained through the present invention.

<110> Industry-University Cooperation Foundation Hanyang University <120> PEPTIDES TARGETING THE BCL-2 PROTEIN AND USES THEREOF <160> 15 <170> Kopatentin 1.71 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 1 targeting the bcl-2 <400> 1 Ala Phe Thr His Glu Ala Pro Lys Arg Arg Asp Ser   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 2 targeting the bcl-2 <400> 2 Ser Phe Pro Gly Pro Ala Ser Ala Arg Ala Gly Ala   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 3 targeting the bcl-2 <400> 3 Gln Ile Glu Glu Ser Phe Val Arg Gly His Thr Thr   1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 4 targeting the bcl-2 <400> 4 Ser Leu Tyr Lys Leu Arg Asn Val Ala Ala Gly Arg   1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 5 targeting the bcl-2 <400> 5 Cys Arg Trp Ser Thr Pro Thr Thr Ser Gln Cys Pro   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> peptide 6 targeting the bcl-2 <400> 6 Ile His Trp Ser Trp Thr Thr Val Glu Arg Pro His   1 5 10 <210> 7 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> peptide 7 targeting the bcl-2 <400> 7 Trp Pro Ala Asn Ile Ile Leu Ser His Gly Pro Lys His   1 5 10 <210> 8 <211> 144 <212> DNA <213> Homo sapiens <400> 8 aattcccttc tgaaagaaac gaaagcaaca ggaacactca tttgtgccat tgtgttttgc 60 cccctcctcg acccctgggg ccagggaacc ctggtcaccg tctcctcagg tgagtcctca 120 ccaccccctc tgagtccact tagg 144 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucloetide of peptide 1 <400> 9 agaatccagc ctcttaggcg cctcatgcgt aaacgc 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 2 <400> 10 agccccagcc cgagcagaag ccggaccagg aaacga 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 3 <400> 11 agtagtatgc ccacgaacaa acgactcctc aatctg 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 4 <400> 12 cctacccgca gccacattag gaagcttata cagaga 36 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 5 <400> 13 aggacactga gacgtcgtcg gcgtactcca cagaca 36 <210> 14 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 6 <400> 14 atgaggccgc tccacagtcg tccaagacca atgaat 36 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> nucleotide of peptide 7 <400> 15 atgcttagga ccatgagaca aaatattcgc aggcca 36

Claims (12)

Peptide for targeting bcl-2 protein consisting of the amino acid sequence of SEQ ID NO: 6. According to claim 1, wherein the bcl-2 protein is a protein targeting peptide, characterized in that the protein encoded from the gene having the nucleotide sequence of SEQ ID NO: 8. A polynucleotide encoding the amino acid sequence of claim 1. According to claim 3, wherein the polynucleotide is a polynucleotide, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 14. Recombinant expression vector comprising the polynucleotide of claim 3. Transformant transformed with the expression vector of claim 5 delete delete delete delete delete delete

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