KR20120132455A - Peptides targeting the bcl-2 protein and uses thereof - Google Patents

Abstract

PURPOSE: A Bcl-2 protein target peptide and a kit for diagnosing cancer using the same are provided to identify excessive activation of Bcl-2 proteins and to diagnose cancer. CONSTITUTION: A Bcl-2 protein target peptide contains an amino acid sequence selected from sequence numbers 1-5 and 7. The Bcl-2 protein contains a gene with a base of sequence number 8. A polynucleotide encoding the amino acid has a base selected from sequence numbers 9-13 and 15. A recombinant expression vector contains the polynucleotide. A transformant is prepared by transforming with the expression vector containing the polynucleotide. [Reference numerals] (AA) Number; (BB) Repeat frequency; (CC) Peptide sequence; (DD) Dissociation constant

Description

ｂｃｌ?2 Protein targeting peptide and its use {PEPTIDES TARGETING THE BCL-2 PROTEIN AND USES THEREOF}

The present invention relates to a peptide for targeting bcl-2 protein, a cancer diagnostic kit and a drug delivery composition using the same. More specifically, the present invention relates to a diagnostic probe low-molecular peptide specifically binding to a cancer-inducing marker bcl-2 protein, a cancer diagnostic kit and a drug delivery material using the same.

Cells, which are the smallest units that make up the human body, divide, grow, die and disappear by intracellular regulatory functions when normal, maintaining the balance of the number of cells. If a cell is damaged for some reason, it recovers through treatment and plays a role as a normal cell, but if it is not recovered, it dies on its own. However, for various reasons, a condition in which abnormal cells that are not controlled for such proliferation and inhibition not only proliferate excessively, but also invade surrounding tissues and organs, resulting in the formation of masses and destruction of normal tissues, is defined as cancer. Cancer is such a proliferation of cells that cannot be suppressed, destroying the structure and function of normal cells and organs, so the importance of diagnosis and treatment is very great.

Apoptosis, called “cell death”, is distinguished from necrosis, in which cells die from necrosis or pathological causes in a way that cells are controlled by genes and die. Apoptosis plays a role in the composition of the body in the process of development, and after growing up, unnecessary parts are removed to maintain homeostasis. In other words, it refers to a natural process in which unwanted cells are removed due to physiologically scheduled cell death. However, if apoptosis does not occur in the area where apoptosis should occur, or excessive cell death occurs, it leads to various diseases. Currently, studies on the relationship with apoptosis in the development and progression of tumors are actively being conducted.

Among the proteins involved in this apoptosis is the bcl-2 protein. When this bcl-2 protein has excessive activity, apoptosis is suppressed and the mutant cell (tumor) proliferates and eventually develops into cancer cells. In the early stages of cancer, there are often no special symptoms, and because the symptoms are non-specific, it is difficult to distinguish them from other diseases.

For the above reasons, there is a need for research on proteins that regulate apoptosis pathways. The bcl-2 family, a protein that regulates the apoptosis pathway, exists in various types as proteins that act on the intrinsic apoptosis pathway by mitochondria. Depending on the type of protein, the bcl-2 family may cause apoptosis or anti-apoptosis.

More specifically, apoptosis is mainly regulated by bcl-2 family proteins, and the bcl-2 family largely inhibits apoptosis (bcl2, bclxl, bclw, Mcl1, A1) and apoptosis-inducing proteins. It can be divided into proteins (bad, PUMA, H, bcl-G, Noxa, bim, bcl-rambo, bax, bik, bid, bmf). These bcl-2 family members bind to 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 determining the degree of apoptosis response. They mainly act in the mitochondria, and the pro-apoptosis regulator causes apoptosis by being involved in the outflow of cytochrome C. Pro-survival, on the contrary, plays a role in preventing apoptosis by preventing leakage. In other words, it plays the opposite role depending on which protein acts by which signal.

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

As such, apoptosis is completed by a series of activities and reactions of various regulatory factors within the cell. Among them, bcl-2 protein, which inhibits apoptosis, exists in the endoplamic reticulum, mitochondria and nuclear membrane, along with cytokines. It inhibits apoptosis and plays an important role in the replacement of normal cells. The bcl-2 family protein is normally bound to the BH3 subfamily bid/bim, and bax and bak are present in the outer mitochondrial membrane in a state in which nothing is bound. When apoptosis signal comes in, bik or bad binds to bcl-2, and bid/bim separated from bcl-2 binds to bak and Bax to induce conformational change and induce apoptosis. Let it. In other words, bcl-2 blocks apoptosis by blocking the apoptosis mechanism of the bax subfamily and the BH3 subfamily. When apoptosis is suppressed, the chance of clonal expansion, secondary genetic changes and malignant transformation increases, resulting in tumors.

The association between cancer prognosis and apoptosis has been studied in several types of carcinoma. In most carcinomas such as breast cancer and lung cancer, abnormal expression of bcl-2 protein, which inhibits apoptosis, increases resistance to anticancer drugs, and in many cases, it has been reported that the patient's survival time is short and the tumor is progressing. Although it is known that there is no significant structural abnormality of the bcl-2 gene, the bcl-2 protein expression is increased in solid cancer, so studies on the correlation between the bcl-2 protein expression and carcinoma are ongoing.

In other words, cancer cells over-express the bcl-2 protein to inhibit cell death, resulting in excessive proliferation. In many solid cancers, increased expression of bcl-2 protein is known to protect cancer cells from apoptosis-inducing mechanisms such as anticancer agents of tumor cells, so further studies are needed to support this part. Therefore, this bcl-2 protein corresponds to a cancer-causing biomarker, and it is necessary to discover a peptide as a diagnostic probe that effectively recognizes it. Through these peptides, a diagnostic kit for early cancer diagnosis can be developed, and it can be used as an independent prognostic factor for cancer. In addition, when a drug that actually blocks cancer cells is used for development, a new concept of broad-spectrum anticancer drugs is expected to be prepared.

The combination of target-recognizing probes, imaging technology through various new imaging, and anti-cancer substances provides an opportunity to improve treatment for early detection of cancer and develop potential diagnostic methods. In order to develop a probe for early detection of cancer, it is necessary to discover a probe with high affinity and selectively attached to early cancer cells and use it for early diagnosis of cancer.

Also, many studies are being conducted in the field of genes. Based on these genetic analysis results, genetic modification studies are underway to disrupt the function of cancer genes or prevent them from being expressed.

According to the prior art, there is a method of using PCR (Polymerase Chain Reaction) as the most common method for diagnosing a subject to be diagnosed. This method analyzes the unique gene possessed by the diagnosed subject, and then analyzes it in vitro ( in In vitro ) amplification is a method of analyzing a small amount of a diagnosis subject. In addition, this method uses the unique nucleotide sequence of the gene to analyze the unique nucleotide sequence of the subject to be diagnosed. This PCR method can expand its application range by securing an extensive genetic information database through recent human genome projects. In addition, the PCR method includes SSCP (Single-strand conformation polymorphism), RFLP (Restriction fragment length polymorphism), and AFLP (Amplified fragment length polymorphism) methods, which identify genetic variations of genes obtained by amplification using various probes. It provides useful information in connection.

However, in the case of the diagnostic method using PCR, it has the advantage of having high sensitivity in obtaining a small amount of the unique nucleotide sequence possessed by only the subject to be diagnosed, but there are problems such as difficulty in pretreatment and field adaptability of the sample. There are limits to use.

Cancer markers, which can be screened for cancer and detected early, are being conducted through studies on antibodies that can identify the early occurrence of tumors. Blood is the most representative body fluid, and it is like a kind of'barometer in living body' whose components change sensitively during disease onset and progression. Plasma proteins are predicted to contain more than 50,000 constituents, and the concentration of each protein component is also very dynamic (1-10 ^-12 mg/mL), so the detection limit is about 10 ^-6 -10 ^-9 mg/mL. It is very difficult to detect and quantify biomarker proteins present in low/medium concentrations by antigen-antibody reaction. However, a diagnostic kit with high sensitivity, rapidity and field adaptability, and a diagnostic probe having characteristics such as high physicochemical stability, sensitivity, specificity, and applicability, have not yet been proposed.

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

The recent ELISA (Enzyme-Linked Immunosorbent Assay) is a method to determine the increase and change in the expression level of a specific protein caused by a disease. In order to recognize the specific protein, an antibody that specifically binds to these specific proteins is used as a diagnostic probe to isolate a diagnosis subject from a biological sample such as blood or urine, and for this purpose, an enzyme-antibody reaction (ECL) is used. However, despite the advantage that the nanomolar (10 ^-9 M) level of sensitivity can target proteins, which are active points in vivo, there is a disadvantage in that it has a lower sensitivity than the PCR diagnostic method.

As part of a related research that can overcome the above-mentioned disadvantages, surface-enhanced Raman spectroscopy (SERS) and bio-barcode technology with nanotechnology (NT) based on the current diagnostic methods such as PCR and ELISA As it was developed, it was proved that the analysis of diagnostic subjects at the level of ^{femtomol (10 -15 M) is experimentally possible.} Both methods have signal characteristics of Raman spectroscopy (narrow Raman band; low sensitivity to moisture, oxygen and soakers; and low photobleaching degree) and high sensitivity corresponding to fluorescence analysis. Since the amplification enabled recognition of a diagnosis subject at a femtomole level, an approach to cancer diagnosis (Prostate specific antigen, PSA) using a sample sample such as blood was proposed. However, these methods are based on a system using an antibody that recognizes a biomarker (diagnostic object), and all of these methods not only limit the number of binding but also various chemical reactions due to size limitations due to the physical and chemical structure of the antibody. There is a significant drawback of showing instability due to structural degeneration under

Therefore, there is a need to develop a new small molecule diagnostic probe differentiated from the above-described antibody diagnostic system technology. Low molecular weight peptides can be used in such a low molecular weight diagnostic system.

Accordingly, the present inventors have come to develop a new peptide as a new diagnostic material that can replace the antibody currently used for cancer diagnosis.

An object of the present invention is to provide a novel peptide that recognizes and specifically binds to a cancer-inducing protein 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 is to provide a composition for drug delivery comprising the peptide.

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

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

In the present invention, a low molecular weight peptide is used as a target material in order to overcome the aforementioned disadvantages of the antibody. These low-molecular peptides have the advantage of being three-dimensionally stabilized because of their small size, easily passing through mucous membranes, and being able to recognize molecular targets even deep in tissues. In addition, since the low-molecular peptide according to the present invention can secure stability and minimize immune reactivity through local injection, there is an advantage in that cancer can be diagnosed early. And the low-molecular peptide according to the present invention is relatively simple in mass production compared to the antibody and has weak toxicity.

In addition, the low-molecular peptide according to the present invention has the advantage of having higher binding power to the target substance than the antibody, and does not denature even during thermal/chemical treatment. In addition, because the molecular size is small, it can be attached to other proteins and used as a fusion protein. Specifically, since it can be used by attaching it 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, and consists of 12 amino acids. This amino acid is a peptide having high affinity for the bcl-2 protein, and includes the amino acids set forth in SEQ ID NO: 1 to SEQ ID NO: 7.

The peptides of the present 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 a genetic engineering method. 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, for example, using an automatic DNA synthesizer (eg, those sold by Biosearch or AppliedBiosystems). The constructed DNA sequence is a vector containing one or more expression control sequences (e.g., promoters, enhancers, etc.) that are operatively linked to this DNA sequence to control the expression of the DNA sequence. And transformed into a host cell with a recombinant expression vector formed therefrom. The resulting transformant is cultured under an appropriate medium and conditions so that the DNA sequence is expressed, and substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using a method known in the art (eg, chromatography). In the above, "substantially pure peptide" means that the peptide according to the present invention does not substantially contain any other protein derived from a host. The genetic engineering method for synthesizing the peptide of the present invention may be referred to the following literature: 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 is bound to a cancer cell tissue, the peptide of the present invention may be provided in a labeled state. That is, it can be provided by linking to a detectable label (eg, covalently bonded or crosslinked). The detectable label is a chromogenic enzyme (e.g., peroxidase, alkaline phosphatase), a radioactive isotope (e.g. ^{, 124} I, ¹²⁵ I, ¹¹¹ In, ^{99 m} Tc, ³² P, ³⁵ S) , Chromophore, luminescent material or fluorescent material (e.g. FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dot dots)), etc.

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

If a fluorescent substance 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 substance 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 an amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 7, a recombinant expression vector containing the polynucleotide, and a transformant transformed with the expression vector.

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

The expression vector refers to a plasmid, viral vector, or other medium known in the art capable of expressing the inserted nucleic acid in a host cell, and the peptide of the present invention is encoded in a conventional expression vector known in the art. The polynucleotide may be operably linked. In general, the expression vector is a replication origin that can proliferate in a host cell, one or more expression control sequences that control expression (eg, promoters, enhancers, etc.), a selective marker, and a bone operably linked to an expression control sequence. It may include a polynucleotide encoding the peptide of the invention.

The transformant may be transformed by the expression vector. Preferably, the transformant uses an expression vector containing a polynucleotide encoding the peptide of the present invention by a method known in the art, for example, but not limited thereto, transient transfection, microinjection, Transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection ( polybrene-mediated transfection), by other known methods for introducing a nucleic acid into electrical invasion process (electropora tion), gene gun (gene gun), and the cells can be obtained by introducing into a host cell (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, and insect cells, It is not limited thereto and all possible cells can be used as host cells.

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

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

The polyclonal antibody can be produced by a method well known in the art by injecting a peptide for targeting bcl-2 protein having the amino acid sequence of SEQ ID NO: 1 to 7 into an animal and collecting blood from the animal to obtain serum containing the antibody. have. For example, it can be prepared by injecting the peptide with Complete Freund's Adjuvant (CFA) by subcutaneous injection into a goat or rabbit, and injecting a booster with CFA subcutaneously or intraperitoneally. Such polyclonal antibodies can be prepared from any animal species host such as sheep, monkey, horse, pig, and cow dog other than goat rabbit.

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

In addition, the present invention provides a kit for diagnosis of 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 that encodes it, and may be obtained by a chemical or genetic engineering method as described above. . The diagnostic kit of the present invention may further include a buffer or reaction solution for stably maintaining the structure or physiological activity of the peptide. In addition, in order to maintain stability, it may be provided in a powder state, dissolved in an appropriate buffer, or maintained at 4°C.

At this time, in the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis is to confirm the presence or character 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 without being labeled, the kit for diagnosis of cancer of the present invention is used to detect whether or not the peptide of the present invention binds to a cancer-causing site, particularly cancer cells, in vitro or in vivo. Ingredients may be further included. The component is a known compound for labeling the peptide of the present invention, or an antibody against a specific receptor binding to the peptide of the present invention or a specific receptor binding to the peptide of the present invention, or a secondary antibody against these, for screening through 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 reacting under appropriate conditions, cancer can be diagnosed by observing the binding of the cancer cells and the peptides on the surface of the plate.

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

In addition, the present invention provides a drug delivery composition comprising the peptide. The peptide of the present invention can be used as an intelligent drug delivery system that selectively delivers drugs such as anticancer agents to cancer tissues. If the peptide of the present invention is linked to a conventional anticancer agent and used for cancer treatment, the effect of the drug can be increased because the anticancer agent is selectively delivered only to cancer cells by the peptide of the present invention, and at the same time, the side effects of the anticancer agent on normal tissues can be significantly reduced. I can.

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

The linkage of the anticancer agent and the peptide of the present invention may be performed through a method known in the art, such as covalent bonding, crosslinking, and the like. To this end, the peptide of the present invention can be chemically modified, if necessary, within a range in which its activity is not lost.

The amount of the peptide of the present invention contained 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 sufficiently delivered to the cancer tissue. However, the effective dosage for the patient is determined by considering various factors such as the age, weight, health condition, sex, severity of the disease, diet and excretion rate of the drug, as well as the route of administration and the number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective amount of the peptide of the present invention in order to treat cancer by combining the peptide with an anticancer agent.

The composition of the present invention may contain an appropriate buffer solution for maintaining and/or preserving the stability of the peptide. The composition for drug delivery comprising the peptide according to the present invention is not particularly limited in its formulation, route of administration, and method of administration, as long as it exhibits the effect of the present invention. For example, the peptide of the present invention may be administered orally or parenterally. When administered orally, the peptide of the present invention is preferably composed of L-type amino acids in order to prevent degradation by digestive enzymes in the gastrointestinal tract. The parenteral administration includes subcutaneous injection, intramuscular injection, intravenous injection, and urethral injection.

In addition, the composition of the present invention may further include a pharmaceutically acceptable carrier that is usually added to a general pharmaceutical composition. The term "pharmaceutically acceptable" means that it is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans or animals. As a pharmaceutically acceptable carrier, in the case of injection, for example, a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, and a stabilizer may be used.

As described above, the formulation of the composition comprising the peptide of the present invention can be prepared in various ways as described above. For example, in the case of injection, it can be prepared in the form of a unit dosage ampoule or a multi-dose dosage form. The drug delivery composition comprising the peptide of the present invention can be administered by a conventional drug administration method 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 substance. When the peptide of the present invention is linked to a drug such as an anticancer agent and used for cancer treatment, the anticancer agent is selectively delivered to the cancer tissue to increase the effect of the drug, and there is almost no side effects such as an immune response or a degenerative reaction.

1 shows a map of the recombinant expression vector of pGEX-KG according to an embodiment of the present invention.
Figure 2 shows the production, induction, isolation and purification results of the bcl-2 protein cloned into the 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, cultured and sonicated cells; FT, Frowthrough; W1, washed with PBST buffer; Washed with W2, PBS buffer; Fractions containing F1-8 and bcl-2 proteins.
3 is an electrophoresis result showing that GST-free protein was obtained prior to phage display: M, molecular weight marker; P, pellet; Thr, no protein (1 U/ml).
Figure 4 shows the experimental results 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 antibodies, and the delivered products were indicated by arrows.
5 is a schematic diagram of M13 phage screening for screening for a peptide that binds to bcl-2.
FIG. 6 is a diagram showing a five-step bio-panning condition for detecting a phage expressing a peptide having high specificity and avidity for bcl-2.
7 is amplified the phage binding to bcl-2 obtained through the 5-step biopanning of FIG. 6, separated and purified DNA, followed by sequencing to compare and analyze the amino acid sequence of the peptide expressed on the phage surface, and determine homology. It shows the result of creating a similar tree diagram based on it.
8 is a diagram showing a conserved region in each of the peptides as a result of performing amino acid sequence analysis on the seven peptides of the present invention classified as a result of homology analysis.
9 is a graph showing the amplification of the seven peptide-expressing phages of the present invention and then measuring the concentration to analyze the binding strength with bcl-2.
10 is a summary of the binding peptide sequence and avidity of the peptide binding to bcl-2 obtained through the present invention.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to the engineer, and it is natural that such modifications and modifications fall within the appended claims.

Example One: bcl -2 Mass production and isolation of protein

The bcl-2 gene (SEQ ID NO: 8) was amplified through PCR as follows: 5'-CTGGTGGACAACATCGCTCTG-3' was used as the Bcl-2 sense primer, and 5'-GGTCTGCTGACCTCACTTGTG-3' was used as the Bcl-2 antisense primer. . In a reaction solution containing 5% DMSO containing the synthetic primer and template DNA, pre-denaturation 94° C. 2 minutes, denaturation 95° C. 45 seconds, annealing 55° C. 1 The template DNA obtained after 28 cycles of PCR reaction was confirmed through gene nucleotide sequence with 1 cycle at 68°C for 3 minutes. 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 this expression vector was transformed into E. coli BL21 (DE3) for protein expression as an expression host. After conversion, the production of bcl-2 protein was induced for 5 hours under conditions of 0.5 mM IPTG and 37°C. A brief description of the transformation method is as follows: First, 5 µl of DNA was added to 100 µl of competent cells, mixed well, and placed on ice for 30 minutes. Then, heat shock was applied at 42° C. for 90 seconds. 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 an incubator at 37°C.

And as a result of examining the solubility of proteins by harvesting bacterial cells through centrifugation, it was confirmed that most of the proteins were dissolved. Therefore, for the separation and purification of bcl-2, a GST fusion protein, a water-soluble protein was obtained, passed through a GSH-sepharose column, and bound to the bcl-2 protein, followed by a 20 mM reduced form of glutathione (GSH) linear gradient. gradient).

The obtained protein was removed from excess GSH using a disalting column (Amersham Biosciences, USA), and the degree of purification 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 must be removed in order to proceed with the screening process to find a peptide that binds only to the bcl-2 protein. Peptides that specifically bind only to the bcl-2 protein can be found. For this, the GST-tag was cut using thrombin (5 unit of thrombin/mg of protein). When thrombin treatment was performed, the GST-fused bcl-2 protein was divided into two fragments, GST-tag (26KDa) and bcl-2 (25KDa), so the bcl-2 protein separated and purified by the above method was treated with thrombin. It was confirmed that the same result was obtained. As a result of thrombin treatment, it was confirmed that two protein fragments, GST-tag and bcl-2, were formed. The reaction was terminated by adding 1 mM benzamidine. To remove the GST-tag and obtain only pure GST-free protein, GST-free protein was obtained with a linear gradient of 0.5M NaCl through anion exchange chromatography (Mono Q HR 5/5 column) (F5 in FIG. 3). Since the size of GST-tag and bcl-2 are similar, it is difficult to accurately identify it on the gel. Therefore, to confirm the bcl-2 protein, the reaction product was dissolved in 15% SDS/PAGE, and a Western blot method was used.

Specifically, a 15% SDS/PAGE gel was made. And each sample was hung on the gel. Protein was transferred to the NC membrane (transfer). When the migration was over, the protein bands transferred to the NC membrane were stained with a staining solution (Ponceaus S) and marked with a marker. Then, the membrane was washed with water and blocked with 2% BSA while shaking at 50 rpm at room temperature for 1 hour. The primary antibody (Anti-bcl-2, Rabbit polyclonal Ab (1:2000 dilution; Santacruz, USA)) was applied at room temperature for 1 hour. Then, the membrane was washed several times at room temperature for 1 hour with 1X TBST buffer. A secondary antibody (Anti-Rb IgG HRP conjugate (1:4000 dilution; Santacruz, USA) was applied at room temperature for 1 hour. Once again, the membrane was washed several times at room temperature for 1 hour with X TBST buffer. ECL (Chemiluminescence) solution was sufficiently sprayed on the membrane and reacted with the secondary antibody to emit light Finally, the film was printed to check whether a band of the desired size appeared. 2 It was confirmed that only one protein fragment was seen (FIG. 4).

Example 2: M13 scrap paper Peptide 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 plate surface made of polystyrene was used. By immobilizing the protein on the bottom of the plate surface using hydrophilic interaction between the protein and the plate surface, a random peptide library (made to have about 2.7 billion various amino acid sequences through a random arrangement of 12 amino acids) Peptide library-Ph.D ^TM phage display peptide library kit, purchased and used from New England Biolabs (NEB)) was designed to enable higher specificity and avidity analysis in the screening process. The combination is shown in FIG. 5.

Specifically, in Figure 5, in order to screen for a peptide that binds to the cancer-causing marker bcl-2, after binding bcl-2 to the surface of a plate (Qiagen), an M13 phage peptide library (about 2.7 billion different amino acid sequences Peptide expression that binds with high affinity through a variety of binding times and washing conditions after binding of the peptide consisting of 12 amino acids with the M13 phage gp3 minor coat protein fused library) and binding (the binding method of the M13 phage and peptide library) Phage was selected. The screening plan and reaction conditions related to this are shown in Figs. 5 and 6, respectively.

Specifically, FIG. 5 is a schematic diagram of M13 phage screening for screening for peptides that bind to bcl-2. (1) bcl-2 protein is immobilized on the surface of a 96-well plate, and (2) 12 on the surface of M13 phage. The phage library expressing the peptide library consisting of dog amino acids was combined with the bcl-2 protein, (3) washed under various conditions, and (4) the finally bound phage was eluted to obtain phage.

The process of finding a phage having a stronger binding force by infecting the phage obtained as described above with E. coli and amplifying it, binding it with bcl-2 protein again, and repeating it under conditions such as higher strength washing conditions and shorter reaction time. Was repeated (biopanning).

In addition, FIG. 6 is a diagram showing five-step bio-panning conditions for detecting a phage expressing a peptide having high specificity and avidity for bcl-2 protein. The reaction conditions were varied with strong washing conditions and short reaction times for each step, and accordingly, bio-panning was performed five times each to obtain a phage having a peptide binding to the bcl-2 protein.

The phage obtained by the above was infected with E. coli ER2738 cells, which are host cells, and amplified in LB medium, and then 50 phage plaques were selected to separate and purify M13 phage genomic DNA (single-stranded circular DNA) to obtain the gene sequence. By confirming, the amino acid sequence of the peptide portion produced to bind bcl-2 by being expressed on a phage surface protein (gp3 minor coat protein) was identified. The results are shown in Table 1 below. This is a similar system based on mutual homology through the Clustal X sequence analysis program.

A diagram was prepared to organize all 7 groups, and the results are summarized in FIG. 7.

Sequence number Peptide sequence One AFTHEAPKRRDS 2 SFPGPASARAGA 3 QIEESFVRGHTT 4 SLYKLRNVAAGR 5 CRWSTPTTSQCP 6 IHWSWTTVERPH 7 WPANIILSHGPKH

In addition, as can be seen in FIG. 8 showing the sequence analysis of the seven classified groups as a result of amino acid homology analysis of the peptides of SEQ ID NOs: 1 to 7, high similarity can be observed in the sequence of the peptides belonging to the same group. It was confirmed that the indicated amino acids were conserved in all of the peptides within 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 corresponding peptide.

Example 3: bcl Combined with -2 Peptide Cohesion analysis

In order to analyze the binding strength of the bcl-2 target-specific peptide obtained by screening, amplify the phage expressing each peptide, and then determine the concentration of the phage solution through titering. The binding power of the peptide was inferred by measuring the amount of phage bound by putting it in.

The phage was put in the bcl-2 conjugated plate and washed to perform ELISA (Biotrak multiwell plate reader, Amersham Bioscience, USA) using an antibody that recognizes the phage surface protein. The ELISA signal intensity according to the corresponding concentration was displayed and the Kd value was determined by fitting it to the saturation curve.

The Kd value was obtained using a known method (Kim JM et . al , (2006) Journal of Microbiolgy and Biotechnology 16, 1784-1790.). Specifically, bcl-2 protein (0.5 g/well) was immobilized on a polystyrene plate (SPL), and the amplified phage was added thereto, followed by fixing 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 added for 1 hour. Then, the antibody-attached protein was washed 10 times, and then the secondary antibody was conjugated (anti-mouse IgG-HRP, diluted 1:4000 in TBST, Santascruz), and then TMB substrate solution (3,3' 5,5 '-tetramethylbenzidine (TMB)/H ₂ O ₂ , Chemicon) was added and 1M sulfuric acid was added after 15 minutes to terminate the reaction, and a saturation curve was drawn with the value obtained by measuring at 450 nm, and the Kd value was determined.

As a result, as shown in Figs. 9 and 10, 6 of the 7 peptide groups show avidity in a picomolar concentration (pM) range. In addition, some peptides showed excellent binding power of 10 pM or less, thereby confirming the binding power corresponding to the binding power of the existing antibody-antigen reaction.

<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 (6)

Peptides for bcl-2 protein targeting consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 and 7.
According to claim 1, wherein the bcl-2 protein is a protein targeting peptide, characterized in that consisting of the gene having the nucleotide sequence of SEQ ID NO: 8.
A polynucleotide encoding the amino acid sequence of claim 1.
The polynucleotide of claim 3, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 13 and 15. 5.
Recombinant expression vector comprising the polynucleotide of claim 3.
A transformant transformed with the expression vector of claim 5.

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