KR20150010882A - Fusion protein comprising annexin A2 binding protein, p53 and p18, and composition comprising the fusion protein for preventing or treating cancer - Google Patents
Fusion protein comprising annexin A2 binding protein, p53 and p18, and composition comprising the fusion protein for preventing or treating cancer Download PDFInfo
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- KR20150010882A KR20150010882A KR20130085513A KR20130085513A KR20150010882A KR 20150010882 A KR20150010882 A KR 20150010882A KR 20130085513 A KR20130085513 A KR 20130085513A KR 20130085513 A KR20130085513 A KR 20130085513A KR 20150010882 A KR20150010882 A KR 20150010882A
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Abstract
Description
아넥신 A2 결합단백질, p53 단백질 및 p18 단백질이 결합된 융합 단백질, 및 상기 융합 단백질을 포함하는 암의 예방 또는 치료용 약학 조성물에 관한 것이다.A fusion protein in which an annexin A2 binding protein, a p53 protein and a p18 protein are bound, and a pharmaceutical composition for preventing or treating cancer comprising the fusion protein.
세포가 증식하기 위해서는 G1기(G1 phase), DNA 복제기(S phase), G2기(G2 phase) 및 세포분열기(M phase)를 거치는 일련의 단계를 순환하는데, 이를 세포주기(cell cycle)라 한다. 세포분열기는 유사분열과 세포질분열을 거쳐 세포가 증식하는 실질적인 단계이고, DNA 복제기에서는 DNA가 복제되고 세포수는 변하지 않는다. 따라서, 세포주기에서 세포분열기를 제외한 DNA 복제기, G1기, G2기를 간기(interphase)라고 한다. G1기와 G2기는 세포주기를 조절하는 체크포인트(checkpoint)를 포함하는데, 이는 세포가 비정상적으로 증식하지 않도록 하는 중요한 역할을 한다. 체크포인트에서 세포주기의 다음 단계로 넘어가기 위한 조건이 충족되었는지를 확인한 후, 모든 조건이 만족되면 DNA 복제기 또는 세포분열기가 진행된다. 이러한 체크포인트에서의 세포 증식 조절이 제대로 이루어지지 않으면 암과 같은 비정상적인 세포 증식을 유발한다.In order for the cells to proliferate, they cycle through a series of steps through the G1 phase, the DNA replicator (S phase), the G2 phase and the cell divider (M phase), which is called the cell cycle . A cell divider is a practical stage in which cells multiply through mitosis and cytoskeleton, DNA is replicated in the DNA replicator, and the number of cells does not change. Therefore, in the cell cycle, the DNA replicators except the cell divider, G1 and G2 are called interphase. The G1 and G2 groups contain checkpoints that control the cell cycle, which plays an important role in preventing cells from abnormally proliferating. After confirming that the conditions for progressing from the checkpoint to the next stage of the cell cycle are met, the DNA replicator or cell divider progresses if all conditions are met. Failure to properly control cell proliferation at these checkpoints leads to abnormal cell proliferation, such as cancer.
G1 체크포인트를 지나고, DNA 복제기, G2 체크포인트를 지나서 유사분열을 하기 위해서는 CDK(cyclin-dependent kinase) 단백질의 활성이 필요하다. CDK는 단독으로 활성을 가질 수 없으며 Cyclin 단백질과 결합되어야 키나아제 활성을 갖게 된다. Cyclin-CDK 복합체는 세포증식에 관련된 단백질들을 인산화시켜 세포 분열을 유도한다. DNA 복제기를 유도하는 단백질들은 주로 Cyclin D/E, CDK 4/6이고, CDK 4/6은 Rb(Retinoblastoma) 단백질을 인산화시켜서 전사 인자인 E2F의 전사 활성을 갖게 한다. 세포분열기를 유도하는 단백질들은 주로 Cyclin A/B, CDK 2/1이며, 이들의 Cyclin-CDK 복합체는 MPF(M-phase promoting factor)라고 일컫는다. 유사분열이 완료되거나 DNA 복제가 완료되면 더 이상의 키나아제 활성이 필요하지 않으므로 Cyclin-CDK 복합체의 Cyclin이 분해되어 CDK의 활성이 사라진다.Activation of the cyclin-dependent kinase (CDK) protein is required to cross the G1 checkpoint and to mitigate past the DNA replicator, G2 checkpoint. CDKs can not have activity alone and must be associated with cyclin proteins to have kinase activity. The cyclin-CDK complex induces cell division by phosphorylating proteins involved in cell proliferation. The proteins that induce DNA replication are mainly Cyclin D / E,
세포 주기 조절을 위해 Cyclin-CDK 복합체의 활성을 조절하는 많은 인자들이 존재한다. 대표적인 것으로는 G1 체크포인트에서 DNA가 손상되었을 때 작용하는 p21 단백질이 있다. DNA가 손상되었을 시에 활성되는 종양 억제인자 유전자(tumor suppressor gene)인 p53에 의해 p21 유전자가 발현된다. p21은 DNA 복제기를 유도하는 Cyclin-CDK 복합체에 결합하여 CDK4/6/2의 키나아제 활성을 저해함으로써, Rb 단백질의 인산화를 막는다. 그 결과, 세포는 G1기에 머물면서 손상된 DNA를 수리할 시간을 얻게 된다. 만일 DNA 손상이 세포가 수용하지 못할 정도로 심하면 p53은 아폽토시스 유도 유전자(apoptosis-inducing gene)인 Bax 단백질의 유전자를 발현시킨다. 즉, 세포증식의 조절에 있어서 p53의 작용은 p21 유전자를 발현시켜 세포증식을 일시적으로 정지시키는 것(growth arrest), 또는 p53-결합 단백질인 ASPP 단백질과 결합한 p53 단백질이 Bax 유전자를 발현시켜 아폽토시스를 유발하는 것(apoptosis inducing)의 두 가지로 나누어 볼 수 있다. 아폽토시스는 예정된 세포 죽음(programmed cell death)이라고도 하며 세포가 조직내에서 더 이상 필요 없을 때, 바이러스의 침입을 받았거나 암세포로 변환되었을 때 등, 개체수준에서 세포의 죽음이 오히려 도움이 되면 세포 스스로 자살하는 기작이다. There are many factors that regulate the activity of the cyclin-CDK complex for cell cycle control. Typically, there is a p21 protein that acts when DNA is damaged at the G1 checkpoint. The p21 gene is expressed by p53, a tumor suppressor gene that is activated when DNA is damaged. p21 binds to a cyclin-CDK complex that induces DNA replication, thereby inhibiting the kinase activity of CDK4 / 6/2, thereby preventing phosphorylation of the Rb protein. As a result, the cells stay in the G1 phase and have time to repair the damaged DNA. If the DNA damage is so severe that the cell is unacceptable, p53 expresses the Bax protein gene, an apoptosis-inducing gene. In other words, in the regulation of cell proliferation, the action of p53 is either growth arrest by expressing the p21 gene, or p53 protein coupled with p53-binding protein ASPP protein expresses Bax gene to induce apoptosis And induction of apoptosis (induction of apoptosis can be divided into two. Apoptosis is also called programmed cell death, and when cell death is more helpful at the individual level, such as when the cell is no longer needed in the tissue, when it has been infected with a virus, or when it has been transformed into cancer cells, It is a mechanism.
세포주기를 조절하는 CDK 억제제는 크게 CIP(CDK inhibitor protein) family와 INK4(Inhibitors of cyclin-dependent kinase 4) 두 가지로 분류할 수 있다. CIP 에는 p21과 p27이 있다. INK4는 주로 CDK 4, 6의 활성을 저해하며 p18, p16, p8, p19가 있다. CDK inhibitors that regulate the cell cycle are classified into two classes: CIP (CDK inhibitor protein) family and INK4 (Inhibitors of cyclin-dependent kinase 4). CIP has p21 and p27. INK4 mainly inhibits the activity of
세포 증식을 조절하기 위해서는 종양 억제인자 유전자와 전암 유전자(proto-oncogene)와의 균형이 유지되어야 한다. 종양 억제인자 유전자인 p53 단백질의 유전자가 변이(mutation)되면, 비정상적인 세포의 아폽토시스를 유도할 수 없으므로 비정상적 세포 증식을 유발한다. 또한 정상적인 세포 증식 인자인 전암유전자가 변이(바이러스에 의한 유전자 치환 등)되거나 과발현 되어 암유전자가 되어도 암세포와 같은 비정상적인 세포 증식을 유도한다. 암유전자는 비정상적인 활성을 갖는 성장인자, 세포내 신호전달 단백질을 만들어 잘못된 신호 전달을 유발하기 때문에 외부의 증식 신호가 없어도 세포증식이 일어난다. In order to control cell proliferation, the balance between the tumor suppressor gene and the proto-oncogene must be maintained. Mutation of the p53 protein gene, which is a tumor suppressor gene, can not induce apoptosis of abnormal cells, resulting in abnormal cell proliferation. In addition, the precancerous gene, which is a normal cell proliferative factor, induces abnormal cell proliferation such as cancer cells even if it becomes a cancer gene by mutation (gene substitution by virus, etc.) or overexpression. Cancer genes produce abnormal growth factors, intracellular signaling proteins and cause false signal transduction, so cell proliferation occurs without external proliferation signal.
이러한 사실로 볼 때, 비정상적인 세포 증식으로 인한 암을 저해하기 위해 세포 주기를 조절하는 각종 인자들을 활용한 항암제의 개발이 요구되고 있는 실정이다.In view of this fact, in order to inhibit cancer caused by abnormal cell proliferation, development of an anticancer drug using various factors controlling cell cycle is required.
일 구체예는 아넥신 A2 결합단백질, p53 단백질 또는 그 단편, 및 p18 단백질을 포함하는 융합 단백질을 제공한다.One embodiment provides a fusion protein comprising anexin A2 binding protein, p53 protein or fragment thereof, and p18 protein.
다른 구체예는 상기 융합 단백질을 코딩하는 폴리뉴클레오티드를 제공한다.Another embodiment provides a polynucleotide encoding said fusion protein.
다른 구체예는 상기 폴리뉴클레오티드를 포함하는 재조합 벡터를 제공한다.Another embodiment provides a recombinant vector comprising said polynucleotide.
다른 구체예는 상기 재조합 벡터로 형질전환된 세포를 제공한다.Another embodiment provides a cell transformed with said recombinant vector.
다른 구체예는 상기 형질전환된 세포를 배양시키는 단계를 포함하는 융합 단백질의 제조방법을 제공한다.Another embodiment provides a method of producing a fusion protein comprising culturing the transformed cells.
다른 구체예는 상기 융합 단백질을 유효성분으로 포함하는 암의 예방 및/또는 치료용 약학 조성물을 제공한다.Another embodiment provides a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient.
다른 구체예는 상기 융합 단백질의 치료적 유효량을 개체에 투여하는 단계를 포함하는 암 치료방법을 제공한다.Another embodiment provides a method of treating cancer comprising administering to the individual a therapeutically effective amount of the fusion protein.
본 발명의 일 예는 아넥신 A2 결합단백질, p53 단백질 또는 그 단편, 및 p18 단백질을 포함하는 융합 단백질을 제공한다. 상기 융합 단백질은 생체외 안정화 단백질 (in vitro stabilization protein), 막 투과 서열 (membrane transfer sequence, MTS) 도메인, 핵-세포질 신호 (nucleus-cytoplasm signal) 도메인, ATT 및 STOPx2 로 이루어진 군에서 선택된 1종 이상을 추가로 포함할 수 있다.An example of the present invention provides a fusion protein comprising anexin A2 binding protein, p53 protein or fragment thereof, and p18 protein. The fusion protein may comprise at least one selected from the group consisting of an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nucleus-cytoplasm signal domain, ATT and STOPx2 May be further included.
또 다른 예는 상기 융합 단백질을 코딩하는 폴리뉴클레오티드를 제공한다.Another example provides a polynucleotide encoding the fusion protein.
또 다른 예는 상기 폴리뉴클레오티드를 포함하는 재조합 벡터를 제공한다.Another example provides a recombinant vector comprising the polynucleotide.
또 다른 예는 상기 재조합 벡터로 형질전환된 세포를 제공한다.Another example provides a cell transformed with the recombinant vector.
또 다른 예는 상기 형질전환된 세포를 배양시키는 단계를 포함하는 융합 단백질의 제조방법을 제공한다.Another example provides a method for producing a fusion protein comprising culturing the transformed cells.
또 다른 예는 상기 융합 단백질을 유효성분으로 포함하는 암의 예방 및/또는 치료용 약학 조성물을 제공한다. 상기 약학 조성물은 필요에 따라서 약학적으로 허용되는 담체, 희석제 및/또는 부형제를 통상적으로 사용되는 양으로 추가로 포함할 수 있다.Yet another example provides a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent and / or excipient in an amount normally used.
앞서 설명한 바와 같이, CDK (Cyclin-dependent kinase) 단백질은 암 발생에 주요한 역할을 하고, 이의 억제제로서 CIP(CDK inhibitor protein) 계통 단백질과 INK4(Inhibitors of cyclin-dependent kinase 4) 계통 단백질들이 있다. 본 발명의 일 구체예에 따른 융합 단백질은 CIP 계통 단백질 발현을 조절하는 p53 단백질과 INK4 계통 단백질에 속하는 p18 단백질이 연결된 것으로, CIP 계통 단백질 활성과 INK4 단백질 활성을 동시에 발휘하여 보다 우수한 암의 예방 및/또는 치료 효과를 얻을 수 있다. As described above, CDK (cyclin-dependent kinase) protein plays a major role in cancer development, and its inhibitors include CIP (CDK inhibitor protein) system protein and INK4 (Inhibitors of cyclin-dependent kinase 4) system protein. The fusion protein according to one embodiment of the present invention is a fusion protein of a p53 protein that regulates the expression of CIP lineage protein and a p18 protein that belongs to INK4 lineage protein and exhibits both CIP system protein activity and INK4 protein activity, / / Therapeutic effect can be obtained.
일 구체예에 따르면, 상기 p53 단백질은 NCBI accession number NP_000537에 의하여 제공되는 아미노산 서열을 갖는 폴리펩타이드 또는 그 단편일 수 있다. 본 명세서에서 p53 단백질은 별도의 언급이 없는 한 전장 단백질과 그 단편을 모두 의미하는 것으로 사용된다. According to one embodiment, the p53 protein may be a polypeptide having an amino acid sequence provided by the NCBI accession number NP_000537 or a fragment thereof. As used herein, the p53 protein is used to mean both a full-length protein and a fragment thereof, unless otherwise noted.
또한, 상기 p53 단백질은 서열번호 1(Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn)의 아미노산 서열을 갖는 p53 단백질의 전사활성 도메인일 수 있다. Also, the p53 protein may be a transcriptionally active domain of the p53 protein having the amino acid sequence of SEQ ID NO: 1 (Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn).
상기 서열번호 1의 아미노산 서열을 갖는 p53 단백질 단편은 p53 단백질 중 Mdm2에 결합하는 부위인 인간 p53 단백질의 18 내지 26번째 아미노산 서열을 포함하는 전사활성 도메인을 의미한다. 상기 p53의 전사활성 도메인은 p53 분해 작용을 하는 Mdm2와 결합함으로써 생체 내에서의 p53의 안정성을 크게 증가시키며, p53의 작용으로 CDK 억제제 단백질을 암호화하는 p21 유전자를 발현시켜 세포증식을 일시적으로 정지시키고 p53-결합 단백질인 ASPP(apoptosis-stimulating protein of p53)와 결합한 p53이 Bax 유전자를 발현시켜 세포 죽음을 유발하는 역할을 한다. 또한, 종양 억제제인 p18 단백질은 CDK 4, 6의 활성을 저해하는 역할을 한다. 따라서 p53 의 전사활성 도메인과 p18 단백질이 결합된 융합 단백질과 이를 유효성분으로 함유하는 약학 조성물은 새로운 항암제로 매우 유용하게 사용될 수 있다.The p53 protein fragment having the amino acid sequence of SEQ ID NO: 1 means a transcription activity domain comprising the 18th to 26th amino acid sequences of the human p53 protein, which is a site binding to Mdm2 in the p53 protein. The transcriptional activation domain of p53 binds to Mdm2 which has a function of p53 degradation, thereby greatly increasing the stability of p53 in vivo. By the action of p53, p21 gene encoding the CDK inhibitor protein is expressed to temporarily stop cell proliferation p53, which binds to the p53-binding protein ASPP (apoptosis-stimulating protein of p53), plays a role in inducing cell death by expressing the Bax gene. In addition, the tumor suppressor p18 protein plays a role in inhibiting the activity of CDK4,6. Therefore, a fusion protein in which a transcription activation domain of p53 and a p18 protein are combined and a pharmaceutical composition containing the fusion protein as an active ingredient can be very useful as a novel anticancer agent.
일 구체예에 따르면, 상기 p18 단백질은 서열번호 2의 아미노산 서열을 갖는 폴리펩타이드일 수 있다. 또한, p18 단백질은 용해도(solubility)를 개선하기 위한 목적으로, 전체 단백질 활성에 영향을 미치지 않는 한 일부 아미노산 잔기를 변형(치환, 삭제, 부가, 결실 등)하여 사용할 수 있다.According to one embodiment, the p18 protein may be a polypeptide having the amino acid sequence of SEQ ID NO: 2. In addition, for the purpose of improving the solubility, the p18 protein can be used by modifying (substituting, deleting, addition, deletion, etc.) some amino acid residues unless it affects the whole protein activity.
상기 융합 단백질에서 p53 단백질과 p18 단백질의 연결되는 순서는 제한이 없으며, N 말단-p53 단백질-p18 단백질-C 말단 또는 N 말단-p18 단백질-p53 단백질-C 말단 형태 모두 포함된다. 일 구체예에서, 서열번호 1의 p53 단백질 단편과 서열번호 2의 p18 단백질이 결합된 융합 단백질은 서열번호 3의 아미노산 서열을 갖는 것일 수 있다.The sequence of the p53 protein and the p18 protein in the fusion protein is not limited, and includes both the N-terminal p53 protein-p18 protein-C terminal end or the N terminal p18 protein-p53 protein-C terminal form. In one embodiment, the fusion protein in which the p53 protein fragment of SEQ ID NO: 1 and the p18 protein of SEQ ID NO: 2 are combined may have the amino acid sequence of SEQ ID NO: 3.
또한, 본 발명의 융합 단백질은 아넥신 A2 결합단백질(Annexin A2 binding protein, Annexin A2BP)을 포함한다. 아넥신 A2 결합단백질은 암세포에 특이적으로 발현되는 아넥신 A2에 결합하므로, 융합 단백질이 암세포에만 선택적으로 작용할 수 있도록 암세포 타겟팅이 가능하게 한다. 아넥신 A2 단백질로는 서열번호 4의 아미노산 서열을 가지는 단백질을 예시할 수 있으나, 본 발명의 범위가 이에 제한되는 것은 아니며, 암세포 타겟팅 활성을 달성할 수 있는 한 아넥신 A2 결합단백질의 단편, 유사체 또는 변이체를 사용해도 무방하다.In addition, the fusion protein of the present invention includes an Annexin A2 binding protein (Annexin A2BP). The annexin A2 binding protein binds to annexin A2, which is specifically expressed in cancer cells, so that cancer cells can be targeted so that the fusion protein selectively acts on cancer cells. The annexin A2 protein may be exemplified by a protein having the amino acid sequence of SEQ ID NO: 4, but the scope of the present invention is not limited thereto. As long as the cancer cell targeting activity can be achieved, the annexin A2 binding protein fragment, Or a mutant may be used.
상기 융합 단백질에서 아넥신 A2 결합단백질은 p53 및 p18 융합체의 N-말단 또는 C-말단에 위치할 수 있다. 예를 들어, 본 발명의 융합 단백질은 아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; 아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; p53 단백질, p18 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; 및 p18 단백질, p53 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질로 이루어진 군에서 선택된 것일 수 있다. The annexin A2 binding protein in the fusion protein may be located at the N-terminus or C-terminus of the p53 and p18 fusions. For example, the fusion protein of the present invention includes a fusion protein comprising annexin A2 binding protein, p53 protein and p18 protein in the order of N-terminal to C-terminal; A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal; a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And a fusion protein comprising the p18 protein, the p53 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal.
바람직한 일 구체예에서, 상기 융합 단백질은 생체외 안정화 단백질 (in vitro stabilization protein), 막 투과 서열 (membrane transfer sequence, MTS) 도메인, 핵-세포질 신호 (nucleus-cytoplasm signal) 도메인, 및 생체내 안정화 단백질 (in vivo stabilization protein)로 이루어진 군에서 선택된 1종 이상을 추가로 포함할 수 있다.In a preferred embodiment, the fusion protein comprises an in vitro stabilization protein, a membrane transfer sequence (MTS) domain, a nucleus-cytoplasm signal domain, and an in vivo stabilization protein (in vivo stabilization protein) may be further included.
이 때, 추가되는 폴리펩타이드의 융합 단백질 내 위치는 제한이 없으며, 상기 추가되는 1종 이상의 폴리펩타이드는 각각 독립적으로 융합 단백질의 N 말단, C 말단, 또는 p53 단백질과 p18 단백질 사이에 포함될 수 있다. In this case, the position of the added polypeptide in the fusion protein is not limited, and each of the added one or more polypeptides can be independently included in the N-terminus, C-terminus, or between the p53 protein and the p18 protein of the fusion protein.
바람직하게, 상기 융합 단백질은 아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p53 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p18 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것일 수 있다.Preferably, the fusion protein comprises an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal, wherein the fusion protein comprises an in vitro stabilization protein in the N-terminal direction of the p53 protein, A transmembrane domain, and a nucleus-cytoplasmic signal domain, or comprises at least one selected from the group consisting of transmembrane domain, nuclear-cytoplasmic signal domain and in vivo stabilizing protein in the C-terminal direction of p18 protein , And < / RTI >
또한 바람직하게, 상기 융합 단백질은 아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p18 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p53 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것일 수 있다.Also preferably, the fusion protein is a fusion protein comprising an annexin A2 binding protein, a p18 protein and a p53 protein in the order of N-terminal to C-terminal, wherein the fusion protein comprises an in vitro stabilization protein, A transmembrane domain, a transmembrane domain, a transmembrane domain, a transmembrane domain, a transmembrane domain, and a nuclear-cytoplasmic signal domain, or comprises at least one selected from the group consisting of transmembrane domain, Or a combination thereof.
본원에서 용어, "생체외 안정화 단백질(in vitro stabilization protein)"은 상기 융합 단백질의 생체 외부에서, 즉, 상기 융합 단백질을 실험적으로 정제를 하는 경우, 상기 융합 단백질의 용해도 및 안정성을 증진시키기 위한 단백질을 의미한다. 상기 생체외 안정화 단백질은 상기 융합 단백질의 일부로써, 생체 내에서 면역원성을 유발하지 않아야 한다. As used herein, the term "in vitro stabilization protein" refers to a protein for enhancing the solubility and stability of the fusion protein in vitro, i.e., when experimentally purifying the fusion protein . The ex-vivo stabilizing protein, as part of the fusion protein, should not induce immunogenicity in vivo.
일 구체예에서, 생체외 안정화 단백질이 융합 단백질 내에 도입되는 경우, 융합 단백질의 N 말단 방향에 위치하는 것이 바람직하나, 이에 제한되는 것은 아니다. 또한 바람직하게, 생체내 안정화 단백질은 융합 단백질의 양 말단 외에 추가적으로 p18과 p53 사이에 위치할 수 있다.In one embodiment, when the in vitro stabilizing protein is introduced into the fusion protein, it is preferably, but not limited to, located in the N-terminal direction of the fusion protein. Also preferably, the in vivo stabilizing protein may be additionally located between p18 and p53 in addition to both ends of the fusion protein.
일 구체예에 따르면, 상기 생체외 안정화 단백질은 유비퀴틴 또는 유비퀴틴-유사 단백질일 수 있으나, 이에 한정되지는 않는다. According to one embodiment, the ex vivo stabilizing protein may be, but is not limited to, ubiquitin or ubiquitin-like protein.
유비퀴틴(ubiquitin, Ub)은 자연계에서 발견되는 가장 보존적인 단백질로 76개의 아미노산 서열로 이루어져 있으며, 곤충, 송어 및 인간처럼 진화적으로 다양한 종들간의 완벽한 상동성을 보이는 수용성 단백질이다. 또한, 유비퀴틴은 pH의 변화에 대해 안정하고, 고온에서도 쉽게 변성되지 않으며, 프로테아제에 대해서도 안정성이 있는 단백질로 알려져 있다. 따라서, 유비퀴틴은 상기 융합 단백질의 불용성을 개선할 수 있다. Ubiquitin (Ub) is the most conserved protein found in nature, consisting of 76 amino acid sequences, and is a water-soluble protein that exhibits perfect homology between evolutionary species such as insects, trout, and humans. In addition, ubiquitin is known to be stable to changes in pH, not easily denatured at high temperatures, and stable to proteases. Thus, ubiquitin can improve the insolubility of the fusion protein.
상기 유비퀴틴 또는 유비퀴틴-유사 단백질(ubiquitin-like protein, Ubl)은 야생형 유비퀴틴, 야생형 유비퀴틴-유사 단백질, 돌연변이 유비퀴틴 및 돌연변이 유비퀴틴-유사 단백질로 이루어진 군으로부터 선택되는 것일 수 있다. 상기 돌연변이 유비퀴틴은 야생형 유비퀴틴의 아미노산 서열을 다른 아미노산 서열로 바꾼 것을 의미하며, 예를 들면, 야생형 유비퀴틴(서열번호 5)의 Lys을 Arg으로 치환한 유비퀴틴, 및/또는 야생형 유비퀴틴 C-말단 RGG를 RGA로 변경시킨 (즉 유비퀴틴 야생형 폴리펩타이드의 76번째에 존재하는 Gly이 Ala으로 치환된) 유비퀴틴을 포함한다. 일 구체예에 따르면, 상기 야생형 유비퀴틴의 Lys을 Arg으로 치환한 돌연변이형 유비퀴틴에서, 상기 치환은 야생형 유비퀴틴의 6, 11, 27, 29, 33, 48 및 63번째에 존재하는 Lys 중에서 선택된 1종 이상에서 이루어질 수 있으며, 치환은 상기 Lys의 위치에서 독립적으로 또는 조합하여 이루어질 수 있다. The ubiquitin-like protein (Ubl) may be selected from the group consisting of wild-type ubiquitin, wild-type ubiquitin-like protein, mutant ubiquitin and mutant ubiquitin-like protein. The mutant ubiquitin means that the amino acid sequence of the wild-type ubiquitin is replaced with another amino acid sequence. For example, ubiquitin substituted with Lys of wild type ubiquitin (SEQ ID NO: 5) and / or wild type ubiquitin C- (I.e., the 76th Gly of the ubiquitin wild-type polypeptide is replaced by Ala). According to one embodiment, in the mutant ubiquitin in which the Lys of the wild-type ubiquitin is substituted with Arg, the substitution is preferably one or more selected from Lys existing at 6th, 11th, 27th, 29th, 33th, 48th and 63rd positions of the wild type ubiquitin , And the substitution can be performed independently or in combination with the position of the Lys.
일 구체예에 따르면, 상기 유비퀴틴-유사 단백질은 유비퀴틴과 그 특성이 유사한 단백질로, 예를 들어, Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 및 ISG15로 구성된 군으로부터 선택된 1종 이상일 수 있으나, 이에 한정하지는 않는다.According to one embodiment, the ubiquitin-like protein is a protein whose characteristics are similar to those of ubiquitin, for example, a protein selected from the group consisting of Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 and ISG15 Or more, but is not limited thereto.
일 구체예에 따르면, 상기 유비퀴틴 또는 유비퀴틴-유사 단백질은 C-말단에 프로테아제에 의해 절단 가능한 아미노산 서열 또는 프로테아제에 의해 절단되지 않는 아미노산 서열을 포함할 수 있다. 상기 프로테아제에 의해 절단 가능한 아미노산 서열은 당업계에 공지된 검색 데이터베이스를 통해 확인할 수 있다. 예를 들어, http://www.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html에서 검색되는 프로테아제 및 그의 절단 가능한 아미노산 서열을 이용할 수 있다. 상기 절단 가능한 아미노산 서열이 포함되는 경우, 상기 융합 단백질이 세포 내로 투과된 다음, 세포 내의 프로테아제에 의해 유비퀴틴 또는 유비퀴틴-유사 단백질은 절단이 되어, 아넥신 A2 결합단백질, p53 단백질 및 p16 단백질을 포함하는 융합 단백질이 세포 내에서 그 기능을 할 수 있게 된다. 상기 절단 후, 융합 단백질에는 막 투과 서열 도메인 및/또는 핵-세포질 신호 도메인 등이 포함되어 있을 수 있지만, 이들은 폴리펩타이드의 길이가 매우 짧으므로 융합 단백질의 기능에는 영향을 미치지 않는다. 또한, 세포 내의 프로테아제에 의해 유비퀴틴 또는 유비퀴틴-유사 단백질이 절단되지 않더라도 유비퀴틴 또는 유비퀴틴-유사 단백질은 면역원성이 없으므로 생체내에서 안전할 뿐 아니라, 시스테인을 포함하지 않아서 폴딩이 되지 않으므로 융합 단백질 구조 변화를 유발하지 않고 융합 단백질이 세포 내에서 그 기능을 발휘하는데 영향을 미치지 않는다.According to one embodiment, the ubiquitin or ubiquitin-like protein may comprise an amino acid sequence which is cleavable by a protease at the C-terminus or an amino acid sequence which is not cleaved by a protease. Amino acid sequences cleavable by the protease can be identified through a search database known in the art. For example, the protease that is searched at http://www.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html and its cleavable amino acid sequence can be used. When the cleavable amino acid sequence is included, the fusion protein is permeabilized into a cell, and then the ubiquitin or ubiquitin-like protein is cleaved by the intracellular protease, and the protein containing the annexin A2 binding protein, the p53 protein and the p16 protein Allowing the fusion protein to function in the cell. After the cleavage, the fusion protein may include a transmembrane sequence domain and / or a nucleus-cytoplasmic signal domain, but they do not affect the function of the fusion protein because the length of the polypeptide is very short. Even if the ubiquitin or ubiquitin-like protein is not cleaved by the intracellular protease, the ubiquitin or ubiquitin-like protein is not safe in the living body since it does not have immunogenicity, and since it does not contain cysteine and does not fold, And does not affect the ability of the fusion protein to exert its function in the cell.
용어, "막 투과 (membrane transfer)"는 인 비트로(in vitro) 및/또는 인 비보(in vivo) 상에서 운반 대상인 융합 단백질을 세포 내 또는 핵 내로 운반할 수 있는 능력을 의미한다. The term "transmembrane (membrane transfer)" is in vitro (in vitro) and / or in vivo (in vivo < / RTI > of the fusion protein to be delivered into the cell or into the nucleus.
또한, "막 투과 서열 (membrane transfer sequence, MTS) 도메인"은 그 자체로 인지질 이중막의 세포막을 통과할 수 있는 아미노산 서열을 갖는 폴리펩타이드를 의미한다. 상기 막 투과 서열 도메인은 그 N-말단에 단일 소수성 영역(single hydrophobic region)을 가지고, 헬릭스 구조(helix structure)를 형성하며, 유연성을 나타내고, 상대적으로 짧은 길이의 아미노산(7개 내지 17개 아미노산)을 갖는 것을 특징으로 한다. 따라서, 상기 막 투과 서열 도메인의 물성은 대개 소수성을 나타낸다. 일 구체예에 따르면, 상기 막 투과 서열 도메인은 그 자체로 인지질 이중막의 세포막을 통과할 수 있는 아미노산 서열을 갖는 폴리펩타이드이면 모두 가능하고 특별히 한정되지 않으나, 서열번호 6의 아미노산 서열로 이루어진 것일 수 있다. In addition, "membrane transfer sequence (MTS) domain" refers to a polypeptide having an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer as such. The membrane permeable sequence domain has a single hydrophobic region at its N-terminus, forms a helix structure, exhibits flexibility, and has a relatively short length of amino acids (7 to 17 amino acids) . Thus, the physical properties of the transmembrane sequence domain usually exhibit hydrophobicity. According to one embodiment, the membrane-permeable sequence domain can be any polypeptide as long as it has an amino acid sequence that can pass through the cell membrane of the phospholipid bilayer, and is not particularly limited. However, the polypeptide transmembrane domain having the amino acid sequence of SEQ ID NO: 6 Lt; / RTI >
일 구체예에서, 상기 막투과 서열 도메인이 융합 단백질에 도입되는 경우에, 융합 단백질의 N 말단 방향에 위치하는 것이 바람직하나, 이에 제한되는 것은 아니다.In one embodiment, when the transmembrane sequence domain is introduced into the fusion protein, it is preferably, but not exclusively, located in the N-terminal direction of the fusion protein.
용어, "핵-세포질 신호 (nucleus-cytoplasm signal) 도메인"은 융합 단백질을 핵의 내부로 수송하거나, 핵의 외부로 수송하기 위는 역할을 하는 폴리펩타이드 서열을 의미하는 것으로 해석된다. 일 구체예에 따르면, 상기 핵-세포질 신호 도메인은 NLS(nucleus location sequence) 도메인 또는 NES(nucleus export sequence) 도메인일 수 있다. 즉, 융합 단백질을 핵 내로 이송시키기 위해서는 상기 융합 단백질에 NLS를 포함시키고, 상기 융합 단백질을 세포질에 남아 있도록 하기 위해서는 상기 융합 단백질에 NES를 포함시킬 수 있다.The term "nucleus-cytoplasm signal domain" is interpreted to mean a polypeptide sequence that serves to transport a fusion protein into the interior of the nucleus or to transport it outside of the nucleus. According to one embodiment, the nuclear-cytoplasmic signal domain may be a nucleus location sequence (NLS) domain or a nucleus export sequence (NES) domain. That is, in order to transfer the fusion protein into the nucleus, the fusion protein may include NLS in order to include NLS in the fusion protein and to allow the fusion protein to remain in the cytoplasm.
NLS 도메인은 세포질에서 핵으로 수송되는 단백질들이, NES 도메인은 핵에서 세포질로 수송되는 단백질들이 특징적으로 가지고 있는데, 상기 도메인 모두 핵막을 통과할 수 있는 아미노산 서열을 갖는 폴리펩타이드를 의미하며, 상기 아미노산 서열이 특별히 한정되지는 않는다. 상기 NLS 도메인으로 사용될 수 있는 폴리펩타이드는 예를 들어, KKKRK(서열번호 7), PKKKRKV(서열번호 8), KRPAATKKAGQAKKKK(서열번호 9) 등일 수 있으나, 이에 한정되지는 않는다. The NLS domain is a protein that is transported from the cytoplasm to the nucleus and the NES domain is a protein that is transported from the nucleus to the cytoplasm. The above domain means a polypeptide having an amino acid sequence that can pass through the nuclear membrane, Is not particularly limited. The polypeptide that can be used as the NLS domain can be, for example, but not limited to, KKKRK (SEQ ID NO: 7), PKKKRKV (SEQ ID NO: 8), KRPAATKKAGQAKKKK (SEQ ID NO: 9), and the like.
상기 융합 단백질에서 핵-세포질 신호 도메인은 상기 융합 단백질을 핵 내외로 이동시키는 중요한 일을 하는 이외에도, 단백질의 용해도를 증가시키는데 중요한 역할을 하는데, 융합 단백질에서 C-말단에 가깝게 위치해 있는 경우, 용해도의 증가에 더욱 도움이 된다. The nuclear-cytoplasmic signal domain in the fusion protein plays an important role in increasing the solubility of the protein in addition to the important task of transferring the fusion protein into and out of the nucleus. When the fusion protein is located close to the C-terminal, It is more helpful to increase.
용어, "생체내 안정화 단백질(in vivo stabilization protein)"은 상기 융합 단백질이 실질적으로 작용하는 생체 내부에서, 상기 융합 단백질이 안정적으로 존재할 수 있도록 안정성을 부여하는 단백질을 의미한다. 상기 생체내 안정화 단백질은 상기 융합 단백질의 일부로써, 생체 내에서 면역원성을 유발하지 않아야 하며, 특히, 대상에 투여된 경우, 대상의 혈액 내에서 안정성을 획득할 수 있는 단백질이면 어떠한 단백질이라도 가능하다. 일 구체예에 따르면, 상기 생체내 안정화 단백질은 AAT (alpha 1 antitrypsin), 혈청 알부민, 혈청 알부민 결합 펩타이드(serum albumin binding peptide; SABP), 면역글로불린의 Fc 및 PEG(polyethyleneglycol)로 이루어진 군으로부터 선택된 1종 이상일 수 있으나, 이에 한정되지는 않는다.The term "in vivo stabilization protein" refers to a protein that, within a living body to which the fusion protein substantially acts, confers stability to allow the fusion protein to stably exist. The in vivo stabilizing protein should not induce immunogenicity in vivo as a part of the fusion protein, and in particular, any protein that can obtain stability in the blood of an object when administered to a subject is possible . According to one embodiment, the in vivo stabilizing protein is selected from the group consisting of AAT (
일 구체예에서, 상기 생체내 안정화 단백질이 융합 단백질에 도입되는 경우, 융합 단백질의 N 말단, C 말단, 또는 p53 단백질과 p18 단백질 사이에 포함될 수 있다.In one embodiment, when the in vivo stabilizing protein is introduced into the fusion protein, it may be included between the N-terminus, the C-terminus, or between the p53 protein and the p18 protein of the fusion protein.
또한, 본 발명의 융합 단백질의 말단에는, 종결코돈(stop codon)이 추가로 포함될 수 있다. 종결코돈은 2회 이상 반복하여 삽입될 수 있으며, 예를 들어 TAA 서열이 2회 반복되어 삽입될 수 있다. 본 명세서 내에서는, 종결코돈이 2회 반복 삽입된 경우를 편의상 STOPx2 로 표시하기로 한다.The terminus of the fusion protein of the present invention may further include a stop codon. The termination codon can be inserted repeatedly two or more times, for example, the TAA sequence can be inserted repeatedly twice. In the present specification, the case where the stop codon is inserted two times repeatedly is referred to as STOPx2 for the sake of convenience.
또한, 본 발명은 아넥신 A2 결합단백질, p53 단백질 또는 서열번호 1의 아미노산 서열을 갖는 p53 단백질 단편, 및 p18 단백질을 포함하는 융합 단백질을 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, an annexin A2 binding protein, a p53 protein, or a p53 protein fragment having an amino acid sequence of SEQ ID NO: 1, and a fusion protein comprising a p18 protein do.
상기 융합 단백질은 생체외 안정화 단백질, 막 투과 서열 도메인, 핵-세포질 신호 도메인, 및 생체내 안정화 단백질로 이루어진 군에서 선택된 1종 이상을 추가로 포함하는 것일 수 있으며, 그 구체적 내용은 앞서 설명한 바와 같다.The fusion protein may further comprise at least one member selected from the group consisting of an in vitro stabilization protein, a membrane permeation sequence domain, a nuclear-cytoplasmic signal domain, and a stabilization protein in vivo, .
일 구체예에 따르면, 상기 융합 단백질은 아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; According to one embodiment, the fusion protein comprises a fusion protein comprising an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal;
아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal;
p53 단백질, p18 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; 및a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And
p18 단백질, p53 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질로 이루어진 군에서 선택된 것일 수 있다. p18 protein, p53 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal.
또한 바람직한 일 구현예에 따르면, 상기 융합 단백질은 아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p53 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p18 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질;According to a preferred embodiment, the fusion protein is a fusion protein comprising an annexin A2 binding protein, a p53 protein and a p18 protein in the order of N-terminal to C-terminal, An outer stabilizing protein, a transmembrane sequence domain, and a nucleus-cytoplasmic signal domain, or may further comprise at least one selected from the group consisting of a transmembrane sequence domain in the C-terminal direction of the p18 protein, a nuclear-cytoplasmic signal domain, Wherein the fusion protein further comprises at least one member selected from the group consisting of a fusion protein and a stabilizing protein;
아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p18 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p53 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질; Terminal fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of the N-terminal to the C-terminal, wherein the in vitro stabilization protein, the transmembrane sequence domain and the nuclear- Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain, and in vivo stabilization protein in the C-terminal direction of p53 protein A fusion protein further comprising;
p53 단백질, p18 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, 아넥신 A2 결합단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, 아넥신 A2 결합단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 STOPx2 로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질; 및a fusion protein comprising p53 protein, p18 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a membrane- Nuclear-cytoplasmic signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain and STOPx2 in the C-terminal direction of annexin A2 binding protein A fusion protein comprising at least a heterologous protein; And
p18 단백질, p53 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, 아넥신 A2 결합단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, 아넥신 A2 결합단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질로 이루어진 군에서 선택된 것일 수 있다.a fusion protein comprising p18 protein, p53 protein and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a transmembrane sequence domain and / Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein And a fusion protein which further comprises at least one selected from the group consisting of a fusion protein and a fusion protein.
또한, 본 발명은 상기 융합 단백질을 코딩하는 폴리뉴클레오티드, 상기 폴리뉴클레오티드를 포함하는 재조합 벡터, 상기 재조합 벡터로 형질전환된 세포를 제공한다. 또한, 본 발명은 상기 형질전환된 세포를 배양시키는 단계를 포함하는 융합 단백질의 제조방법을 제공한다.The present invention also provides a polynucleotide encoding the fusion protein, a recombinant vector comprising the polynucleotide, and a cell transformed with the recombinant vector. The present invention also provides a method for producing a fusion protein comprising culturing the transformed cells.
용어 "폴리뉴클레오티드(polynucleotide)" 는 단일가닥 또는 이중가닥 형태로 존재하는 디옥시리보뉴클레오티드 또는 리보뉴클레오티드의 중합체를 말한다. 상기 폴리뉴클레오티드는 RNA 게놈 서열, cDNA 및 이로부터 전사되는 RNA 서열을 포괄하며, 특별하게 다른 언급이 없는 한 천연의 폴리뉴클레오티드의 유사체를 포함한다. 일예로, 본 발명의 단백질 복합체를 코딩하는 폴리뉴클레오티드는 서열번호 11의 핵산 서열을 포함하는 것일 수 있다. The term "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. The polynucleotide encompasses RNA genomic sequences, cDNA and RNA sequences transcribed therefrom, and includes analogs of natural polynucleotides unless otherwise specified. For example, the polynucleotide encoding the protein complex of the present invention may comprise the nucleic acid sequence of SEQ ID NO: 11.
상기 폴리뉴클레오티드는 상기 융합 단백질의 아미노산 서열을 코딩하는 뉴클레오티드 서열뿐만 아니라, 그 서열에 상보적인(complementary) 서열도 포함한다. 상기 상보적인 서열은 완벽하게 상보적인 서열뿐만 아니라, 실질적으로 상보적인 서열도 포함하며, 이는 당업계에 공지된 엄격 조건(stringent conditions) 하에서, 예를 들어, 상기 융합 단백질의 아미노산 서열을 코딩하는 뉴클레오티드 서열의 뉴클레오티드 서열과 혼성화될 수 있는 서열을 의미한다.The polynucleotide includes not only a nucleotide sequence coding for the amino acid sequence of the fusion protein but also a complementary sequence to the sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences, which can be obtained under stringent conditions known in the art, for example, using nucleotides encoding the amino acid sequence of the fusion protein Quot; means a sequence that can hybridize with a nucleotide sequence of a nucleotide sequence.
상기 재조합 벡터는, 숙주 세포 내에서 안정적으로 상기 융합 단백질을 발현시킬 수 있는, 발현용 벡터일 수 있다. 상기 발현용 벡터는 당업계에서 식물, 동물 또는 미생물에서 외래의 단백질을 발현하는 데 사용되는 통상의 것을 사용할 수 있다. 상기 재조합 벡터는 당업계에 공지된 다양한 방법을 통해 구축될 수 있다.The recombinant vector may be an expression vector capable of stably expressing the fusion protein in a host cell. The expression vector may be any conventional vector used in the art to express an exogenous protein in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.
상기 재조합 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다. 예를 들어, 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터 (예를 들어, pLλ프로모터, trp 프로모터, lac 프로모터, tac 프로모터, T7 프로모터 등), 해독의 개시를 위한 리보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 진핵 세포를 숙주로 하는 경우에는, 벡터에 포함되는 진핵 세포에서 작동하는 복제원점은 f1 복제원점, SV40 복제원점, pMB1 복제원점, 아데노 복제원점, AAV 복제원점 및 BBV 복제원점 등을 포함하나, 이에 한정되는 것은 아니다. 또한, 포유동물 세포의 게놈으로부터 유래된 프로모터 (예를 들어, 메탈로티오닌 프로모터) 또는 포유동물 바이러스로부터 유래된 프로모터 (예를 들어, 아데노바이러스 후기 프로모터, 백시니아 바이러스 7.5K 프로모터, SV40 프로모터, 사이토메갈로바이러스 프로모터 및 HSV의 tk프로모터)가 이용될 수 있으며, 전사 종결 서열로서 폴리아데닐화 서열을 일반적으로 포함할 수 있다.The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector is an expression vector and the prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, tac promoter, T7 promoter etc.) Lt; / RTI > sequence and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the origin of replication that functions in eukaryotic cells contained in the vector includes f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus promoter and the tk promoter of HSV) can be used, and can generally include a polyadenylation sequence as a transcription termination sequence.
본 발명에서 형질전환된 세포는 상기 재조합 벡터를 안정되면서 연속적으로 클로닝 또는 발현시킬 수 있는 숙주 세포는 당업계에 공지된 어떠한 숙주 세포도 이용할 수 있으며, 원핵 세포로는, 예를 들어, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있으며, 진핵 세포에 형질 전환시키는 경우에는 숙주 세포로서, 효모(Saccharomyce cerevisiae), 곤충 세포, 식물 세포 및 동물 세포, 예를 들어, CHO 세포주 (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주 등이 이용될 수 있다.The host cells transformed in the present invention can be used to clone or express the recombinant vector in a stable manner. Any host cell known in the art can be used. Examples of prokaryotic cells include E. coli JM109, E. coli Bacillus subtilis, Bacillus subtilis, and Bacillus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Salmonella typhimurium, Yeast ( Saccharomyce cerevisiae ), insect cells, plant cells and animal cells such as a CHO cell line (for example, a yeast cell, a yeast cell, a yeast cell, etc.) Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines.
상기 폴리뉴클레오티드 또는 이를 포함하는 재조합 벡터의 숙주 세포 내로의 운반은, 당업계에 널리 알려진 운반 방법을 사용할 수 있다. 상기 운반 방법은 예를 들어, 숙주 세포가 원핵 세포인 경우, CaCl2 방법 또는 전기 천공 방법 등을 사용할 수 있고, 숙주 세포가 진핵 세포인 경우에는, 미세 주입법, 칼슘 포스페이트 침전법, 전기 천공법, 리포좀-매개 형질감염법 및 유전자 밤바드먼트 등을 사용할 수 있으나, 이에 한정하지는 않는다.The delivery of the polynucleotide or a recombinant vector containing the polynucleotide into a host cell can be carried out by a method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl 2 method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection methods, and gene bombardment, but the present invention is not limited thereto.
상기 형질 전환된 숙주 세포를 선별하는 방법은 선택 표지에 의해 발현되는 표현형을 이용하여, 당업계에 널리 알려진 방법에 따라 용이하게 실시할 수 있다. 예를 들어, 상기 선택 표지가 특정 항생제 내성 유전자인 경우에는, 상기 항생제가 함유된 배지에서 형질전환체를 배양함으로써 형질전환체를 용이하게 선별할 수 있다.The method of selecting the transformed host cells can be easily carried out according to a method widely known in the art by using a phenotype expressed by a selection marker. For example, when the selection mark is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.
형질 전환된 세포의 배양은 당업계에 공지된 다양한 방법을 통하여 실시할 수 있다. 예를 들어, 형질 전환된 세포를 YT 액상 배지에 접종하여 배양을 실시한 다음, 세포 밀도가 일정 수준에 도달한 시점에서 IPTG를 배지에 첨가하여 lacZ 프로모터에 의한 단백질 발현을 유도하고 배양함으로써, 세포 내 또는 배지로 분비된 단백질을 얻을 수 있다.Culturing of the transformed cells can be carried out through various methods known in the art. For example, when the transformed cells are inoculated into a YT liquid medium and cultured, when IPTG is added to the medium at the time when the cell density reaches a certain level, protein expression by the lac Z promoter is induced and cultured, Lt; RTI ID = 0.0 > and / or < / RTI >
세포 내 또는 배지로 분비된 단백질은 당업계에 공지된 다양한 정제 방법에 따라 정제된 형태로 얻을 수 있다. 예를 들어, 암모늄 설페이트에 의한 용해도 분획화(solubility fractionation), 크기 분별 여과 및 다양한 크로마토그래피 (크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 정제 방법을 통하여 정제된 형태로 단백질을 얻을 수 있다. 예를 들어, 융합 단백질이 GST에 융합된 경우에는 글루타티온이 결합된 레진 칼럼, 6x His에 융합된 경우에는 Ni2 +-NTA His-결합 레진 칼럼을 이용하여 원하는 단백질을 용이하게 얻을 수 있다.The protein secreted intracellularly or in the medium can be obtained in a purified form according to various purification methods known in the art. For example, by purification with solubility fractionation by ammonium sulfate, size fractional filtration and purification by various chromatographies (size, charge, hydrophobicity, or affinity-based separation) Proteins can be obtained. For example, when a fusion protein is fused to GST, a glutathione-coupled resin column is used. When the fusion protein is fused to 6x His, a desired protein can be easily obtained using a Ni 2 + -NTA His-bonded resin column.
앞서 설명한 바와 같이, 상기 융합 단백질에 포함된 p53 및 p18의 융합체는 p53의 안정성을 크게 증가시키고, p21 유전자를 발현시켜 세포증식을 일시적으로 정지시키며, Bax 유전자를 발현시켜 세포 죽음을 유발하고, CDK 4, 6의 활성을 저해하는 역할을 하므로, 암의 예방 및/또는 치료에 있어서 유효성분으로 효과적이다. 또한, 상기 융합 단백질에 포함된 또 다른 성분인 아넥신 A2 결합단백질은 암세포에 특이적으로 발현되는 아넥신 A2에 결합하므로 융합 단백질이 암세포에만 선택적으로 작용할 수 있도록 암세포 타겟팅이 가능하게 한다. 따라서, 본 발명의 융합 단백질은 암의 예방 및/또는 치료용 약학적 조성물로 제공될 수 있다.As described above, the fusion of p53 and p18 contained in the fusion protein greatly increases the stability of p53, transiently stops cell proliferation by expressing the p21 gene, induces cell death by expressing the Bax gene, 4, and 6, it is effective as an active ingredient in the prevention and / or treatment of cancer. In addition, the annexin A2 binding protein, another component contained in the fusion protein, binds to annexin A2, which is specifically expressed in cancer cells, so that cancer cells can be targeted so that the fusion protein selectively acts on cancer cells. Accordingly, the fusion protein of the present invention can be provided as a pharmaceutical composition for the prevention and / or treatment of cancer.
따라서, 다른 예에서, 상기 융합 단백질을 유효성분으로 포함하는 암의 예방 및/또는 치료용 약학 조성물이 제공된다. 상기 약학 조성물은 필요에 따라서 약학적으로 허용되는 담체, 희석제 및/또는 부형제를 통상적으로 사용되는 양으로 추가로 포함할 수 있다. Therefore, in another example, there is provided a pharmaceutical composition for preventing and / or treating cancer comprising the fusion protein as an active ingredient. The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent and / or excipient in an amount normally used.
상기 약학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 상기 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
또 다른 예에서, 상기 융합 단백질의 치료적 유효량을 암의 예방 및/또는 치료를 필요로 하는 환자에게 투여하는 단계를 포함하는, 암의 예방 및/또는 치료 방법이 제공된다. 상기 암의 예방 및/또는 치료 방법은 상기 투여 단계 이전에 암의 예방 및/또는 치료를 필요로 하는 환자를 확인하는 단계를 추가로 포함할 수 있다.In another embodiment, there is provided a method of preventing and / or treating cancer, comprising administering a therapeutically effective amount of the fusion protein to a patient in need of such prevention and / or treatment. The method for preventing and / or treating cancer may further comprise the step of identifying a patient in need of prevention and / or treatment of cancer before the administration step.
또 다른 예에서, 상기 융합 단백질의 암의 예방 및/또는 치료에 사용하기 위한 용도, 또는 상기 융합 단백질의 암의 예방 및/또는 치료를 위한 약물 제조에 사용하기 위한 용도가 제공된다. In yet another example, there is provided a use of the fusion protein for use in the prevention and / or treatment of cancer, or for use in the manufacture of a medicament for the prevention and / or treatment of cancer of the fusion protein.
상기 융합 단백질 또는 이를 유효성분으로 포함하는 약학 조성물은 당업자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다. 또한, 상기 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. The fusion protein or the pharmaceutical composition containing it as an active ingredient may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art, As shown in FIG. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent.
상기 융합 단백질 또는 이를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물은 경구 또는 비경구로 투여할 수 있다. 비경구 투여인 경우에는 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 내피 투여, 국소 투여, 비내 투여, 폐내 투여 및 직장내 투여 등으로 투여할 수 있다. 경구 투여시, 단백질 또는 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 되어야 한다. 또한, 상기 조성물은 상기 조성물이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The pharmaceutical composition for preventing or treating the fusion protein or cancer comprising the fusion protein as an active ingredient may be orally or parenterally administered. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the composition to a target cell.
상기 융합 단백질 또는 이를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 상기 조성물의 바람직한 투여량은 성인 기준으로 0.001 내지 100 ㎎/kg 범위 내이다. 용어 "약학적 유효량" 또는 "치료적 유효량"은 암의 예방 또는 치료에 효과를 나타낼 수 있는 양을 의미한다.The appropriate dosage of the pharmaceutical composition for preventing or treating the fusion protein or the cancer comprising the same as an effective ingredient can be determined depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration route, And response sensitivities. ≪ / RTI > The preferred dose of the composition is in the range of 0.001 to 100 mg / kg on an adult basis. The term " pharmaceutically effective amount "or" therapeutically effective amount "means an amount that is effective in preventing or treating cancer.
상기 융합 단백질 또는 이를 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물의 투여 대상 환자는 포유류, 예컨대 인간, 원숭이 등을 포함하는 영장류, 래트, 마우스 등을 포함하는 설치류 등일 수 있다.The subject to be administered with the pharmaceutical composition for preventing or treating the fusion protein or the cancer comprising the same as an active ingredient may be a mammal such as a rodent including a primate, a rat, a mouse and the like including a human, a monkey and the like.
일 구체예에 따르면, 상기 융합 단백질 또는 약학 조성물이 예방 또는 치료 대상으로 하는 암은 고형암 또는 혈액암일 수 있으며, 예컨대, 편평상피세포암, 소세포폐암, 비소세포폐암, 폐의 선암, 폐의 편평상피암, 복막암, 피부암, 피부 또는 안구내 흑색종, 직장암, 항문부근암, 식도암, 소장암, 내분비선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 만성 또는 급성 백혈병, 림프종, 위장암, 췌장암, 교아종, 경부암, 난소암, 간암, 방광암, 유방암 (triple negative breast cancer 포함), 결장암, 대장암, 자궁내막 또는 자궁암, 침샘암, 신장암, 전립선암, 음문암, 갑상선암, 두경부암 등으로 이루어진 군으로부터 선택되는 것일 수 있으니, 이에 한정하지는 않는다.According to one embodiment, the cancer to be prevented or treated by the fusion protein or pharmaceutical composition may be a solid cancer or a blood cancer, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, Endometrioid adenocarcinoma, adenocarcinoma, adenocarcinoma, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphoma, gastrointestinal cancer, pancreatic cancer, endometrioid cancer, pancreatic cancer, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectum cancer, (Including a triple negative breast cancer), colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, renal cancer, prostate cancer, mucin cancer, thyroid cancer, head and neck cancer, etc. But the present invention is not limited thereto.
일 구체예에 따른 융합 단백질 및 이를 유효성분으로 포함하는 약학 조성물은 암의 예방 또는 치료에 효과적으로 사용 가능하다. 또한 상기 융합 단백질은 면역원성이 없기 때문에, 안전한 항암 치료용 단백질로서 유용하고, 암세포만을 타겟팅하여 특이적으로 작용하여 부작용이 적을 뿐 아니라, 거의 모든 종류의 암의 예방 및/또는 예방에 적용 가능하고, 기존의 항암제가 효과를 발휘하지 못하는 고형암에도 우수한 효과를 기대할 수 있다.The fusion protein according to one embodiment and the pharmaceutical composition comprising it as an effective ingredient can be effectively used for prevention or treatment of cancer. In addition, since the fusion protein is free of immunogenicity, it is useful as a safe anticancer therapy protein. It can be used for prevention and / or prevention of almost all kinds of cancer as well as having a specific effect by targeting only cancer cells, , And excellent effects can be expected also for solid cancer which does not exhibit the effect of existing anticancer drugs.
도 1은 일 구체예에 따른 단백질 복합체의 예를 모식적으로 나타낸 것이다.
도 2는 일 구체예에 따른 단백질 복합체가 암 세포에 작용하는 기전을 모식적으로 나타낸 것이다.
도 3은 본 발명에 따른 단백질 복합체 #1 및 #2 를 벡터로부터 발현시켜 정제한 결과를 SDS-PAGE를 통해 나타낸 것이다.
#1: A2BP-Ub-Ub(m1)-MTS-p18(F71N)-Ub(m3)-Ub(m4)-p53F-NLS
#2: A2BP-Ub-Ub(m1)-MTS-p18(F71N)-AAT-p53F-NLS
도 4는 본 발명에 따른 단백질 복합체 #1 및 #2 가 아넥신 A2 와 결합하는지를 확인한 SDS-PAGE 결과를 나타낸 것이다.
#1: A2BP-Ub-Ub(m1)-MTS-p18(F71N)-Ub(m3)-Ub(m4)-p53F-NLS
#2: A2BP-Ub-Ub(m1)-MTS-p18(F71N)-AAT-p53F-NLS
도 3은 HCC1806 세포주에서 단백질 복합체 #2의 처리 농도에 따른 세포 성장 저해 효과를 확인한 결과를 나타낸다.
#2: A2BP-Ub-Ub(m1)-MTS-p18(F71N)-AAT-p53F-NLSFig. 1 schematically shows an example of a protein complex according to one embodiment.
Fig. 2 schematically shows the mechanism by which a protein complex according to one embodiment acts on cancer cells.
FIG. 3 shows the results of SDS-PAGE of the
# 1: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -Ub (m3)
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS
Figure 4 shows SDS-PAGE results confirming whether
# 1: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -Ub (m3)
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS
FIG. 3 shows the results of confirming cell growth inhibition effect according to the treatment concentration of
# 2: A2BP-Ub-Ub (m1) -MTS-p18 (F71N) -AAT-p53F-NLS
이하, 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.
Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.
실시예Example 1: 융합 단백질의 발현 벡터의 제조 1: Preparation of Expression Vector of Fusion Protein
본 실시예에서는 친수성 폴리펩티드의 한 종류인 유비퀴틴 야생형 단백질 또는 유비퀴틴 돌연변이형 단백질, 막 투과 서열 도메인(MTS: membrane translocation sequence), p18, 생체내 안정화 단백질(AAT: alpha 1 antitrypsin), p53 단편 및 핵 위치 신호 도메인(NLS: nucleus localization signal domain)의 순서로 연결된 세포 내 전달용 단백질 복합체를 생산하기 위한 상기 단백질 복합체의 발현 벡터를 제조하였다. 또한, 상기 단백질 복합체와의 비교 실험을 위해 상기 단백질 복합체에서 하나 이상의 구성 요소를 제외한 단백질 복합체를 생산하기 위한 발현 벡터를 제조하였다. 이하, 야생형 유비퀴틴을 이하 Ub7KR로 (서열번호 5), 야생형 유비퀴틴 C-말단의 RGG를 RGA로 변경시킨 돌연변이형 유비퀴틴을 Ub7KR(G76A) (서열번호 12) 라 명명하도록 한다. 또한, 상기 돌연이형 유비퀴틴은 합성을 용이하게 하기 위하여, 아미노산 서열을 같으나 wobble sequence를 사용하여 핵산 서열이 서로 다른 4가지 핵산을 제조하였으며, 이를 Ub(m1), Ub(m3), Ub(m4)로 각각 명명하였다 (서열번호 13 내지 서열번호 16).In this embodiment, a ubiquitin wild-type protein or a ubiquitin mutant type protein, a membrane translocation sequence (MTS), p18, an
총 6가지 종류의 발현 벡터를 ㈜ 제노텍에 의뢰하여 제조하였으며, 단백질 과발현을 위한 벡터는 pET-21b(+)(EMD Biosciences)를 사용하였다. Six types of expression vectors were purchased from Genentech, Inc., and pET-21b (+) (EMD Biosciences) was used as a vector for protein overexpression.
한편, 상기 각각의 인서트 DNA 절편은 5' 말단에 NdeI으로 절단될 수 있는 뉴클레오티드 서열을, 3' 말단에 XhoI으로 절단될 수 있는 뉴클레오티드 서열을 포함함으로써, 상기 pET21b(+) 벡터의 NdeI-XhoI 절단 서열에 삽입될 수 있도록 하였다. Each of the insert DNA fragments includes a nucleotide sequence that can be cleaved with NdeI at the 5'end and a nucleotide sequence that can be cleaved with XhoI at the 3'end so that the NdeI-XhoI cleavage of the pET21b (+) vector Lt; / RTI > sequence.
일 구체예에 따른 단백질 복합체의 가능한 1차 구조를 나타내는 모식도를 도 1에 나타내었다.
A schematic diagram illustrating a possible primary structure of a protein complex according to one embodiment is shown in FIG.
실시예Example 2: 융합 단백질의 발현 및 정제 2: Expression and purification of fusion protein
상기 실시예 1에서 제조한 벡터를 이용하여 단백질 복합체를 과발현시키기 위해, 상기 벡터로 형질 전환된 E. coli BL21(DE3)에서 발현시켰다. 이 때, 배양액으로 YT 배지를 사용하였으며, 흡광도 600 nm에서 O.D.값이 0.5일 때 0.5 mM의 IPTG를 넣고 18 ℃에서 16시간 동안 더 배양하였다. 상기 배양하여 얻은 세포를, 5% 글리세롤, 5 mM β-머캅토에탄올, 0.2% Triton X-100 및 0.2 M NaCl을 포함하는 50 mM Tris-HCl 완충액(pH 8.0) 하에서 초음파로 분쇄한 후, 원심분리기(10,000 g)를 이용하여 상층액을 얻었다. 상기 상층액을 상기 완충액으로 평형화된 Ni2 +-NTA superflow 칼럼(Qiagen)에 적용하고, 컬럼 부피의 5배에 해당하는 세척 완충액(50 mM Tris-HCl, pH 8.0, 5% 글리세롤, 5mM β- 머캅토에탄올, 0.2% Triton X-100 및 1 M NaCl)로 세척한 다음, 용출 완충액(50 mM Tris-HCl, pH 8.0, 5% 글리세롤, 5 mM β-머캅토에탄올, 0.2% Triton X-100 및 0.2 M NaCl)으로 상기 단백질 복합체를 용출하였다. 단백질 복합체가 포함된 분획들을 모아 Amicon Ultra-15 Centrifugal Filter(Milipore)를 이용하여 분획 중에 포함된 염을 제거하고 농축하였다. 정제된 단백질 농도는 BSA를 표준 물질로 사용하여 측정하였다.To over-express the protein complex using the vector prepared in Example 1, E. coli transformed with the vector BL21 (DE3). At this time, YT medium was used as a culture medium. When the OD value was 0.5 at an absorbance of 600 nm, 0.5 mM IPTG was added and further cultured at 18 ° C for 16 hours. The cultured cells were ultrasonically pulverized in 50 mM Tris-HCl buffer (pH 8.0) containing 5% glycerol, 5 mM? -Mercaptoethanol, 0.2% Triton X-100 and 0.2 M NaCl, Separator (10,000 g) was used to obtain the supernatant. The supernatant was applied to a Ni 2 + -NTA superflow column (Qiagen) equilibrated with the buffer and eluted with a washing buffer (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM β- (50 mM Tris-HCl, pH 8.0, 5% glycerol, 5 mM [beta] -mercaptoethanol, 0.2% Triton X-100 And 0.2 M NaCl) to elute the protein complex. Protein complex-containing fractions were collected and the salts contained in the fractions were removed using an Amicon Ultra-15 Centrifugal Filter (Milipore) and concentrated. The purified protein concentration was measured using BSA as a standard.
도 3은 실시예 1에서 제작한 벡터로부터 발현된 단백질 복합체들을 상기 방법에 의해 정제한 결과를 SDS-PAGE를 통해 나타낸 것이다. 도 3에서, 단백질 복합체 #1은 A2-Ub-Ub(m1)-p18-Ub(m3)-Ub(m4)-p53 의 순서로 결합된 단백질 복합체이고, 단백질 복합체 #2는 A2-Ub-Ub(m1)-p18-AAT-p53의 순서로 결합된 단백질 복합체이다. 아넥신 A2 단백질과의 결합 여부를 확인하기 위해, 아넥신 A2는 따로 정제하였다. M은 사이즈 마커를 의미한다.
FIG. 3 shows the result of purification of protein complexes expressed from the vector prepared in Example 1 by SDS-PAGE. In Figure 3,
실시예Example 3: 융합 단백질의 3: of the fusion protein 아넥신Annexin A2에 대한 결합성 확인 Confirmation of binding to A2
우선 실시예 2에서 수득한 단백질 복합체가 실제로 아넥신 A2에 결합하는 지 알아보기 위해 in vitro에서 결합 테스트를 실시하였다. 먼저, 아넥신 A2 결합 펩타이드를 포함하는 단백질 복합체 #1인 A2-Ub-Ub(m1)-p18-Ub(m3)-Ub(m4)-p53, 단백질 복합체 #2인 A2-Ub-Ub(m1)-p18-AAT-p53(#2) 500 ug와 아넥신 A2 단백질 1500 ug을 1000 ul의 PBS 완충액(1 x PBS, 0.2 % Tween 20, 100 mM arginine, 0.2 % glutathione reduced) 에 18℃에서 120분 동안 방치하여 결합 시간을 충분히 제공한 다음, 이를 HiLoad Superdex S-75 16/60 컬럼 (GE healthcare)에 로딩하여 용출하였다. 이 때 용출 조건은 flow rate 1 ml/min, PBS 완충액 (1 x PBS, 0.2 % Tween 20, 100 mM arginine, 0.2 % glutathione reduced)이며, 2 ml fraction으로 120분 동안 수득하였다. 이후, 상기 용출액을 50 mM Tris 완충액 (50 mM Tris-HCl, pH 8.0, 5 % 글리세롤, 5 mM β- 머캅토에탄올, 0.2 % Triton X-100 및 1 M NaCl)로 평형화된 Ni2 +-NTA superflow 컬럼(Qiagen)에 적용하여, 실시예 2와 동일한 방법으로 단백질 복합체를 용출하였다.First, a binding test was conducted in vitro to determine whether the protein complex obtained in Example 2 actually binds to Annexin A2. First, A2-Ub-Ub (m1) -ub (m3) -Ub (m4) -p53, which is a
도 4은 상기 과정에 의해 용출된 단백질 복합체를 SDS-PAGE를 통해 나타낸 것이다. 도 4에서 보는 바와 같이, 아넥신 A2 결합 펩타이드를 포함하는 단백질인 단백질 복합체 #1, #2 모두 아넥신 A2 와 결합하였음을 확인할 수 있었다.
FIG. 4 shows SDS-PAGE of the protein complex eluted by the above procedure. As shown in FIG. 4,
실시예Example 4: 4: 암세포주를Cancer cells 이용한 융합 단백질의 항암효과 확인 Identification of antitumor effect of fusion protein used
실시예 2에서 제조한 단백질 복합체 #2의 세포 내 투과 및 암세포의 치료 효능을 확인하기 위하여, 삼중음성(triple negative) 유방암 세포주(HCC1806, ATCC)를 대상으로 상기 단백질 복합체의 투여에 의한 p18-p53 단백질의 항암 효과를 확인하였다.In order to confirm the therapeutic effect of intracellular permeation and cancer cells of the
상기 세포들을 각각 웰당 5×103개가 포함되도록 10%의 FBS가 포함된 RPMI 배지(Gibco BL)에 넣고, 단백질 복합체를 각각 0, 0.05625, 0.1125, 0.225, 0.45, 0.9uM의 농도로 처리한 다음, 96시간 동안 CO2 배양기에서 온도 37oC, 습도 85%, CO2 5%의 조건으로 배양하였다. 비교 실험으로는 상기 단백질 복합체 대신 단백질 복합체가 없는 buffer(0.1% arginine, 0.2% Tween20, 0.2% L-Glutathione, 1XPBS(pH 7.4))를 동일 양으로 처리하고 상기와 동일한 조건에서 배양하였다.The cells were each inoculated into RPMI medium (Gibco BL) containing 10% FBS so as to contain 5 × 10 3 cells per well. The protein complexes were treated at concentrations of 0, 0.05625, 0.1125, 0.225, 0.45 and 0.9 μM, respectively , And incubated for 96 hours in a CO 2 incubator at 37 ° C, 85% humidity and 5% CO 2 . As a comparative experiment, a protein-free buffer (0.1% arginine, 0.2
그 결과, 도 5에서 나타낸 바와 같이, 핵 위치 신호 도메인(NLS) 및 인 비보 안정 단백질(AAT)을 모두 포함하는 단백질 복합체 #2는 0.9uM의 농도에서 상기 모든 세포주의 세포 성장을 50% 억제함을 확인할 수 있었다.As a result, as shown in FIG. 5, the
<110> SAMSUNG ELECTRONICS CO., LTD. <120> Fusion protein comprising annexin A2 binding protein, p53 and p18, and composition comprising the fusion protein for preventing or treating cancer <130> DPP20125838KR <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> p53 fragment <400> 1 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn 1 5 10 <210> 2 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> P18 protein <400> 2 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly 1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn 20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly 35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp 50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala 65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val 85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys 100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser 115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala 130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Gly Ala Thr Asn Leu Gln 165 <210> 3 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> p18-p53 fragment fusion protein <400> 3 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly 1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn 20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly 35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp 50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala 65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val 85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys 100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser 115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala 130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Gly Ala Thr Asn Leu Gln Gly Ser Glu Thr Phe Ser Asp Leu 165 170 175 Trp Lys Leu Leu Pro Glu Asn 180 <210> 4 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Annexin A2 binding protein <400> 4 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Thr Gly Pro Gln 20 25 30 Cys Leu Cys Arg 35 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> wild type Ubiquitin <400> 5 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> membrane transfer sequence (MTS) <400> 6 Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 7 Lys Lys Lys Arg Lys 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 8 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 9 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 10 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> AAT <400> 10 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu 1 5 10 15 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser 20 25 30 Pro Val Ser Ile Ala Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys 35 40 45 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr 50 55 60 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg 65 70 75 80 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly 85 90 95 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp 100 105 110 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp 115 120 125 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr 130 135 140 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 145 150 155 160 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro 165 170 175 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val 180 185 190 Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile 195 200 205 Gln His Ala Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu 210 215 220 Gly Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu Gly Lys Leu Gln 225 230 235 240 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu 245 250 255 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile 260 265 270 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr 275 280 285 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala 290 295 300 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 305 310 315 320 Glu Lys Gly Thr Glu Ala Ala Pro Asp Asp Asp Asp Glu Ala Ile Pro 325 330 335 Arg <210> 11 <211> 2327 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide encoding fusion protein (A2-Ub-Ub(m1)-p18-AAT-p53) <400> 11 catatgagag gatcgcatca ccatcaccat cacgattacg atatcccaac gaccgaaaac 60 ctgtattttc agggatccgg cagcggcagc atgtgcatgc cgtgcttcac caccgaccac 120 cagatggctc gtaaatgcga cgactgctgc ggtggtaaag gtcgtggtaa atgcaccggt 180 ccgcagtgcc tgtgccgtgg cagcatgcag atcttcgtta aaaccctgac cggtaaaacc 240 atcaccctgg aagttgaacc gtctgacacc atcgaaaacg ttaaagctaa aatccaggac 300 aaagaaggta tcccgccgga ccagcagcgt ctgatcttcg ctggtaaaca gctggaagac 360 ggtcgtaccc tgtctgacta caacatccag aaagaatcta ccctgcacct ggttctgcgt 420 ctgcgtggtg gtatgcaaat ctttgttcgt accctgaccg gtcgtaccat caccctggaa 480 gttgaaccgt cggataccat agaaaatgta cgtgcgcgta tccaggatcg tgaaggtata 540 ccgccggatc agcagcgtct gatctttgcg ggtcgtcagc tggaagatgg tcgcaccctg 600 tcggattaca atattcaacg tgaatcgacc ctgcatctgg tgctgcgtct gcgcggtgcg 660 gaatttgccg cggtagcgct gctcccggcg gtcctgctgg ccttgctggc gcccggcagc 720 atggccgagc cttgggggaa cgagttggcg tccgcagctg ccagggggga cctagagcaa 780 cttactagtt tgttgcaaaa taatgtaaac gtcaatgcac aaaatggatt tggaaggact 840 gcgctgcagg ttatgaaact tggaaatccc gagattgcca ggagactgct acttagaggt 900 gctaatcccg atttgaaaga ccgaactggt aatgctgtca ttcatgatgc ggccagagca 960 ggtttcctgg acactttaca gactttgctg gagtttcaag ctgatgttaa catcgaggat 1020 aatgaaggga acctgccctt gcacttggct gccaaagaag gccacctccg ggtggtggag 1080 ttcctggtga agcacacggc cagcaatgtg gggcatcgga accataaggg ggacaccgcc 1140 tgtgatttgg ccaggctcta tgggaggaat gaggttgtta gcctgatgca ggcaaacggg 1200 gctgggggag ccacaaatct tcaaggcagc accttcaaca aaatcacccc gaacctggct 1260 gaattcgctt tctctctgta ccgtcagctg gctcaccagt ctaactctac caacatcttc 1320 ttctctccgg tttctatcgc taccgctttc gctatgctgt ctctgggtac caaagctgac 1380 acccacgacg aaatcctgga aggtctgaac ttcaacctga ccgaaatccc ggaagctcag 1440 atccacgaag gtttccagga actgctgcgt accctgaacc agccggactc tcagctgcag 1500 ctgaccaccg gtaacggtct gttcctgtct gaaggtctga aactggttga caaattcctg 1560 gaagacgtta aaaaactgta ccactctgaa gctttcaccg ttaacttcgg tgacaccgaa 1620 gaagctaaaa aacagatcaa cgactacgtt gaaaaaggta cccagggtaa aatcgttgac 1680 ctggttaaag aactggaccg tgacaccgtt ttcgctctgg ttaactacat cttcttcaaa 1740 ggtaaatggg aacgtccgtt cgaagttaaa gacaccgaag aagaagactt ccacgttgac 1800 caggttacca ccgttaaagt tccgatgatg aaacgtctgg gtatgttcaa catccagcac 1860 gctaaaaaac tgtcttcttg ggttctgctg atgaaatacc tgggtaacgc taccgctatc 1920 ttcttcctgc cggacgaagg taaactgcag cacctggaaa acgaactgac ccacgacatc 1980 atcaccaaat tcctggaaaa cgaagaccgt cgttctgctt ctctgcacct gccgaaactg 2040 tctatcaccg gtacctacga cctgaaatct gttctgggtc agctgggtat caccaaagtt 2100 ttctctaacg gtgctgacct gtctggtgtt accgaagaag ctccgctgaa actgtctaaa 2160 gctgttcaca aagctgttct gaccatcgac gaaaaaggta ccgaagctgc tccggacgac 2220 gacgacgaag ctatcccgcg tgaaacattt tcagacctat ggaaactact tcctgaaaac 2280 aaaaagaaga gaaagtaata actcgagcac caccaccacc accactg 2327 <210> 12 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mutant type Ubiquitin <400> 12 Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp 20 25 30 Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Ala 65 70 75 <210> 13 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub(m1) <400> 13 atgcaaatct ttgttcgtac cctgaccggt cgtaccatca ccctggaagt tgaaccgtcg 60 gataccatag aaaatgtacg tgcgcgtatc caggatcgtg aaggtatacc gccggatcag 120 cagcgtctga tctttgcggg tcgtcagctg gaagatggtc gcaccctgtc ggattacaat 180 attcaacgtg aatcgaccct gcatctggtg ctgcgtctgc gcggtgcg 228 <210> 14 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub(m2) <400> 14 atgcagattt tcgtgcgcac gctgacgggc cgcacgatca cgctggaggt tgagccgtcc 60 gacacgatag agaacgtacg cgcccgcatc caggaccgcg agggcatacc gccggaccag 120 cagcgcctga tctttgccgg ccgccagctg gaggacggcc gtacgctgtc cgactacaac 180 attcaacgcg agtccacgct gcatctggtg ctgagactgc gtggcgcc 228 <210> 15 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub(m3) <400> 15 atgcagatct tcgttcgcac cctgaccggc cgtacgatca cgctggaggt tgagccgtcc 60 gatacgatag agaatgtacg cgcgcgcatc caggatcgcg aaggcatacc gccggaccag 120 cagcgtctga tctttgcggg ccgtcagctg gaagacggtc gtaccctgtc ggactacaac 180 attcaacgtg agtcgacgct gcatctggtg ctgagactgc gtggtgcc 228 <210> 16 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub(m4) <400> 16 atgcaaattt ttgtgcgtac gctgacgggt cgcaccatca ccctggaagt tgaaccgtcg 60 gacaccatag aaaacgtacg tgcccgtatc caggaccgtg agggtatacc gccggatcag 120 cagcgcctga tctttgccgg tcgccagctg gaggatggcc gcacgctgtc cgattacaat 180 attcaacgcg aatccaccct gcatctggtg ctgcgtctgc gcggcgcg 228 <110> SAMSUNG ELECTRONICS CO., LTD. <120> Fusion protein comprising annexin A2 binding protein, p53 and p18, and composition comprising the fusion protein for preventing or treating cancer <130> DPP20125838 <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> p53 fragment <400> 1 Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn 1 5 10 <210> 2 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> P18 protein <400> 2 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly 1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn 20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly 35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp 50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala 65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val 85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys 100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser 115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala 130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Aly Thr Asn Leu Gln 165 <210> 3 <211> 183 <212> PRT <213> Artificial Sequence <220> <223> p18-p53 fragment fusion protein <400> 3 Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly 1 5 10 15 Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn 20 25 30 Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly 35 40 45 Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp 50 55 60 Leu Lys Asp Arg Thr Gly Asn Ala Val Ile His Asp Ala Ala Arg Ala 65 70 75 80 Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val 85 90 95 Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys 100 105 110 Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Thr Ala Ser 115 120 125 Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala 130 135 140 Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly 145 150 155 160 Ala Gly Aly Gly Aly Thr Asn Aly Gly Gly Gly Aly Gly Thr Phe Ser Asp Leu 165 170 175 Trp Lys Leu Leu Pro Glu Asn 180 <210> 4 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> Annexin A2 binding protein <400> 4 Met Cys Met Pro Cys Phe Thr Thr Asp His Gln Met Ala Arg Lys Cys 1 5 10 15 Asp Asp Cys Cys Gly Gly Lys Gly Arg Gly Lys Cys Thr Gly Pro Gln 20 25 30 Cys Leu Cys Arg 35 <210> 5 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> wild type Ubiquitin <400> 5 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> membrane transfer sequence (MTS) <400> 6 Ala Ala Val Ala Leu Ala Leu Pro Ala Val Leu Leu Ala Leu 1 5 10 15 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 7 Lys Lys Lys Arg Lys 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 8 Pro Lys Lys Lys Arg Lys Val 1 5 <210> 9 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> NLS domain <400> 9 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 10 <211> 337 <212> PRT <213> Artificial Sequence <220> <223> AAT <400> 10 Thr Phe Asn Lys Ile Thr Pro Asn Leu Ala Glu Phe Ala Phe Ser Leu 1 5 10 15 Tyr Arg Gln Leu Ala His Gln Ser Asn Ser Thr Asn Ile Phe Phe Ser 20 25 30 Pro Val Ser Ile Thr Ala Phe Ala Met Leu Ser Leu Gly Thr Lys 35 40 45 Ala Asp Thr His Asp Glu Ile Leu Glu Gly Leu Asn Phe Asn Leu Thr 50 55 60 Glu Ile Pro Glu Ala Gln Ile His Glu Gly Phe Gln Glu Leu Leu Arg 65 70 75 80 Thr Leu Asn Gln Pro Asp Ser Gln Leu Gln Leu Thr Thr Gly Asn Gly 85 90 95 Leu Phe Leu Ser Glu Gly Leu Lys Leu Val Asp Lys Phe Leu Glu Asp 100 105 110 Val Lys Lys Leu Tyr His Ser Glu Ala Phe Thr Val Asn Phe Gly Asp 115 120 125 Thr Glu Glu Ala Lys Lys Gln Ile Asn Asp Tyr Val Glu Lys Gly Thr 130 135 140 Gln Gly Lys Ile Val Asp Leu Val Lys Glu Leu Asp Arg Asp Thr Val 145 150 155 160 Phe Ala Leu Val Asn Tyr Ile Phe Phe Lys Gly Lys Trp Glu Arg Pro 165 170 175 Phe Glu Val Lys Asp Thr Glu Glu Glu Asp Phe His Val Asp Gln Val 180 185 190 Thr Thr Val Lys Val Pro Met Met Lys Arg Leu Gly Met Phe Asn Ile 195 200 205 Gln His Ala Lys Lys Leu Ser Ser Trp Val Leu Leu Met Lys Tyr Leu 210 215 220 Gly Asn Ala Thr Ala Ile Phe Leu Pro Asp Glu Gly Lys Leu Gln 225 230 235 240 His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr Lys Phe Leu Glu 245 250 255 Asn Glu Asp Arg Arg Ser Ala Ser Leu His Leu Pro Lys Leu Ser Ile 260 265 270 Thr Gly Thr Tyr Asp Leu Lys Ser Val Leu Gly Gln Leu Gly Ile Thr 275 280 285 Lys Val Phe Ser Asn Gly Ala Asp Leu Ser Gly Val Thr Glu Glu Ala 290 295 300 Pro Leu Lys Leu Ser Lys Ala Val His Lys Ala Val Leu Thr Ile Asp 305 310 315 320 Glu Lys Gly Thr Glu Ala Ala Pro Asp Asp Asp Asp Glu Ala Ile Pro 325 330 335 Arg <210> 11 <211> 2327 <212> DNA <213> Artificial Sequence <220> The polynucleotide encoding fusion protein (A2-Ub-Ub (m1) -p18-AAT-p53) <400> 11 catatgagag gatcgcatca ccatcaccat cacgattacg atatcccaac gaccgaaaac 60 ctgtattttc agggatccgg cagcggcagc atgtgcatgc cgtgcttcac caccgaccac 120 cagatggctc gtaaatgcga cgactgctgc ggtggtaaag gtcgtggtaa atgcaccggt 180 ccgcagtgcc tgtgccgtgg cagcatgcag atcttcgtta aaaccctgac cggtaaaacc 240 atcaccctgg aagttgaacc gtctgacacc atcgaaaacg ttaaagctaa aatccaggac 300 aaagaaggta tcccgccgga ccagcagcgt ctgatcttcg ctggtaaaca gctggaagac 360 ggtcgtaccc tgtctgacta caacatccag aaagaatcta ccctgcacct ggttctgcgt 420 ctgcgtggtg gtatgcaaat ctttgttcgt accctgaccg gtcgtaccat caccctggaa 480 gttgaaccgt cggataccat agaaaatgta cgtgcgcgta tccaggatcg tgaaggtata 540 ccgccggatc agcagcgtct gatctttgcg ggtcgtcagc tggaagatgg tcgcaccctg 600 tcggattaca atattcaacg tgaatcgacc ctgcatctgg tgctgcgtct gcgcggtgcg 660 gaatttgccg cggtagcgct gctcccggcg gtcctgctgg ccttgctggc gcccggcagc 720 atggccgagc cttgggggaa cgagttggcg tccgcagctg ccagggggga cctagagcaa 780 cttactagtt tgttgcaaaa taatgtaaac gtcaatgcac aaaatggatt tggaaggact 840 gcgctgcagg ttatgaaact tggaaatccc gagattgcca ggagactgct acttagaggt 900 gctaatcccg atttgaaaga ccgaactggt aatgctgtca ttcatgatgc ggccagagca 960 ggtttcctgg acactttaca gactttgctg gagtttcaag ctgatgttaa catcgaggat 1020 aatgaaggga acctgccctt gcacttggct gccaaagaag gccacctccg ggtggtggag 1080 ttcctggtga agcacacggc cagcaatgtg gggcatcgga accataaggg ggacaccgcc 1140 tgtgatttgg ccaggctcta tgggaggaat gaggttgtta gcctgatgca ggcaaacggg 1200 gctgggggag ccacaaatct tcaaggcagc accttcaaca aaatcacccc gaacctggct 1260 gaattcgctt tctctctgta ccgtcagctg gctcaccagt ctaactctac caacatcttc 1320 ttctctccgg tttctatcgc taccgctttc gctatgctgt ctctgggtac caaagctgac 1380 acccacgacg aaatcctgga aggtctgaac ttcaacctga ccgaaatccc ggaagctcag 1440 atccacgaag gtttccagga actgctgcgt accctgaacc agccggactc tcagctgcag 1500 ctgaccaccg gtaacggtct gttcctgtct gaaggtctga aactggttga caaattcctg 1560 gaagacgtta aaaaactgta ccactctgaa gctttcaccg ttaacttcgg tgacaccgaa 1620 gaagctaaaa aacagatcaa cgactacgtt gaaaaaggta cccagggtaa aatcgttgac 1680 ctggttaaag aactggaccg tgacaccgtt ttcgctctgg ttaactacat cttcttcaaa 1740 ggtaaatggg aacgtccgtt cgaagttaaa gacaccgaag aagaagactt ccacgttgac 1800 caggttacca ccgttaaagt tccgatgatg aaacgtctgg gtatgttcaa catccagcac 1860 gctaaaaaac tgtcttcttg ggttctgctg atgaaatacc tgggtaacgc taccgctatc 1920 ttcttcctgc cggacgaagg taaactgcag cacctggaaa acgaactgac ccacgacatc 1980 atcaccaaat tcctggaaaa cgaagaccgt cgttctgctt ctctgcacct gccgaaactg 2040 tctatcaccg gtacctacga cctgaaatct gttctgggtc agctgggtat caccaaagtt 2100 ttctctaacg gtgctgacct gtctggtgtt accgaagaag ctccgctgaa actgtctaaa 2160 gctgttcaca aagctgttct gaccatcgac gaaaaaggta ccgaagctgc tccggacgac 2220 gacgacgaag ctatcccgcg tgaaacattt tcagacctat ggaaactact tcctgaaaac 2280 aaaaagaaga gaaagtaata actcgagcac caccaccacc accactg 2327 <210> 12 <211> 76 <212> PRT <213> Artificial Sequence <220> <223> mutant type Ubiquitin <400> 12 Met Gln Ile Phe Val Arg Thr Leu Thr Gly Arg Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Arg Ala Arg Ile Gln Asp 20 25 30 Arg Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Arg 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Arg Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Ala 65 70 75 <210> 13 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m1) <400> 13 atgcaaatct ttgttcgtac cctgaccggt cgtaccatca ccctggaagt tgaaccgtcg 60 gataccatag aaaatgtacg tgcgcgtatc caggatcgtg aaggtatacc gccggatcag 120 cagcgtctga tctttgcggg tcgtcagctg gaagatggtc gcaccctgtc ggattacaat 180 attcaacgtg aatcgaccct gcatctggtg ctgcgtctgc gcggtgcg 228 <210> 14 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m2) <400> 14 atgcagattt tcgtgcgcac gctgacgggc cgcacgatca cgctggaggt tgagccgtcc 60 gacacgatag agaacgtacg cgcccgcatc caggaccgcg agggcatacc gccggaccag 120 cagcgcctga tctttgccgg ccgccagctg gaggacggcc gtacgctgtc cgactacaac 180 attcaacgcg agtccacgct gcatctggtg ctgagactgc gtggcgcc 228 <210> 15 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m3) <400> 15 atgcagatct tcgttcgcac cctgaccggc cgtacgatca cgctggaggt tgagccgtcc 60 gatacgatag agaatgtacg cgcgcgcatc caggatcgcg aaggcatacc gccggaccag 120 cagcgtctga tctttgcggg ccgtcagctg gaagacggtc gtaccctgtc ggactacaac 180 attcaacgtg agtcgacgct gcatctggtg ctgagactgc gtggtgcc 228 <210> 16 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Ub (m4) <400> 16 atgcaaattt ttgtgcgtac gctgacgggt cgcaccatca ccctggaagt tgaaccgtcg 60 gacaccatag aaaacgtacg tgcccgtatc caggaccgtg agggtatacc gccggatcag 120 cagcgcctga tctttgccgg tcgccagctg gaggatggcc gcacgctgtc cgattacaat 180 attcaacgcg aatccaccct gcatctggtg ctgcgtctgc gcggcgcg 228
Claims (15)
An annexin A2 binding protein, a p53 protein or a p53 protein fragment consisting of the amino acid sequence of SEQ ID NO: 1, and a p18 protein.
생체외 안정화 단백질 (in vitro stabilization protein),
막 투과 서열 (membrane transfer sequence, MTS) 도메인,
핵-세포질 신호 (nucleus-cytoplasm signal) 도메인, 및
생체내 안정화 단백질 (in vivo stabilization protein)
로 이루어진 군에서 선택된 1종 이상의 폴리펩타이드를 추가로 포함하는 것인, 융합 단백질.
2. The method of claim 1, wherein the fusion protein comprises
An in vitro stabilization protein,
The membrane transfer sequence (MTS) domain,
A nucleus-cytoplasm signal domain, and
In vivo stabilization protein
≪ / RTI > wherein the fusion polypeptide further comprises at least one polypeptide selected from the group consisting of:
상기 야생형 유비퀴틴은 서열번호 5의 아미노산 서열로 이루어진 것이고,
상기 돌연변이 유비퀴틴은 상기 야생형 유비퀴틴의 아미노산 서열의 6, 11, 27, 29, 33, 48 및 63번째에 존재하는 Lys 중 하나 이상이 Arg으로 치환된 것, 상기 야생형 유비퀴틴의 76번째에 존재하는 Gly이 Ala으로 치환된 것, 또는 상기 야생형 유비퀴틴의 아미노산 서열의 6, 11, 27, 29, 33, 48 및 63번째에 존재하는 Lys 중 하나 이상이 Arg으로 치환되고 76번째에 존재하는 Gly이 Ala으로 치환된 것이고,
상기 유비퀴틴-유사 단백질은 Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 및 ISG15로 구성된 군으로부터 선택된 것인,
융합 단백질.
3. The method according to claim 2, wherein the exo-stabilizing protein is at least one selected from the group consisting of wild-type ubiquitin, mutant ubiquitin, and ubiquitin-
The wild-type ubiquitin comprises the amino acid sequence of SEQ ID NO: 5,
Wherein the mutant ubiquitin is one in which at least one of the Lys present at positions 6, 11, 27, 29, 33, 48 and 63 of the amino acid sequence of the wild-type ubiquitin is substituted with Arg, Gly present at the 76th position of the wild- Ala, or at least one of Lys present at positions 6, 11, 27, 29, 33, 48 and 63 of the amino acid sequence of wild-type ubiquitin is substituted with Arg and Gly existing at the 76th position is substituted with Ala However,
Wherein said ubiquitin-like protein is selected from the group consisting of Nedd8, SUMO-1, SUMO-2, NUB1, PIC1, UBL3, UBL5 and ISG15.
Fusion protein.
3. The fusion protein of claim 2, wherein the transmembrane sequence domain is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7.
3. The fusion protein of claim 2, wherein the nuclear-cytoplasmic signal domain is a nucleus location sequence (NLS) domain or a nucleus export sequence (NES) domain.
6. The fusion protein according to claim 5, wherein the NLS domain is a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO:
The method according to claim 2, wherein the at least one member selected from the group consisting of the in vivo stabilizing protein AAT (alpha 1 antitrypsin), serum albumin, serum albumin binding peptide (SABP), Fc and PEG (polyethyleneglycol) , Fusion protein.
아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질;
아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질;
p53 단백질, p18 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질; 및
p18 단백질, p53 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질
로 이루어진 군에서 선택된 것인, 융합 단백질.
2. The method of claim 1, wherein the fusion protein comprises
A fusion protein comprising the annexin A2 binding protein, the p53 protein and the p18 protein in the order of N-terminal to C-terminal;
A fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of N-terminal to C-terminal;
a fusion protein comprising the p53 protein, the p18 protein and the annexin A2 binding protein in the order of N-terminal to C-terminal; And
p18 protein, p53 protein and annexin A2 binding protein in the order of N-terminal to C-terminal
≪ / RTI >
아넥신 A2 결합단백질, p53 단백질 및 p18 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p53 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p18 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질;
아넥신 A2 결합단백질, p18 단백질 및 p53 단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, p18 단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, p53 단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질;
p53 단백질, p18 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, 아넥신 A2 결합단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, 아넥신 A2 결합단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질; 및
p18 단백질, p53 단백질 및 아넥신 A2 결합단백질을 N-말단에서 C-말단의 순서로 포함하는 융합 단백질에 있어서, 아넥신 A2 결합단백질의 N-말단 방향에 생체외 안정화 단백질, 막 투과 서열 도메인 및 핵-세포질 신호 도메인으로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하거나, 아넥신 A2 결합단백질의 C-말단 방향에 막 투과 서열 도메인, 핵-세포질 신호 도메인 및 생체내 안정화 단백질로 이루어진 군에서 선택되는 1종 이상을 추가로 포함하는 것인 융합 단백질
로 이루어진 군에서 선택된 것인, 융합 단백질.
9. The method of claim 8, wherein the fusion protein comprises
Terminal fusion protein comprising the annexin A2 binding protein, the p53 protein and the p18 protein in the order of the N-terminal to the C-terminal in the N-terminal direction of the p53 protein, the in vitro stabilization protein, the transmembrane sequence domain, Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain and in vivo stabilizing protein in the C-terminal direction of p18 protein A fusion protein further comprising;
Terminal fusion protein comprising the annexin A2 binding protein, the p18 protein and the p53 protein in the order of the N-terminal to the C-terminal, wherein the in vitro stabilization protein, the transmembrane sequence domain and the nuclear- Signal domain, or at least one selected from the group consisting of transmembrane sequence domain, nuclear-cytoplasmic signal domain, and in vivo stabilization protein in the C-terminal direction of p53 protein A fusion protein further comprising;
a fusion protein comprising p53 protein, p18 protein, and annexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a membrane- Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein A fusion protein which further comprises at least one selected; And
a fusion protein comprising p18 protein, p53 protein and anexin A2 binding protein in the order of N-terminal to C-terminal, wherein an exo-stabilizing protein, a transmembrane sequence domain and / Nuclear-cytoplasmic signal domain, or in the group consisting of the transmembrane sequence domain, the nuclear-cytoplasmic signal domain and the in vivo stabilizing protein in the C-terminal direction of the annexin A2 binding protein Wherein the fusion protein further comprises one or more selected < RTI ID = 0.0 >
≪ / RTI >
10. A polynucleotide encoding the fusion protein of any one of claims 1 to 9.
10. A recombinant vector comprising the polynucleotide of claim 10.
A cell transformed with the recombinant vector of claim 11.
14. A method for producing the fusion protein of claim 1 comprising culturing the cell of claim 12.
9. A pharmaceutical composition for preventing or treating cancer comprising the fusion protein of any one of claims 1 to 9 as an active ingredient.
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PCT/KR2014/006510 WO2015009082A1 (en) | 2013-07-19 | 2014-07-17 | Fusion protein comprising annexin a2 binding protein, p53 protein, and p18 protein, and composition containing same for preventing or treating cancer |
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