KR100980315B1 - Method for large scale preparation of procaspases, which can be artificially activated, in E. coli expression system and their activation - Google Patents

Method for large scale preparation of procaspases, which can be artificially activated, in E. coli expression system and their activation Download PDF

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KR100980315B1
KR100980315B1 KR1020080059645A KR20080059645A KR100980315B1 KR 100980315 B1 KR100980315 B1 KR 100980315B1 KR 1020080059645 A KR1020080059645 A KR 1020080059645A KR 20080059645 A KR20080059645 A KR 20080059645A KR 100980315 B1 KR100980315 B1 KR 100980315B1
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정상전
강효진
이영미
김문일
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Abstract

본 발명은 재조합 카스파제 및 그의 제조방법에 관한 것으로, 구체적으로 카스파제의 자가활성 인식부위를 비시스테인계 단백질 가수분해효소에 의해 인식될 수 있도록 치환함으로써 대장균을 이용한 과발현시 자가활성화를 나타내지 않아 세포독성 없이 과발현될 수 있는 재조합 카스파제 발현벡터에 관한 것이다. 본 발명의 자가활성화 방지형 재조합 카스파제 전구체에 의해 대량생산된 카스파제는 신약개발 및 효소의 생물학적 기능 연구 등을 위하여 널리 사용될 수 있다.The present invention relates to a recombinant caspase and a method for preparing the same. Specifically, by replacing a self-activating recognition site of caspase so that it can be recognized by a bicysteine-based proteolytic enzyme, the cell does not exhibit self-activation upon overexpression using E. coli. A recombinant caspase expression vector that can be overexpressed without toxicity. Caspases mass-produced by the anti-activation recombinant caspase precursor of the present invention can be widely used for drug discovery and biological function research of enzymes.

카스파제, 대량 생산, 돌연변이, 활성화, 신약개발 Caspase, mass production, mutation, activation, drug development

Description

대장균시스템을 이용한 인위적 활성화가 가능한 카스파제 전구체 대량생산 및 활성화 방법{Method for large scale preparation of procaspases, which can be artificially activated, in E. coli expression system and their activation}Method for large scale preparation of procaspases, which can be artificially activated, in E. coli expression system and their activation}

본 발명은 재조합 카스파제 및 그의 제조방법에 관한 것이다.The present invention relates to recombinant caspases and methods for their preparation.

세포사멸(apoptosis) 과정은 불필요한 세포의 제거를 위해 세포내의 효소들이 활발하게 관여하는 능동적인 죽음기작이다. 이러한 기작은 각종 기관의 형태 형성 과정에서 불필요한 세포를 제거하는 데 있어 필수적인 현상이다. 또한 자가면역질환(autoimmune disease)이 세포사멸의 장애로 인하여 발생한다는 보고가 있었으며(Miller LJ & Marx J, Science 281:1301-1326, 1998), 항암 치료를 위한 유전자 요법에서 세포사멸을 이용하여 암세포의 사멸을 유도시키는 방법들이 보고된 바 있다(Zhang XM et al., EMBO J 16:2271-2281, 1997).Apoptosis is an active death mechanism in which enzymes in cells are actively involved in the removal of unnecessary cells. This mechanism is essential for removing unnecessary cells in the formation of various organs. In addition, autoimmune disease has been reported to be caused by apoptosis disorder (Miller LJ & Marx J, Science 281: 1301-1326, 1998), and cancer cells using apoptosis in gene therapy for chemotherapy Methods have been reported to induce the killing of (Zhang XM et al. , EMBO J 16: 2271-2281, 1997).

카스파제(Caspase)는 세포사멸에 관련된 신호전달 과정의 마지막 단계에서 일련의 다른 세포사멸 단백질들을 활성화하여 DNA와 핵을 분해하여 단편화시키고, 염색체를 응축시켜 실제로 세포 사멸을 일으키는 주된 단백분해 효소이다. 카스파제는 파킨슨 질환과 알츠하이머병과 같은 자가면역질환과 관련이 있을 것으로 추측되고 있으며(대한민국 공개특허: 제 2002-0092042 호), 현재 세포사멸 기전 연구 및 신약개발연구의 대상이 되고 있는 중요한 효소이다. 카스파제는 프로도메인(prodomain), 대단위체(large subunit), 소단위체(small subunit)의 세 개의 도메인으로 구성되며, 세포내에서 비활성형 프로카스파제(procaspase)로 발현이 된다. 세포사멸 신호가 전달되면 비활성형의 카스파제는 자가절단(autolysis) 반응을 거쳐 세 개의 도메인으로 잘려서 프로도메인을 제거하게 된다. 각각 2개씩의 대단위체와 소단위체가 결합하여 4개의 단위체(subunit)가 합쳐지면서 활성형 카스파제로 바뀌게 된다(도 2 참조). 카스파제의 활성 부위에는 시스테인(cysteine)이 있으며, 기질이 되는 단백질의 아미노산 서열 중 아스파틱산(aspartic acid)을 특이적으로 인식하여 펩티드 결합을 절단하는 효소이다. 현재까지 11개 종류의 카스파제들이 발견되었는데, 그 중 1, 4, 5 및 11번 카스파제는 염증을 유발하는 사이토카인(cytokine)의 활성과 관련이 있고, 그 외의 카스파제들은 세포사멸에서 중요한 역할을 하는 것으로 알려져 있다(Kumar S, Trends Biochem Sci 20:198-202, 1995; Martin SJ & Green DR, Cell 82:349-352, 1995; Kumar S & Lavin MF, Cell Death Differ 3:255-267, 1996; Nicholson DW & Thornberry NA, Trends Biochem Sci 22:299-306, 1997; Chinnaiyan AM & Dixit VM, Semin Immunol 9:69-76, 1997).Caspase is the main proteolytic enzyme that activates a series of other apoptosis proteins to break down DNA and nuclei, fragment them, condense chromosomes and actually cause cell death at the end of the signaling process involved in cell death. Caspase is thought to be associated with autoimmune diseases such as Parkinson's disease and Alzheimer's disease (Korean Patent Publication No. 2002-0092042), and is an important enzyme that is currently being studied for apoptosis mechanism research and drug development research. Caspase consists of three domains: prodomain, large subunit, and small subunit, and is expressed in the cell as inactive procaspase. When apoptosis signal is transmitted, the inactive caspase is cut into three domains through an autolysis reaction to remove the prodomain. Two large units and two subunits each combine to change the active caspase as four subunits are combined (see FIG. 2). The active site of caspase is cysteine, an enzyme that cleaves peptide bonds by specifically recognizing aspartic acid in the amino acid sequence of a protein that is a substrate. To date, eleven types of caspases have been discovered, of which caspases 1, 4, 5 and 11 are associated with cytokine activity that causes inflammation, and other caspases are important in apoptosis. (Kumar S, Trends Biochem Sci 20: 198-202, 1995; Martin SJ & Green DR, Cell 82: 349-352, 1995; Kumar S & Lavin MF, Cell Death Differ 3: 255-267). , 1996; Nicholson DW & Thornberry NA, Trends Biochem Sci 22: 299-306, 1997; Chinnaiyan AM & Dixit VM, Semin Immunol 9: 69-76, 1997).

카스파제가 퇴행성 중추신경계 질환과 면역질환 등에 관여하는 것이 알려지면서 신약개발의 표적으로 연구되고 있고, 종전의 세포사멸 관련 연구와 더불어 많 은 연구자가 이 효소를 활용함에 따라 그 수요가 급증하고 있다. 따라서 카스파제를 재조합 단백질 형태로 대량생산하는 것이 필요하나, 카스파제와 같이 세포사멸에 관여하는 단백질을 대장균 시스템에서 전체 유전자를 이용하여 과발현시킬 경우, 프로카스파제의 미약한 활성에 의하여 특정부위가 가수분해되면서 자가 활성화(Autoactivation 또는 automaturation)되어 세포독성을 보이고 아직 그 자세한 작용기작이 규명되지 않아 대량 발현 및 정제가 매우 어렵다.As it is known that caspase is involved in degenerative central nervous system diseases and immune diseases, it is being studied as a target of drug development. As a result, many researchers have used this enzyme along with previous apoptosis studies, and the demand is rapidly increasing. Therefore, it is necessary to mass-produce caspase in the form of recombinant protein.However, when overexpressing a protein involved in apoptosis such as caspase by using the entire gene in E. coli system, the specific site may be lost due to the weak activity of procaspase. Hydrolysis results in cytotoxicity due to autoactivation or automaturation, and its detailed mechanism of action has not been elucidated, making mass expression and purification very difficult.

현재 카스파제의 대량발현 및 정제를 위하여 가장 널리 사용되는 방법은 대게 서브유닛별로 각각 발현시켜서 불용성 단백질을 변성 재생(renaturation or refolding)하거나, 아주 낮은 수준으로 발현시킨 후 소량씩 정제하여 사용하고 있다(Nancy A et al., J Biol Chem 272:17907-17911, 1997; Paul R et al., J Biol Chem 270:9378-9383, 1995; Henning R et al., J Biol Chem 272:25719-25723, 1997). 그러나 이러한 단백질 재생 방법은 전체 단백질을 수용성으로 한 번에 발현시키는 방법에 비해 많은 비용과 노력이 요구되는 과정으로써 산업화에는 적당하지 않은 방법으로 인식되고 있다. 또한 어렵게 수득한 활성형 카스파제는 장기간 보존할 경우 자가 가수분해되어 활성을 잃어버리기 쉽다.Currently, the most widely used methods for mass expression and purification of caspases are usually expressed by subunits to regenerate or refold insoluble proteins, or to express them at very low levels and to purify them in small amounts. Nancy A et al. , J Biol Chem 272: 17907-17911, 1997; Paul R et al. , J Biol Chem 270: 9378-9383, 1995; Henning R et al. , J Biol Chem 272: 25719-25723, 1997 ). However, this protein regeneration method is a process that requires a lot of cost and effort compared to the method of expressing the entire protein at once in water solubility is recognized as an unsuitable method for industrialization. In addition, the hardly obtained active caspase is prone to self-hydrolysis and loss of activity when stored for a long time.

이에, 본 발명자들은 유전공학적 방법을 이용하여 카스파제의 활성화 과정에서 카스파제를 포함하는 시스테인계 단백질 가수분해효소(Cysteine proteases)에 의해 가수분해되는 아미노산 서열을 다른 계열의 특정 가수분해 효소에 의해 가수분해되는 아미노산 서열로 변화시킨 카스파제 전구체를 발현하는 대장균 발현시스 템을 개발함으로써, 상기 시스템으로부터 발현된 카스파제 전구체가 발현과정에서 자가활성화되는 것이 방지되고, 수용성 단백질로 과발현되며, 정제 및 보관이 용이하고 특정 가수분해 효소에 의해 활성형 카스파제가 되었을 때에 천연 카스파제와 동일한 활성을 나타내므로, 본 발명이 카스파제 전구체 대량생산에 유용하게 이용될 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors hydrolyzed the amino acid sequence hydrolyzed by cysteine proteases including caspase during the activation of caspase using a genetic engineering method by a specific hydrolase of another series. By developing an E. coli expression system expressing a caspase precursor changed to an amino acid sequence to be degraded, the caspase precursor expressed from the system is prevented from self-activating during expression, overexpressed as a water soluble protein, purified and stored The present invention was completed by confirming that the present invention can be usefully used for mass production of caspase precursors, since it exhibits the same activity as natural caspase when it is easily and becomes an active caspase by a specific hydrolase.

본 발명의 목적은 카스파제(Caspase) 내에 존재하는 시스테인계 단백질 가수분해효소의 인식부위가 비시스테인계 단백질 가수분해효소(Non-Cysteine proteases)의 인식부위로 치환된 재조합 카스파제 전구체 및 이를 제조하는 방법을 제공하는 것이다.An object of the present invention is to prepare a recombinant caspase precursor in which a recognition site of cysteine-based proteolytic enzymes present in Caspase is replaced with a recognition site of non-Cysteine proteases, and a preparation thereof. To provide a way.

본 발명의 다른 목적은 카스파제 내에 존재하는 시스테인계 단백질 가수분해효소의 인식부위의 아미노산 잔기가 상기 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산으로 치환되고, 상기 시스테인계 단백질 가수분해효소의 인식부위의 주변에 비시스테인계 단백질 가수분해효소 인식부위가 삽입된 재조합 카스파제 전구체 및 이를 제조하는 방법을 제공하는 것이다.Another object of the present invention is to replace the amino acid residues of the recognition site of the cysteine-based proteolytic enzymes present in the caspase to amino acids that the cysteine-based proteolytic enzymes cannot recognize, and to recognize the cysteine-based proteolytic enzymes. The present invention provides a recombinant caspase precursor in which a bicysteine-based protease recognition site is inserted around a site, and a method of manufacturing the same.

본 발명의 다른 목적은 상기 재조합 카스파제 전구체를 비시스테인계 단백질 가수분해효소로 활성화하는 방법 및 상기 방법에 의해 활성화된 재조합 카스파제를 제공하는 것이다.Another object of the present invention is to provide a method for activating the recombinant caspase precursor with a bicysteine-based proteolytic enzyme and a recombinant caspase activated by the method.

본 발명의 다른 목적은 상기 활성화된 재조합 카스파제를 이용하여 카스파제 활성 저해제 또는 촉진제의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a caspase activity inhibitor or promoter using the activated recombinant caspase.

상기 목적을 달성하기 위하여, 본 발명은 카스파제(Caspase) 내에 존재하는 시스테인계 단백질 가수분해효소의 인식부위가 비시스테인계 단백질 가수분해효 소(Non-Cysteine proteases)의 인식부위로 치환된 재조합 카스파제 전구체를 제공한다.In order to achieve the above object, the present invention provides a recombinant caspa in which a recognition site of cysteine-based proteolytic enzymes present in caspase is replaced with a recognition site of non-cysteine proteases. It provides a first precursor.

또한, 본 발명은 카스파제 3의 28번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산으로 치환하고, 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산이 삽입된 재조합 카스파제 3 전구체를 제공한다.In addition, the present invention substitutes a peptide consisting of six consecutive amino acids including the 28th amino acid of caspase 3 with 6 consecutive amino acids recognized and cleaved by thrombin, and thrombin between 180 and 181 amino acids. It provides a recombinant caspase 3 precursor inserted with six consecutive amino acids recognized and cleaved by.

또한, 본 발명은 카스파제 내에 존재하는, 적어도 하나 이상의 시스테인계 단백질 가수분해효소의 인식부위 내의 하나 이상의 아미노산 잔기가 상기 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산으로 치환되고, 상기 시스테인계 단백질 가수분해효소의 인식부위의 주변에 비시스테인계 단백질 가수분해효소 인식부위가 삽입된 재조합 카스파제 전구체를 제공한다.In addition, the present invention is the one or more amino acid residues in the recognition site of at least one cysteine-based proteolytic enzyme present in the caspase is replaced with an amino acid that the cysteine-based proteolytic enzyme is not recognized, the cysteine-based protein The present invention provides a recombinant caspase precursor in which a bicysteine-based protease recognition site is inserted around a recognition site of a hydrolase.

또한, 본 발명은 상기 재조합 카스파제 전구체 중 어느 하나를 암호화하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding any one of the recombinant caspase precursors.

또한, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현벡터를 제공한다.In addition, the present invention provides an expression vector comprising the polynucleotide.

또한, 본 발명은 상기 발현벡터가 형질도입된 형질전환체를 제공한다.The present invention also provides a transformant to which the expression vector is transduced.

또한, 1) 상기 재조합 카스파제 전구체 중 어느 하나를 암호화하는 폴리뉴클레오티드를 포함하는 발현벡터를 제조하는 단계;In addition, 1) preparing an expression vector comprising a polynucleotide encoding any one of the recombinant caspase precursor;

2) 상기 발현벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계; 및,2) preparing a transformant by introducing the expression vector into a host cell; And,

3) 상기 형질전환체를 배양하여 재조합 단백질의 발현을 유도하고 이를 수득 하는 단계로 구성되는 재조합 카스파제 전구체의 제조방법을 제공한다.3) culturing the transformant to induce the expression of a recombinant protein and to provide a method for producing a recombinant caspase precursor consisting of obtaining the same.

또한, 본 발명은 상기 재조합 카스파제 전구체를 비시스테인계 단백질 가수분해효소로 처리하는 단계를 포함하는 상기 재조합 카스파제 전구체를 활성화하는 방법을 제공한다.The present invention also provides a method of activating the recombinant caspase precursor, comprising treating the recombinant caspase precursor with a bicysteine-based proteolytic enzyme.

또한, 본 발명은 상기 방법에 의해 활성화된 재조합 카스파제를 제공한다.The present invention also provides a recombinant caspase activated by the above method.

아울러, 본 발명은 1) 상기 활성화된 재조합 카스파제에 카스파제 특이적 기질 및 후보 물질을 처리하는 단계;In addition, the present invention comprises the steps of: 1) treating the caspase specific substrate and the candidate substance to the activated recombinant caspase;

2) 상기 활성화된 재조합 카스파제의 기질에 대한 효소 활성을 측정하는 단계; 및,2) measuring enzyme activity on the substrate of the activated recombinant caspase; And,

3) 후보 물질을 처리하지 않은 대조군과 비교하여 상기 활성화된 재조합 카스파제의 효소 활성을 감소시키거나 증진시킨 후보 물질을 선별하는 단계를 포함하는 카스파제 활성 저해제 또는 촉진제의 스크리닝 방법을 제공한다.3) A method for screening a caspase activity inhibitor or promoter comprising the step of selecting a candidate substance that reduces or enhances the enzymatic activity of the activated recombinant caspase compared to a control that has not been treated with the candidate substance.

이하, 본 발명에서 사용한 용어를 설명한다.Hereinafter, the term used by this invention is demonstrated.

"시스테인계 단백질 가수분해효소(Cysteine proteases)"는 카스파제를 포함하는 활성 부위에 시스테인(cysteine)이 있으며, 기질이 되는 단백질의 아미노산 서열 중 아스파틱산(aspartic acid)을 특이적으로 인식하여 아미노산 결합을 절단하는 단백질 분해 효소를 의미한다."Cysteine proteases" have cysteine at the active site including caspase and bind amino acids by specifically recognizing aspartic acid in the amino acid sequence of the protein that becomes the substrate. Means proteolytic enzymes that cleave it.

"비시스테인계 단백질 가수분해효소(Non-Cysteine proteases)"는 시스테인계 단백질 가수분해효소(Cysteine proteases)를 제외한 모든 단백질 분해 효소를 의미한다."Non-Cysteine proteases" means all proteolytic enzymes except for cysteine proteases.

"메가프라이머(megaprimer) 방법"은 PCR 방법의 일종으로 돌연변이를 유도하고자 하는 위치가 단백질의 중간 부분에 있어서 한 번에 돌연변이를 완성할 수 없는 경우 2단계에 걸친 PCR을 진행하여 돌연변이를 완성할 때 사용하는 방법이다. 예를 들면, 유발하고자 하는 돌연변이의 뉴클레오티드를 포함한 정방향 프라이머와 단백질 C-말단의 뉴클레오티드를 포함하는 역방향 프라이머를 이용하여 PCR증폭을 유도하면 정방향 프라이머 보다 앞 부분에 있는 N-말단에 대한 정보가 없는 DNA를 얻게 된다. 이때 생성된 DNA를 메가프라이머라고 칭한다. 다음 단계에서 메가프라이머를 역방향 프라이머로 사용하고 단백질의 N-말단을 포함하는 올리고 DNA를 정방향 프라이머로 사용하여 PCR을 진행하면 원하는 위치에 돌연변이가 유도된 전장 단백질을 암호화하는 DNA를 얻을 수 있다. 만약, 초기에 돌연변이를 포함한 역방향 프라이머와 단백질 N-말단정보를 포함하는 역방향 프라이머를 이용하여 메가프라이머를 얻게 되면 다음 단계에서는 C-말단 정보를 포함하는 역방향 프라이머를 함께 사용하여 돌연변이 된 전장 단백질을 암호화하는 DNA를 얻을 수 있다. 이와 같은 방법을 메가프라이머 방법이라고 한다.The "megaprimer method" is a PCR method. When the position to induce a mutation is not able to complete the mutation at once in the middle of the protein, the PCR is performed in two stages to complete the mutation. How to use. For example, inducing PCR amplification using a forward primer containing the nucleotide of the mutant to be induced and a reverse primer comprising the nucleotide of the protein C-terminal results in DNA without information about the N-terminal preceding the forward primer. You get The DNA produced at this time is called a megaprimer. In the next step, PCR is performed using a megaprimer as a reverse primer and oligo DNA including the N-terminus of the protein as a forward primer to obtain DNA encoding a full-length protein in which a mutation is induced at a desired position. If a megaprimer is obtained using a reverse primer containing a mutation and a reverse primer containing protein N-terminal information, the next step is to encode a mutated full-length protein using a reverse primer containing C-terminal information. DNA can be obtained. This method is called the megaprimer method.

"발현벡터"는 상기 발현벡터의 전사에 제공되는 추가단편에 작동가능하게 연결된 관심의 폴리펩티드를 암호화하는 단편으로 구성되는 선형 또는 원형의 DNA 분자이다. 그와 같은 추가단편은 프로모터 및 종료암호 서열을 포함한다. 또한 발현벡터는 하나 이상의 복제 개시점, 하나 이상의 선택마커, 폴리아데닐화 신호 등 을 포함한다. 발현벡터는 일반적으로 플라스미드 또는 바이러스 DNA로부터 유도되거나, 또는 둘 다의 요소를 함유한다.An "expression vector" is a linear or circular DNA molecule consisting of fragments encoding polypeptides of interest operably linked to additional fragments provided for transcription of said expression vector. Such additional fragments include promoter and termination code sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both.

이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.

본 발명은 카스파제(Caspase) 내에 존재하는 시스테인계 단백질 가수분해효소의 인식부위가 비시스테인계 단백질 가수분해효소(Non-Cysteine proteases)의 인식부위로 치환된 재조합 카스파제 전구체를 제공한다.The present invention provides a recombinant caspase precursor in which a recognition site of cysteine-based proteolytic enzymes present in Caspase is replaced with a recognition site of non-Cysteine proteases.

본 발명의 구체적인 실시예에서, 카스파제 3의 28번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 78: ESMDSG; 아미노산 25번째에서 30번째까지) 및/또는 175번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 80: IETDSG; 아미노산 172번째에서 177번째까지)가 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 79: LVPRGS)로 치환된 재조합 카스파제 3 전구체(28TS, Δ28TS, 175TS, 28TS/175TS, Δ28TS/175TS Caspase 3; Δ: 1번째에서 28번째 아미노산 제거)의 발현벡터를 제작하였다. 상기 발현벡터를 대장균에 형질도입하여 상기 재조합 카스파제 3 전구체를 발현하고 히스티딘 태그(hexa-his tag) 인식부위에 결합할 수 있는 흡착 컬럼을 이용하여 정제한 결과, 재조합 카스파제 3 전구체의 28번째 아미노산을 포함하는 6개의 아미노산으로 구성된 인식부위 및 175번째 아미노산을 포함하는 6개의 아미노산으로 구성된 인식부위를 트롬빈 인식부위로 치환함으로써 재조합 카스파제 3 전구체의 발현량이 높아지는 것을 확인하였다(도 3 참조). 상기 정제된 재조합 카스파제 3 전구체(서열번호 53)를 활성화하기 위한 트롬빈 처리 조건은 18℃에서 8시간 또는 4℃에서 24시간 동안 가수분해하여 활성형 카스파제로 변환시키는 것이 적당한 것을 확인하였다(도 4 참조). 또한, 본 발명에서 트롬빈의 사용량은 반응 온도와 상관없이 40 NIH 유닛 이상이 바람직하였다. 또한, 본 발명의 구체적인 실시예에서, 카스파제 7의 23번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 81: EDSVDAKPDR; 아미노산 19번째에서 28번째까지)를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 82: EDLVPRGSDR)로 치환 및/또는 175번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 83: GIQADSGPIN; 아미노산 194번째에서 203번째까지)를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 98: GLVPRGSPIN)로 치환된 재조합 카스파제 7 전구체(23TS, 198TS, 23TS/198TS Caspase 7)의 발현벡터를 제작하였다. 상기 발현벡터를 대장균에 형질도입하여 상기 재조합 카스파제 7 전구체를 발현 및 정제한 결과, 재조합 카스파제 7 전구체(서열번호 97 및 64)가 4℃ 또는 18℃에서 활성형 카스파제로 변환되는 것을 확인하였다(도 5 참조). 상기 조건에서 수득한 활성형 카스파제 3의 효소반응속도를 확인한 결과, 본 발명의 재조합 카스파제 3의 kcat/Km(효소반응속도)는 야생형 카스파제 3(서열번호 49)에 비해 낮으나(표 2 참조), 야생형 카스파제 3의 자가활성 인식부위의 아스파틱산(aspartic acid)를 중심으로 6개의 연 속적인 아미노산으로 구성된 펩타이드를 트롬빈 인식부위의 아미노산 서열로 치환함으로써, 본 발명의 재조합 카스파제 3 전구체는 대장균에서 과발현 시 자가절단에 의한 활성화 없이 대량생산이 가능할 수 있다(도 3 참조).In a specific embodiment of the invention, a peptide consisting of six consecutive amino acids comprising the 28th amino acid of Caspase 3 (SEQ ID NO: 78: ESMDSG; amino acids 25-30) and / or 6 comprising the 175th amino acid Recombinant caspase 3 precursor substituted with a peptide consisting of consecutive amino acids (SEQ ID NO: 80: IETDSG; amino acids 172-177) replaced with a peptide consisting of consecutive amino acids that are recognized and cleaved by thrombin (SEQ ID NO: 79: LVPRGS) Expression vectors of (28TS, Δ28TS, 175TS, 28TS / 175TS, Δ28TS / 175TS Caspase 3; Δ: 1st to 28th amino acid removal) were prepared. The expression vector was transfected into E. coli to express the recombinant caspase 3 precursor and purified using an adsorption column capable of binding to a histidine tag (hexa-his tag) recognition site. As a result, the 28th of the recombinant caspase 3 precursor was purified. It was confirmed that the expression level of the recombinant caspase 3 precursor was increased by replacing the recognition site consisting of six amino acids including amino acids and the recognition site consisting of six amino acids including the 175th amino acid with a thrombin recognition site (see FIG. 3). The thrombin treatment conditions for activating the purified recombinant caspase 3 precursor (SEQ ID NO: 53) confirmed that it is appropriate to convert the activated caspase by hydrolysis at 18 ° C. for 8 hours or at 4 ° C. for 24 hours (FIG. 4). Reference). In addition, the amount of thrombin used in the present invention is preferably at least 40 NIH units regardless of the reaction temperature. In a specific embodiment of the present invention, a peptide consisting of 10 consecutive amino acids including the 23rd amino acid of Caspase 7 (SEQ ID NO: 81: EDSVDAKPDR; amino acids 19-28) is recognized and cleaved by thrombin. Substituting a peptide consisting of 10 amino acids (SEQ ID NO: 83: GIQADSGPIN; amino acids 194-203) with thrombin substituted with a peptide consisting of consecutive amino acids (SEQ ID NO: 82: EDLVPRGSDR) and / or including the 175th amino acid Expression vectors of recombinant caspase 7 precursors (23TS, 198TS, 23TS / 198TS Caspase 7) substituted with a peptide consisting of consecutive amino acids recognized and cleaved (SEQ ID NO: 98: GLVPRGSPIN) were prepared. The expression vector was transduced into E. coli to express and purify the recombinant caspase 7 precursor, and it was confirmed that the recombinant caspase 7 precursors (SEQ ID NOs: 97 and 64) were converted into active caspase at 4 ° C or 18 ° C. (See Figure 5). As a result of confirming the enzyme reaction rate of the active caspase 3 obtained in the above conditions, k cat / K m (enzyme reaction rate) of the recombinant caspase 3 of the present invention is lower than that of the wild type caspase 3 (SEQ ID NO: 49) ( Table 2), the recombinant caspase of the present invention by substituting the amino acid sequence of the thrombin recognition site for a peptide consisting of six consecutive amino acids around the aspartic acid of the wild type caspase 3 The three precursors may be mass-produced without activation by self-cutting when overexpressed in E. coli (see FIG. 3).

본 발명의 카스파제는 자가활성 인식부위가 치환됨으로써 단백질 발현과 동시에 활성형으로 바뀌지 않기 때문에 자가활성화에 의한 숙주세포에 대한 세포독성을 나타내지 않을 수 있고, 분리·정제 후 비활성형의 형태로 존재하기 때문에 장기 보관이 가능할 수 있다. 또한 기존의 가수분해 되는 인식부위를 새로운 인식부위로 대체하면서 상업적으로 널리 사용되는 단백질 가수분해 효소를 사용하면 간단하게 활성화할 수 있다.The caspase of the present invention may not exhibit cytotoxicity to host cells by self-activation because the caspase does not change to the active form at the same time as the expression of the protein by the substitution of the self-activated recognition site, and exists in an inactive form after separation and purification. Long-term storage may therefore be possible. In addition, it is possible to simply activate the proteolytic enzymes widely used commercially while replacing the existing hydrolysis recognition sites with new recognition sites.

상기 카스파제는 카스파제 2, 카스파제 3, 카스파제 6, 카스파제 7 및 카스파제 9로 구성된 군으로부터 선택된 어느 하나일 수 있다. 또한, 상기 시스테인계 단백질 가수분해효소의 인식부위는 아스파틱산(aspartic acid)을 중심으로 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드 일 수 있다. 상기 시스테인계 단백질 가수분해효소의 인식부위는 다음과 같다:The caspase may be any one selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. In addition, the recognition site of the cysteine-based hydrolase may be a peptide consisting of 4 to 10 consecutive amino acids around aspartic acid. Recognition sites of the cysteine proteinase are as follows:

(1) 카스파제 2(서열번호 73)의 경우 전장 카스파제 2의 162번째에서 171번째 아미노산(서열번호 84), 297번째에서 306번째 아미노산(서열번호 85) 및 319번째에서 328번째 아미노산까지(서열번호 86);(1) For caspase 2 (SEQ ID NO: 73), from 162 th to 171 th amino acids (SEQ ID NO: 84), 297 th to 306 th amino acids (SEQ ID NO: 85), and 319 th to 328 th amino acids of full length caspase 2 ( SEQ ID NO: 86);

(2) 카스파제 3(서열번호 74)의 경우 전장 카스파제 3의 23번째에서 32번째 아미노산(서열번호 95) 및 170번째에서 179번째 아미노산까지(서열번호 96);(2) for caspase 3 (SEQ ID NO: 74) from 23rd to 32nd amino acids (SEQ ID NO: 95) and 170th to 179th amino acids (SEQ ID NO: 96) of full length caspase 3;

(3) 카스파제 6(서열번호 75)의 경우 전장 카스파제 6의 19번째에서 28번째 아미노산까지(서열번호 87);(3) for caspase 6 (SEQ ID NO: 75), from the 19th to 28th amino acids of full length caspase 6 (SEQ ID NO: 87);

(4) 카스파제 7(서열번호 76)의 경우 전장 카스파제 7의 19번째에서 28번째 아미노산(서열번호 81) 및 194번째에서 203번째 아미노산까지(서열번호 83); 및,(4) for caspase 7 (SEQ ID NO: 76), 19 th to 28 th amino acids (SEQ ID NO: 81) and 194 th to 203 th amino acids (SEQ ID NO: 83) of full length caspase 7; And,

(5) 카스파제 9(서열번호 77)의 경우 전장 카스파제 9의 134번째에서 143번째 아미노산(서열번호 88), 301번째에서 310번째 아미노산(서열번호 89) 및 311번째에서 320번째 아미노산까지(서열번호 90).(5) For caspase 9 (SEQ ID NO: 77), from 134 th to 143 th amino acids (SEQ ID NO: 88), 301 th to 310 th amino acids (SEQ ID NO: 89) and 311 th to 320 th amino acids of full length caspase 9 ( SEQ ID NO: 90).

또한, 상기 비시스테인계 단백질 가수분해효소 인식부위는 카스파제를 포함하는 시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 것이 아니면 모두 가능하며, 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드 일 수 있다. 상업적으로 널리 이용되는 상기 비시스테인계 단백질 가수분해효소의 인식부위를 도입함으로써 상기 인식부위를 이용한 활성화방법을 통해 본 발명의 카스파제를 특이적으로 활성화할 수 있다. 상기 비시스테인계 단백질 가수분해효소 인식부위에는 트롬빈 인식부위(Thrombin; 서열번호 79: LVPRGS), 엔테로키나제 인식부위(Enterokinase; 서열번호 91: DDDDK), TEV 인식부위(서열번호 92: ENLYFQG) 및 Factor Xa 인식부위(서열번호 93: IEGR 또는 서열번호 94: IDGR) 등이 사용될 수 있으며, 이에 제한되지 않고 당업자에게 알려진 비시스테인계 효소 인식부위는 무엇이든지 사용 가능하고, 본 발명의 바람직한 실시예에서는 트롬빈 인식부위를 사용하였다. 또한, 상기 비시스테인계 단백질 가수분해효소에는 트롬빈, 엔테로키나제, TEV 및 Factor Xa등이 가능할 수 있고, 본 발명의 바람직한 실시예에서는 트롬빈을 사용하였다.In addition, the bicysteine-based protease recognition site may be any one that is not recognized and cleaved by a cysteine-based protease including caspase, and may be a peptide composed of 4 to 10 consecutive amino acids. By introducing a recognition site of the bicysteine-based protease which is widely used commercially, the caspase of the present invention can be specifically activated through an activation method using the recognition site. The bicysteine protease recognition site includes a thrombin recognition site (Thrombin; SEQ ID NO: 79: LVPRGS), an enterokinase recognition site (Enterokinase; SEQ ID NO: 91: DDDDK), a TEV recognition site (SEQ ID NO: 92: ENLYFQG), and a factor. Xa recognition sites (SEQ ID NO: 93: IEGR or SEQ ID NO: 94: IDGR) and the like can be used, and not limited to any non-cysteine-based enzyme recognition sites known to those skilled in the art can be used, in a preferred embodiment of the present invention thrombin A recognition site was used. In addition, the bicysteine-based protease may be thrombin, enterokinase, TEV and Factor Xa, and the like, thrombin was used in the preferred embodiment of the present invention.

또한, 본 발명은 카스파제 3의 28번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 78: ESMDSG; 아미노산 25번째에서 30번째까지)를 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산(서열번호 79: LVPRGS)으로 치환하고, 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산(서열번호 79: LVPRGS)이 삽입된 재조합 카스파제 3 전구체를 제공한다.In addition, the present invention provides six consecutive amino acid peptides (SEQ ID NO: 78: ESMDSG; amino acids 25 to 30 amino acids) consisting of six consecutive amino acids including the 28th amino acid of caspase 3, which are recognized and cleaved by thrombin. Providing a recombinant caspase 3 precursor substituted with amino acid (SEQ ID NO: 79: LVPRGS) and inserted with six consecutive amino acids (SEQ ID NO: 79: LVPRGS) that are recognized and cleaved by thrombin between the 180 and 181 amino acids do.

본 발명의 구체적인 실시예에서는 카스파제 3의 28번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산으로 치환하고, 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산이 삽입된 재조합 카스파제 3 전구체 28TS/180TI caspase 3(서열번호 54)의 발현벡터를 제작하였다. 상기 발현벡터를 대장균에 형질도입하여 상기 재조합 카스파제 3 전구체를 발현 및 정제하고, 트롬빈을 처리하여 활성형으로 바꾸어 효소반응속도를 측정한 결과, 카스파제 3의 28번째 및 175번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산으로 치환된 재조합 카스파제 3 전구체 28TS/175TS caspase 3(서열번호 53)보다 효소활성이 높은 것을 확인하였다(표 2 참조).In a specific embodiment of the present invention, a peptide consisting of six consecutive amino acids including the 28th amino acid of caspase 3 is replaced with six consecutive amino acids recognized and cleaved by thrombin, and between 180 and 181 amino acids. An expression vector of recombinant caspase 3 precursor 28TS / 180TI caspase 3 (SEQ ID NO: 54) inserted with six consecutive amino acids recognized and cleaved by thrombin was prepared. The expression vector was transfected into Escherichia coli to express and purify the recombinant caspase 3 precursor, and treated with thrombin to change the active form to measure the enzyme reaction rate. As a result, the 28th and 175th amino acids of caspase 3 were included. Peptides consisting of six consecutive amino acids were found to have higher enzymatic activity than recombinant caspase 3 precursor 28TS / 175TS caspase 3 (SEQ ID NO: 53) substituted with six consecutive amino acids recognized and cleaved by thrombin (Table 2). Reference).

본 발명의 카스파제는 자가활성 인식부위가 치환됨으로써 단백질 발현과 동시에 활성형으로 바뀌지 않기 때문에 자가활성화에 의한 숙주세포에 대한 세포독성 을 나타내지 않을 수 있고, 분리·정제 후 비활성형의 형태로 존재하기 때문에 장기 보관이 가능할 수 있다. 또한 기존의 가수분해 되는 인식부위를 새로운 인식부위로 대체하면서 상업적으로 널리 사용되는 단백질 가수분해 효소를 사용하면 간단하게 활성화할 수 있다.The caspase of the present invention may not exhibit cytotoxicity to host cells by self-activation because the caspase does not change to the active form at the same time as the expression of the protein by the substitution of the self-activated recognition site, and exists in an inactive form after separation and purification. Long-term storage may therefore be possible. In addition, it is possible to simply activate the proteolytic enzymes widely used commercially while replacing the existing hydrolysis recognition sites with new recognition sites.

또한, 본 발명은 카스파제 내에 존재하는, 적어도 하나 이상의 시스테인계 단백질 가수분해효소의 인식부위 내의 하나 이상의 아미노산 잔기가 상기 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산으로 치환되고, 상기 시스테인계 단백질 가수분해효소의 인식부위의 주변에 비시스테인계 단백질 가수분해효소 인식부위가 삽입된 재조합 카스파제 전구체를 제공한다.In addition, the present invention is the one or more amino acid residues in the recognition site of at least one cysteine-based proteolytic enzyme present in the caspase is replaced with an amino acid that the cysteine-based proteolytic enzyme is not recognized, the cysteine-based protein The present invention provides a recombinant caspase precursor in which a bicysteine-based protease recognition site is inserted around a recognition site of a hydrolase.

본 발명의 구체적인 실시예에서는 28TS/175TS Caspase 3의 168번째 아미노산인 류신(Leucine)을 페닐알라닌(Phenylalanine)으로 치환한 L168F/28TS/175TS caspase 3(서열번호 56)의 발현벡터를 제작하였다. 상기 발현벡터를 대장균에 형질도입하여 상기 재조합 카스파제 3 전구체를 발현 및 정제하고, 트롬빈을 처리하여 활성형으로 바꾸어 효소반응속도를 측정한 결과, 28TS/175TS caspase 3(서열번호 53)보다 효소활성이 높은 것을 확인하였다(표 2 참조). 또한, 본 발명의 구체적인 실시예에서는 28TS/180TI caspase 3(서열번호 54)의 168번째 아미노산인 류신(Leucine)을 페닐알라닌(Phenylalanine) 또는 트립토판(Tryptophan)으로 치환하고, 175번째 아미노산인 아스파틱산(Aspartic acid)을 알라닌(Alanine)으로 치환한 L168F/28TS/D175A/180TI caspase 3(서열번호 59) 및 L168W/28TS/D175A/180TI caspase 3(서열번호 60)의 발현벡터를 제작하였다. 상기 발현벡터를 대장균에 형질도입하여 상기 재조합 카스파제 3 전구체를 발현 및 정제하고, 트롬빈을 처리하여 활성형으로 바꾸어 효소반응속도를 측정한 결과, 28TS/180TI caspase 3(서열번호 54)보다 효소활성이 높은 것을 확인하였다(표 2 참조).In a specific embodiment of the present invention, an expression vector of L168F / 28TS / 175TS caspase 3 (SEQ ID NO: 56) in which leucine (Leucine), 168th amino acid of 28TS / 175TS Caspase 3, was substituted with phenylalanine (Phenylalanine) was prepared. The expression vector was transfected into Escherichia coli to express and purify the recombinant caspase 3 precursor, and treated with thrombin to change the active form to measure the enzyme reaction rate. The enzyme activity was higher than that of 28TS / 175TS caspase 3 (SEQ ID NO: 53). This high thing was confirmed (refer Table 2). In a specific embodiment of the present invention, leucine (Leucine), the 168th amino acid of 28TS / 180TI caspase 3 (SEQ ID NO: 54), is substituted with phenylalanine (Phenylalanine) or tryptophan (Tryptophan), and aspartic acid (Aspartic acid), the 175th amino acid, is used. acid) was prepared with the expression vector of L168F / 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 59) and L168W / 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 60) substituted with alanine. The expression vector was transfected into Escherichia coli to express and purify the recombinant caspase 3 precursor, and then treated with thrombin to be converted into an active form, and the enzyme reaction rate was measured. This high thing was confirmed (refer Table 2).

상기 카스파제는 카스파제 2, 카스파제 3, 카스파제 6, 카스파제 7 및 카스파제 9로 구성된 군으로부터 선택된 어느 하나일 수 있다. 또한, 상기 시스테인계 단백질 가수분해효소의 인식부위는 아스파틱산(aspartic acid)을 중심으로 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드 일 수 있다. 상기 시스테인계 단백질 가수분해효소의 인식부위는 다음과 같다:The caspase may be any one selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. In addition, the recognition site of the cysteine-based hydrolase may be a peptide consisting of 4 to 10 consecutive amino acids around aspartic acid. Recognition sites of the cysteine proteinase are as follows:

(1) 카스파제 2(서열번호 73)의 경우 전장 카스파제 2의 162번째에서 171번째 아미노산(서열번호 84), 297번째에서 306번째 아미노산(서열번호 85) 및 319번째에서 328번째 아미노산까지(서열번호 86);(1) For caspase 2 (SEQ ID NO: 73), from 162 th to 171 th amino acids (SEQ ID NO: 84), 297 th to 306 th amino acids (SEQ ID NO: 85), and 319 th to 328 th amino acids of full length caspase 2 ( SEQ ID NO: 86);

(2) 카스파제 3(서열번호 74)의 경우 전장 카스파제 3의 23번째에서 32번째 아미노산(서열번호 95) 및 170번째에서 179번째 아미노산까지(서열번호 96);(2) for caspase 3 (SEQ ID NO: 74) from 23rd to 32nd amino acids (SEQ ID NO: 95) and 170th to 179th amino acids (SEQ ID NO: 96) of full length caspase 3;

(3) 카스파제 6(서열번호 75)의 경우 전장 카스파제 6의 19번째에서 28번째 아미노산까지(서열번호 87);(3) for caspase 6 (SEQ ID NO: 75), from the 19th to 28th amino acids of full length caspase 6 (SEQ ID NO: 87);

(4) 카스파제 7(서열번호 76)의 경우 전장 카스파제 7의 19번째에서 28번째 아미노산(서열번호 81) 및 194번째에서 203번째 아미노산까지(서열번호 83); 및,(4) for caspase 7 (SEQ ID NO: 76), 19 th to 28 th amino acids (SEQ ID NO: 81) and 194 th to 203 th amino acids (SEQ ID NO: 83) of full length caspase 7; And,

(5) 카스파제 9(서열번호 77)의 경우 전장 카스파제 9의 134번째에서 143번째 아미노산(서열번호 88), 301번째에서 310번째 아미노산(서열번호 89) 및 311번 째에서 320번째 아미노산까지(서열번호 90).(5) In case of caspase 9 (SEQ ID NO: 77), the 134th to 143th amino acids (SEQ ID NO: 88), the 301st to 310th amino acids (SEQ ID NO: 89), and the 311rd to 320th amino acids of full length caspase 9; (SEQ ID NO: 90).

또한, 상기 비시스테인계 단백질 가수분해효소 인식부위는 카스파제를 포함하는 시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 것이 아니면 모두 가능하며, 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드 일 수 있다. 상업적으로 널리 이용되는 상기 비시스테인계 단백질 가수분해효소의 인식부위를 도입함으로써 상기 인식부위를 이용한 활성화방법을 통해 본 발명의 카스파제를 특이적으로 활성화할 수 있다. 상기 비시스테인계 단백질 가수분해효소 인식부위에는 트롬빈 인식부위(Thrombin; 서열번호 79: LVPRGS), 엔테로키나제 인식부위(Enterokinase; 서열번호 91: DDDDK), TEV 인식부위(서열번호 92: ENLYFQG) 및 Factor Xa 인식부위(서열번호 93: IEGR 또는 서열번호 94: IDGR) 등이 사용될 수 있으며, 이에 제한되지 않고 당업자에게 알려진 비시스테인계 효소 인식부위는 무엇이든지 사용 가능하고, 본 발명의 바람직한 실시예에서는 트롬빈 인식부위를 사용하였다. 또한, 상기 비시스테인계 단백질 가수분해효소에는 트롬빈, 엔테로키나제, TEV 및 Factor Xa등이 가능할 수 있고, 본 발명의 바람직한 실시예에서는 트롬빈을 사용하였다.In addition, the bicysteine-based protease recognition site may be any one that is not recognized and cleaved by a cysteine-based protease including caspase, and may be a peptide composed of 4 to 10 consecutive amino acids. By introducing a recognition site of the bicysteine-based protease which is widely used commercially, the caspase of the present invention can be specifically activated through an activation method using the recognition site. The bicysteine protease recognition site includes a thrombin recognition site (Thrombin; SEQ ID NO: 79: LVPRGS), an enterokinase recognition site (Enterokinase; SEQ ID NO: 91: DDDDK), a TEV recognition site (SEQ ID NO: 92: ENLYFQG), and a factor. Xa recognition sites (SEQ ID NO: 93: IEGR or SEQ ID NO: 94: IDGR) and the like can be used, and not limited to any non-cysteine-based enzyme recognition sites known to those skilled in the art can be used, in a preferred embodiment of the present invention thrombin A recognition site was used. In addition, the bicysteine-based protease may be thrombin, enterokinase, TEV and Factor Xa, and the like, thrombin was used in the preferred embodiment of the present invention.

또한, 상기 시스테인계 단백질 가수분해효소의 인식부위 내의 하나 이상의 아미노산 잔기를 치환하기 위해 사용되는, 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산은, 아스파틱산(Aspartic acid)을 제외한 아미노산을 의미하며, 페닐알라닌, 알라닌, 류신 및 트립토판 등이 사용될 수 있으며, 이에 제한되지 않는다.In addition, the amino acid that is not recognized by the cysteine protease, which is used to replace one or more amino acid residues in the recognition site of the cysteine protease, means an amino acid except for aspartic acid. , Phenylalanine, alanine, leucine and tryptophan may be used, but is not limited thereto.

아울러, 본 발명의 아미노산 잔기가 치환되지 않은 시스테인계 단백질 가수분해효소 인식부위는 비시스테인계 단백질 가수분해효소 인식부위로 치환될 수 있다.In addition, the cysteine-based protease recognition site in which the amino acid residue of the present invention is not substituted may be substituted with the bicysteine-based protease recognition site.

본 발명의 카스파제는 자가활성 인식부위가 치환됨으로써 단백질 발현과 동시에 활성형으로 바뀌지 않기 때문에 자가활성화에 의한 숙주세포에 대한 세포독성을 나타내지 않을 수 있고, 분리·정제 후 비활성형의 형태로 존재하기 때문에 장기 보관이 가능할 수 있다. 또한 기존의 가수분해 되는 인식부위를 새로운 인식부위로 대체하면서 상업적으로 널리 사용되는 단백질 가수분해 효소를 사용하면 간단하게 활성화할 수 있다.The caspase of the present invention may not exhibit cytotoxicity to host cells by self-activation because the caspase does not change to the active form at the same time as the expression of the protein by the substitution of the self-activated recognition site, and exists in an inactive form after separation and purification. Long-term storage may therefore be possible. In addition, it is possible to simply activate the proteolytic enzymes widely used commercially while replacing the existing hydrolysis recognition sites with new recognition sites.

또한, 본 발명은 상기 재조합 카스파제 전구체 중 어느 하나를 암호화하는 폴리뉴클레오티드를 제공한다.The present invention also provides a polynucleotide encoding any one of the recombinant caspase precursors.

카스파제의 시스테인계 단백질 가수분해효소의 인식부위를 비시스테인계 단백질 가수분해효소의 인식부위로 치환하는 것과 시스테인계 단백질 가수분해효소의 인식부위의 주변에 비시스테인계 단백질 가수분해효소 인식부위를 삽입하는 것은 메가프라이머 PCR 법을 수행함으로써 실행될 수 있다. 상기 메가프라이머 PCR 법에 사용되는 프라이머 쌍은 정방향 프라이머와 역방향 프라이머 중의 하나가 비시스테인계 단백질 가수분해효소의 인식부위를 암호화하는 뉴클레오티드의 서열을 포함하고 있어서, PCR 반응 산물에 비시스테인계 단백질 가수분해효소의 인식부위를 암호화하는 뉴클레오티드 서열을 포함할 수 있는 것이면 모두 사용할 수 있다.Substituting the recognition site for the cysteine-based proteolytic enzyme of caspase with the recognition site for the non-cysteine-based proteolytic enzyme, and inserting the non-cysteine-based protease recognition site around the recognition site of the cysteine-based proteolytic enzyme Can be performed by performing a megaprimer PCR method. The primer pair used in the megaprimer PCR method includes a sequence of nucleotides in which one of the forward primer and the reverse primer encodes the recognition site of the bicysteine proteolytic enzyme, and thus the bicysteine proteolytic hydrolysis to the PCR reaction product. Any may be used as long as it can include a nucleotide sequence encoding an enzyme recognition site.

본 발명의 구체적인 실시예에서 카스파제 3(서열번호 49)의 28번째 및/또는 175번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈 인식부위로 치환하기 위해서는 각각 서열번호 5 내지 6으로 기재되는 프라이머쌍 및/또는 서열번호 7 내지 8로 기재되는 프라이머쌍을 이용하였다. 또한, 카스파제 7(서열번호 62)의 23번째 및/또는 198번째 아미노산을 포함하는 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈(Thrombin) 인식부위로 치환하기 위해서는 서열번호 24 내지 25로 기재되는 프라이머쌍 및/또는 서열번호 26 내지 27로 기재되는 프라이머쌍을 이용하였다.In specific embodiments of the present invention, to replace a peptide consisting of six consecutive amino acids including the 28th and / or 175th amino acids of caspase 3 (SEQ ID NO: 49) with thrombin recognition sites, respectively, SEQ ID NOs: 5 to 6 Primer pairs described and / or primer pairs set forth in SEQ ID NOS: 7-8 were used. In addition, in order to substitute a thrombin recognition site for a peptide consisting of 10 consecutive amino acids including the 23rd and / or 198th amino acids of caspase 7 (SEQ ID NO: 62), the primers represented by SEQ ID NOs: 24 to 25 Pairs and / or primer pairs set forth in SEQ ID NOs: 26-27 were used.

또한, 시스테인계 단백질 가수분해효소의 인식부위의 하나 이상의 아미노산 서열을 상기 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산으로 치환하는 것은 Site Directed Mutagenesis 법을 수행함으로써 실행될 수 있다. 상기 Site Directed Mutagenesis 법을 수행하기 위한 키트가 이미 상용화되어 있으므로, 본 발명의 재현하는 것이 용이한 것은 당업자에게 자명한 사실이다.In addition, the substitution of one or more amino acid sequences of the recognition sites of the cysteine-based proteolytic enzymes with amino acids that the cysteine-based proteolytic enzymes cannot recognize may be performed by performing a Site Directed Mutagenesis method. Since a kit for performing the Site Directed Mutagenesis method has already been commercialized, it is obvious to those skilled in the art that it is easy to reproduce the present invention.

또한, 본 발명은 상기 폴리뉴클레오티드를 포함하는 발현벡터를 제공한다.In addition, the present invention provides an expression vector comprising the polynucleotide.

본 발명의 치환된 재조합 카스파제를 암호화하는 폴리뉴클레오티드가 뼈대 벡터에 제한효소 부위를 통해 클로닝되어 포함되며, 본 발명의 방법에서 사용될 수 있는 뼈대 벡터는 특별히 이에 제한되는 것은 아니나, pET21a, pET28a, pET/Rb, pGEX, pET-22b(+) 및 pGEX로 이루어진 군으로부터 선택되는 대장균에 형질전환 가능한 다양한 벡터를 사용할 수 있다. 본 발명의 상기 발현벡터는 pET21a 뼈대 벡 터에 상기 치환된 카스파제 유전자를 포함함으로써 상기 재조합 카스파제 전구체로 발현할 수 있다.Polynucleotides encoding the substituted recombinant caspase of the present invention are cloned into the skeletal vector via restriction enzyme sites, and the skeletal vectors that can be used in the methods of the present invention are not particularly limited, but may be pET21a, pET28a, pET Various vectors capable of transforming E. coli selected from the group consisting of / Rb, pGEX, pET-22b (+) and pGEX can be used. The expression vector of the present invention can be expressed as the recombinant caspase precursor by including the substituted caspase gene in the pET21a skeleton vector.

또한, 본 발명은 상기 발현벡터가 형질도입된 형질전환체를 제공한다.The present invention also provides a transformant to which the expression vector is transduced.

또한, 본 발명은 1) 상기 재조합 카스파제 전구체 중 어느 하나를 암호화하는 폴리뉴클레오티드를 포함하는 발현벡터를 제조하는 단계;In addition, the present invention comprises the steps of 1) preparing an expression vector comprising a polynucleotide encoding any one of the recombinant caspase precursors;

2) 상기 발현벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계; 및,2) preparing a transformant by introducing the expression vector into a host cell; And,

3) 상기 형질전환체를 배양하여 재조합 단백질의 발현을 유도하고 이를 수득하는 단계로 구성되는 재조합 카스파제 전구체의 제조방법을 제공한다.3) culturing the transformant to induce the expression of a recombinant protein and to provide a method for producing a recombinant caspase precursor consisting of the same.

본 발명의 제조방법에 의해 재조합 카스파제 전구체의 대량생산이 가능하고(도 3 및 5 참조), 또한, 상기 재조합 카스파제 전구체에 비시스테인계 가수분해 효소로 처리하는 간단한 방법으로 상기 재조합 카스파제 전구체가 카스파제 활성을 나타낼 수 있으므로(도 4, 도 5 및 표 2 참조), 기존에 신약개발 및 효소의 생물학적 기능 연구 등을 위하여 널리 사용되는 천연 카스파제의 수요를 충족할 수 있다.The production method of the present invention enables the mass production of recombinant caspase precursors (see FIGS. 3 and 5), and the recombinant caspase precursor is also a simple method of treating the recombinant caspase precursor with a bicysteine-based hydrolase. Since it may exhibit caspase activity (see FIGS. 4, 5 and Table 2), it is possible to meet the demand of natural caspase, which is widely used for drug discovery and biological function research of enzymes.

또한, 본 발명은 상기 재조합 카스파제 전구체를 비시스테인계 단백질 가수분해효소로 처리하는 단계를 포함하는 상기 재조합 카스파제 전구체를 활성화하는 방법을 제공한다.The present invention also provides a method of activating the recombinant caspase precursor, comprising treating the recombinant caspase precursor with a bicysteine-based proteolytic enzyme.

또한, 본 발명은 상기 방법에 의해 활성화된 재조합 카스파제를 제공한다.The present invention also provides a recombinant caspase activated by the above method.

본 발명의 제조방법에 의해 재조합 카스파제 전구체의 대량생산이 가능하고(도 3 및 5 참조), 또한, 상기 재조합 카스파제 전구체에 비시스테인계 가수분해 효소로 처리하는 간단한 방법으로 상기 재조합 카스파제 전구체가 카스파제 활성을 나타낼 수 있으므로(도 4, 도 5 및 표 2 참조), 기존에 신약개발 및 효소의 생물학적 기능 연구 등을 위하여 널리 사용되는 천연 카스파제의 수요를 충족할 수 있다.The production method of the present invention enables the mass production of recombinant caspase precursors (see FIGS. 3 and 5), and the recombinant caspase precursor is also a simple method of treating the recombinant caspase precursor with a bicysteine-based hydrolase. Since it may exhibit caspase activity (see FIGS. 4, 5 and Table 2), it is possible to meet the demand of natural caspase, which is widely used for drug discovery and biological function research of enzymes.

본 발명의 구체적인 실시예에서 재조합 카스파제 3 전구체(서열번호 53)를 활성화하기 위한 트롬빈 처리 조건은 18℃에서 8시간 또는 4℃에서 24시간 동안 가수분해하여 활성형 카스파제로 변환시키는 것이 적당한 것을 확인하였다(도 4 참조). 또한, 본 발명에서 트롬빈의 사용량은 반응 온도와 상관없이 40 NIH 유닛 이상이 바람직하였다.The thrombin treatment conditions for activating the recombinant caspase 3 precursor (SEQ ID NO: 53) in a specific embodiment of the present invention confirm that it is appropriate to convert to active caspase by hydrolysis at 18 ° C. for 8 hours or at 4 ° C. for 24 hours. (See FIG. 4). In addition, the amount of thrombin used in the present invention is preferably at least 40 NIH units regardless of the reaction temperature.

상기 비시스테인계 단백질 가수분해효소에는 트롬빈, 엔테로키나제, TEV 및 Factor Xa등이 가능할 수 있고, 본 발명의 바람직한 실시예에서는 트롬빈을 사용하였다.The bicysteine proteinase may be thrombin, enterokinase, TEV, Factor Xa, and the like, and thrombin was used in a preferred embodiment of the present invention.

아울러, 본 발명은 1) 상기 활성화된 재조합 카스파제에 카스파제 특이적 기질 및 후보 물질을 처리하는 단계;In addition, the present invention comprises the steps of: 1) treating the caspase specific substrate and the candidate substance to the activated recombinant caspase;

2) 상기 활성화된 재조합 카스파제의 기질에 대한 효소 활성을 측정하는 단계; 및,2) measuring enzyme activity on the substrate of the activated recombinant caspase; And,

3) 후보 물질을 처리하지 않은 대조군과 비교하여 상기 활성화된 재조합 카스파제의 효소 활성을 감소시키거나 증진시킨 후보 물질을 선별하는 단계를 포함하 는 카스파제 활성 저해제 또는 촉진제의 스크리닝 방법을 제공한다.3) A method for screening a caspase activity inhibitor or promoter comprising the step of selecting a candidate substance that reduces or enhances the enzymatic activity of the activated recombinant caspase compared to a control that has not been treated with the candidate substance.

상기 단계 1)의 카스파제 특이적 기질에는 Ac-DEVD-pNA(pNA:para-nitroanilide), Ac-DEVD-AFC (7-amino-4-trifluoromethylcoumarin) 등이 사용될 수 있으며, 본 발명의 구체적인 실시예에서는 Ac-DEVD-pNA를 이용하였다.As the caspase specific substrate of step 1), Ac-DEVD-pNA (pNA: para-nitroanilide), Ac-DEVD-AFC (7-amino-4-trifluoromethylcoumarin) and the like may be used. Ac-DEVD-pNA was used.

또한, 상기 단계 1)의 후보물질은 천연화합물, 합성화합물, RNA, DNA, 폴리펩티드, 효소, 단백질, 리간드, 항체, 항원, 박테리아 또는 진균의 대사산물 및 생활성 분자인 것이 바람직하나 이에 한정되는 것은 아니다.In addition, the candidate material of step 1) is preferably a natural compound, synthetic compound, RNA, DNA, polypeptide, enzymes, proteins, ligands, antibodies, antigens, bacteria or fungi metabolites and bioactive molecules, but is not limited thereto. no.

아울러, 상기 단계 2)의 효소 활성은 상기 활성화된 재조합 카스파제가 기질을 분해하는 정도를 UV/VIS 스펙트로포토미터를 이용하여 측정함으로써 결정될 수 있다.In addition, the enzymatic activity of step 2) can be determined by measuring the extent to which the activated recombinant caspase degrades the substrate using a UV / VIS spectrophotometer.

본 발명의 자가활성화 방지형 재조합 카스파제 전구체에 의해 대량생산된 카스파제는 신약개발 및 효소의 생물학적 기능 연구 등을 위하여 널리 사용될 수 있다.Caspases mass-produced by the anti-activation recombinant caspase precursor of the present invention can be widely used for drug discovery and biological function research of enzymes.

이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by way of examples.

단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

<< 실시예Example 1> 천연  1> natural 카스파제Caspase 전구체 과발현벡터 Precursor overexpression vector

시스테인계 단백질 가수분해효소에 의해 활성화될 수 있는 천연 카스파제 전구체 발현벡터를 제작하였다.A natural caspase precursor expression vector was constructed that can be activated by cysteine-based proteolytic enzymes.

우선, 21C Human Gene Bank, Genome Research Center(KRIBB, 한국)에서 caspase 3(서열번호 49), caspase-2(서열번호 70), caspase-6(서열번호 71), Caspase 7(서열번호 62) 및 caspase-9(서열번호 72)를 암호화하는 유전자의 주형 DNA를 구입하였다. 표 1에서 나타내는 제한효소 인식부위를 포함하도록, 상기 5개의 카스파제의 전체 유전자를 각각 증폭하기 위한 프라이머 쌍을 설계하여 바이오니아사(한국)에 의뢰하여 제작하였다. 설계한 프라이머의 서열은 표 1과 같다.First, caspase 3 (SEQ ID NO: 49), caspase-2 (SEQ ID NO: 70), caspase-6 (SEQ ID NO: 71), Caspase 7 (SEQ ID NO: 62) and the 21C Human Gene Bank, Genome Research Center (KRIBB, Korea); The template DNA of the gene encoding caspase-9 (SEQ ID NO: 72) was purchased. To include the restriction enzyme recognition sites shown in Table 1, primer pairs for amplifying the entire genes of the five caspases, respectively, were designed and commissioned by Bioneer (Korea). The designed primer sequences are shown in Table 1.

카스파제 전체 유전자 증폭을 위한 프라이머 쌍Primer Pairs for Caspase Whole Gene Amplification 유전자명Gene name 프라이머primer 제한효소Restriction enzyme 서열번호SEQ ID NO: 염기서열Sequence
카스파제 3
Caspase 3 정방향Forward NdeI Nde I 1One gggaattcca tatggagaac actgaaaact caggggaattcca tatggagaac actgaaaact cag 역방향 1Reverse 1 XhoI Xho I 22 ccgctcgagt tagtgataaa aatagagttcccgctcgagt tagtgataaa aatagagttc 역방향 2Reverse 2 XhoI Xho I 33 ccgctcgagg tgataaaaat agagttcttt tgtccgctcgagg tgataaaaat agagttcttt tgt
카스파제-2
Caspase-2 정방향Forward NdeI Nde I 4040 gggaattcca tatggcggcg ccgagcgcgg ggtcttgggaattcca tatggcggcg ccgagcgcgg ggtctt 역방향 1Reverse 1 XhoI Xho I 4141 ccgctcgagt tatgtgggag ggtgtcctgg gaaccgctcgagt tatgtgggag ggtgtcctgg gaa 역방향 2Reverse 2 XhoI Xho I 4242 ccgctcgagt gtgggagggt gtcctgggaaccgctcgagt gtgggagggt gtcctgggaa
카스파제 6
Caspase 6 정방향Forward NheI Nhe I 4343 ctagctagca gctcggcctc ggggctccgcctagctagca gctcggcctc ggggctccgc 역방향 1Reverse 1 SalI Sal I 4444 acgcgtcgac ttaattagat tttggaaaga aatgacgcgtcgac ttaattagat tttggaaaga aatg 역방향 2Reverse 2 SalI Sal I 4545 acgcgtcgac attagatttt ggaaagaaat gacgcgtcgac attagatttt ggaaagaaat g
카스파제 7
Caspase 7 정방향Forward NdeI Nde I 2121 gggaattcca tatggcagat gagcagggct gtattgaaggggaattcca tatggcagat gagcagggct gtattgaag 역방향 1Reverse 1 XhoI Xho I 2222 ccgctcgagt tattgactga agtagagttc cttggtccgctcgagt tattgactga agtagagttc cttggt 역방향 2Reverse 2 XhoI Xho I 2323 ccgctcgagt tgactgaagt agagttcctt ggtccgctcgagt tgactgaagt agagttcctt ggt
카스파제 9
Caspase 9 정방향Forward NheI Nhe I 4646 ctagctagcg acgaagcgga tcggcggctc ctgctagctagcg acgaagcgga tcggcggctc ctg 역방향 1Reverse 1 SalI Sal I 4747 acgcgtcgac ttatgatgtt ttaaagaaaa gtttacgcgtcgac ttatgatgtt ttaaagaaaa gttt 역방향 2Reverse 2 SalI Sal I 4848 acgcgtcgac tgatgtttta aagaaaagtt tacgcgtcgac tgatgtttta aagaaaagtt t

상기 주형 DNA 1 ㎕, 각 유전자의 20 μΜ 정방향 프라이머 및 역방향 프라이머 1 또는 역방향 프라이머 2 각각 1 ㎕, 중합효소 활성 완충액 2.5 ㎕, dNTP 2 ㎕, Taq 중합효소 1 ㎕, 멸균증류수 16.5 ㎕를 첨가하여 최종 25 ㎕가 되도록 혼합한 뒤, 하기조건으로 PCR 반응을 수행하였다: 초기변성 95℃ 5분, 변성 95℃ 30초, 어닐링 55℃ 1분, 신장 68℃ 2분, 25회 반복, 최종 신장 72℃ 10분, 반응 종결 및 보관 4℃. 역방향 프라이머는 종결 코돈의 포함 여부에 따라 역방향 1 프라이머 또는 역방향 2 프라이머를 사용하였다. 상기 PCR 반응 산물을 1% 아가로즈 젤에서 전기영동한 결과, 각각 800 내지 1300 bp의 DNA 단편을 확인하였다.1 μl of the template DNA, 20 μΜ forward primer and reverse primer 1 or reverse primer 2 of each gene, 2.5 μl of polymerase activity buffer, 2 μl of dNTP, 1 μl of Taq polymerase, and 16.5 μl of sterile distilled water were added. After mixing to 25 μl, the PCR reaction was carried out under the following conditions: initial denaturation 95 ℃ 5 minutes, denaturation 95 ℃ 30 seconds, annealing 55 ℃ 1 minutes, elongation 68 ℃ 2 minutes, 25 repetitions, final elongation 72 ℃ 10 min, reaction termination and storage 4 ° C. The reverse primer used a reverse 1 primer or a reverse 2 primer depending on whether the stop codon was included. The PCR reaction product was subjected to electrophoresis on 1% agarose gel, and DNA fragments of 800 to 1300 bp were identified, respectively.

상기 최종 PCR 반응 산물 및 pET28a 벡터(Novagen, USA)를 표 1에서 나타낸 각각의 제한효소(New England Biolabs, UK)로 37℃에서 2 시간 동안 반응 후, DNA Ligation kit(Takara, 일본)를 이용하여 연결해 주었고, 상기 벡터의 염기서열을 시퀀싱하여 확인함으로써 천연 카스파제 전구체를 과발현하는 pET28a-Cas3, pET28a-Cas2, pET28a-Cas6, pET28a-Cas7 및 pET28a-Cas9 벡터의 제작을 완료하였다.The final PCR reaction product and the pET28a vector (Novagen, USA) were reacted at 37 ° C. for 2 hours with the respective restriction enzymes (New England Biolabs, UK) shown in Table 1, using a DNA Ligation kit (Takara, Japan). By sequencing and confirming the nucleotide sequence of the vector, the preparation of pET28a-Cas3, pET28a-Cas2, pET28a-Cas6, pET28a-Cas7 and pET28a-Cas9 vectors overexpressing natural caspase precursors was completed.

<실시예 2> 치환된 재조합 카스파제 전구체 발현벡터Example 2 Substituted Recombinant Caspase Precursor Expression Vector

시스테인계 단백질 가수분해효소에 의해 활성화될 수 없고, 트롬빈에 의해 활성을 나타낼 수 있는 치환된 재조합 카스파제 전구체 발현벡터를 제작하였다.Substituted recombinant caspase precursor expression vectors, which cannot be activated by cysteine-based proteolytic enzymes and can be activated by thrombin, were constructed.

<2-1> 28<2-1> 28 TSTS caspasecaspase 3(서열번호 51) 발현벡터 3 (SEQ ID NO: 51) Expression Vector

카스파제 3의 28번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 78: ESMDSG; 아미노산 25번째에서 30번째까지)가 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 79: LVPRGS)로 치환된 재조합 카스파제 3 전구체를 발현하는 발현벡터를 제작하였다.Peptide consisting of six consecutive amino acids, including the 28th amino acid of Caspase 3 (SEQ ID NO: 78: ESMDSG; amino acids 25-30) is a peptide consisting of consecutive amino acids that are recognized and cleaved by thrombin : An expression vector expressing recombinant caspase 3 precursor substituted with LVPRGS) was constructed.

주형 DNA(pET28a-Cas3 벡터), 20 μΜ 정방향 프라이머(서열번호 1) 및 역방향 프라이머(서열번호 6: AAT GGA GCT GCC GCG CGG CAC CAG TTC AGT GTT TTC AGT GTT CTC) 각각 1 ㎕, 중합효소 활성 완충액 2.5 ㎕, dNTP 2 ㎕, Taq 중합효소 1 ㎕, 멸균증류수 16.5 ㎕를 첨가하여 최종 25 ㎕가 되도록 혼합한 뒤, 하기의 조건으로 1차 PCR 반응을 수행하였다: 초기변성 95℃ 5분, 변성 95℃ 30초, 어닐링 55℃ 1분, 신장 68℃ 2분, 25회 반복, 최종 신장 72℃ 10분, 반응 종결 및 보관 4℃. 반응 종료 후 정제과정 없이 상기 PCR 반응 산물을 메가프라이머(megaprimer)로 사용하였다. 이어서, 주형 DNA 1 ㎕, 메가프라이머 및 20 μΜ 역방향 프라이머(서열번호 3) 각각 1 ㎕, 중합효소 활성 완충액 2.5 ㎕, dNTP 2 ㎕, Taq 중합효소 1 ㎕, 멸균증류수 16.5 ㎕를 첨가하여 최종 25 ㎕가 되도록 혼합한 뒤, 하기의 조건으로 2차 PCR 반응을 수행하여 목표유전자를 제조하였다: 초기변성 95℃ 5분, 변성 95℃ 30초, 어닐링 55℃ 1분, 신장 68℃ 2분, 25회 반복, 최종 신장 72℃ 10분, 반응 종결 및 보관 4℃.Template DNA (pET28a-Cas3 vector), 20 μΜ forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 6: AAT GGA GCT GCC GCG CGG CAC CAG TTC AGT GTT TTC AGT GTT CTC), respectively, 1 μL, polymerase activity buffer 2.5 μl, 2 μl of dNTP, 1 μl of Taq polymerase, 16.5 μl of sterile distilled water were added and mixed to the final 25 μl. 30 ° C., annealing 55 ° C. 1 min, elongation 68 ° C. 2 min, 25 repetitions, final elongation 72 ° C. 10 min, reaction termination and storage 4 ° C. After completion of the reaction, the PCR reaction product was used as a megaprimer without purification. Then, 1 μl of template DNA, 1 μl of megaprimer and 20 μM reverse primer (SEQ ID NO: 3), 2.5 μl of polymerase activity buffer, 2 μl of dNTP, 1 μl of Taq polymerase, and 16.5 μl of sterile distilled water were added. After mixing so as to perform a second PCR reaction under the following conditions, the target gene was prepared: initial denaturation 95 ° C. 5 minutes, denaturation 95 ° C. 30 seconds, annealing 55 ° C. 1 minute, elongation 68 ° C. 2 minutes, 25 times Repeat, final elongation 72 ° C. 10 min, reaction termination and storage 4 ° C.

또 다른 방법으로는 20 μΜ 정방향 프라이머(서열번호 5: ACT GAA CTG GTG CCG CGC GGC AGC TCC ATT AAA AAT TTG GAA CCA AAG) 및 역방향 프라이머(서열번호 3)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머로 사용하고 정방향 프라이머(서열번호 1)을 사용하여 2차 PCR 반응을 수행하였다.Another method was the same method as Example 2-1 using 20 μΜ forward primer (SEQ ID NO: 5: ACT GAA CTG GTG CCG CGC GGC AGC TCC ATT AAA AAT TTG GAA CCA AAG) and reverse primer (SEQ ID NO: 3) After performing the first PCR reaction, the PCR reaction product was used as a mega primer, and the second PCR reaction was performed using a forward primer (SEQ ID NO: 1).

상기 최종 PCR 반응 산물 및 pET21a 벡터(Novagen, USA)를 표 1에서 나타낸 바와 같이 제한효소 NdeI 및 XhoI으로 37℃에서 2 시간 동안 처리한 후, DNA Ligation kit를 이용하여 연결해 주었고, 상기 벡터의 염기서열을 시퀀싱하여 확인하였다. 염기서열분석 결과를 Blast(//www.ncbi.nlm.nih.gov)를 통해 천연 카스파제 유전자의 염기서열과 비교함으로써 28번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드를 암호화하는 부위의 폴리뉴클레오티드 염기서열의 변화를 확인함으로써 28번째 아미노산이 트롬빈 인식 부위로 치환된 재조합 카스파제 3 전구체인 28TS caspase 3(서열번호 51)을 과발현하는 벡터의 제작을 완료하였다.The final PCR reaction product and the pET21a vector (Novagen, USA) were treated with restriction enzymes Nde I and Xho I for 2 hours at 37 ° C. as shown in Table 1, and then linked using a DNA Ligation kit. The sequence was confirmed by sequencing. The sequencing results were compared with the sequencing of the natural caspase gene via Blast (//www.ncbi.nlm.nih.gov) to identify peptides encoding six consecutive amino acids including the 28th amino acid. By confirming the change in the polynucleotide sequence, the production of a vector overexpressing 28TS caspase 3 (SEQ ID NO: 51), a recombinant caspase 3 precursor in which the 28th amino acid was substituted with a thrombin recognition site, was completed.

<2-2> Δ28 <2-2> Δ28 caspasecaspase 3(서열번호 50) 발현벡터 3 (SEQ ID NO: 50) Expression Vector

카스파제 3의 1 내지 28번째 아미노산을 제거한 재조합 카스파제 3 전구체를 발현하는 발현벡터를 제작하였다.An expression vector was prepared expressing the recombinant caspase 3 precursor from which the 1st to 28th amino acids of caspase 3 were removed.

20 μΜ 정방향 프라이머(서열번호 4: GGG AAT TCC ATA TGT CTG GAA TAT CCC TGG ACA ACA GT)와 역방향 프라이머(서열번호 8: ATC AAC GCT GCC GCG CGG CAC CAG GCC ACA GTC CAG TTC TGT ACC ACG)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머로 사용하고 역방향 프라이머(서열번호 3)를 사용하여 2차 PCR 반응을 수행하였다.Using 20 μΜ forward primer (SEQ ID NO: 4: GGG AAT TCC ATA TGT CTG GAA TAT CCC TGG ACA ACA GT) and reverse primer (SEQ ID NO: 8: ATC AAC GCT GCC GCG CGG CAC CAG GCC ACA GTC CAG TTC TGT ACC ACG) By performing the first PCR reaction in the same manner as in Example 2-1, using the PCR reaction product as a mega primer and a second PCR reaction using a reverse primer (SEQ ID NO: 3).

실시예 2-1의 방법을 이용하여 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 175번째 아미노산이 트롬빈 인식 부위로 치환된 재조합 카스파제 3 전구체인 Δ28 caspase 3(서열번호 50)을 과발현하는 벡터의 제작을 완료하였다.Preparation of a vector overexpressing Δ28 caspase 3 (SEQ ID NO: 50), a recombinant caspase 3 precursor in which the 175th amino acid was substituted with a thrombin recognition site by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1 Completed.

<2-3> 175TS caspase 3(서열번호 52) 발현벡터<2-3> 175TS caspase 3 (SEQ ID NO: 52) Expression Vector

카스파제 3의 175번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 80: IETDSG; 아미노산 172번째에서 177번째까지)가 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 79: LVPRGS)로 치환된 재조합 카스파제 3 전구체인 175TS caspase 3의 발현벡터를 제작하였다.Peptides consisting of six consecutive amino acids including the 175th amino acid of Caspase 3 (SEQ ID NO: 80: IETDSG; amino acids 172 through 177) are peptides consisting of consecutive amino acids that are recognized and cleaved by thrombin : Expression vector of 175TS caspase 3, a recombinant caspase 3 precursor substituted with LVPRGS), was prepared.

20 μΜ 정방향 프라이머(서열번호 1) 및 역방향 프라이머(서열번호 8: ATC AAC GCT GCC GCG CGG CAC CAG GCC ACA GTC CAG TTC TGT ACC ACG)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머로 사용하고 역방향 프라이머(서열번호 3)을 사용하여 2차 PCR 반응을 수행하였다.Primary PCR reaction in the same manner as in Example 2-1 using 20 μΜ forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 8: ATC AAC GCT GCC GCG CGG CAC CAG GCC ACA GTC CAG TTC TGT ACC ACG) After the execution, the PCR reaction product was used as a megaprimer and a second PCR reaction was performed using a reverse primer (SEQ ID NO: 3).

또 다른 방법으로는 20 μΜ 정방향 프라이머(서열번호 7: TGT GGC CTG GTG CCG CGC GGC AGC GTT GAT GAT GAC ATG GCG TGT CAT) 및 역방향 프라이머(서열번호 3)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머로 사용하고 정방향 프라이머(서열번호 1)을 사용하여 2차 PCR 반응을 수행하였다.Another method was the same as in Example 2-1 using 20 μΜ forward primer (SEQ ID NO: 7: TGT GGC CTG GTG CCG CGC GGC AGC GTT GAT GAT GAC ATG GCG TGT CAT) and reverse primer (SEQ ID NO: 3) After performing the first PCR reaction, the PCR reaction product was used as a mega primer, and the second PCR reaction was performed using a forward primer (SEQ ID NO: 1).

실시예 2-1의 방법을 이용하여 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 175번째 아미노산이 트롬빈 인식 부위로 치환된 재조합 카스파제 3 전구체인 175TS caspase 3(서열번호 52)을 과발현하는 벡터의 제작을 완료하였다.Preparation of a vector overexpressing 175TS caspase 3 (SEQ ID NO: 52), a recombinant caspase 3 precursor in which the 175th amino acid was substituted with a thrombin recognition site by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1 Completed.

<2-4> Δ28/175TS caspase 3(서열번호 55) 발현벡터<2-4> Δ28 / 175TS caspase 3 (SEQ ID NO: 55) expression vector

카스파제 3의 1 내지 28번째 연속적인 아미노산으로 구성된 펩타이드가 제거되고, 175번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드가 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 79)로 치환된 재조합 카스파제 3 전구체를 발현하는 발현벡터를 제작하였다.Peptides consisting of consecutive amino acids wherein the peptide consisting of the 1 to 28th consecutive amino acids of caspase 3 is removed and the peptide consisting of 6 consecutive amino acids including the 175th amino acid is recognized and cleaved by thrombin (SEQ ID NO: 79) An expression vector was prepared expressing the recombinant caspase 3 precursor.

20 μΜ 정방향 프라이머(서열번호 4) 및 역방향 프라이머(서열번호 8)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응을 수행하였고, 상기 PCR 결과물을 메가프라이머로 사용하고 역방향 프라이머(서열번호 3)를 이용하여 2차 PCR 반응을 수행하였다.The first PCR reaction was carried out in the same manner as in Example 2-1 using 20 μΜ forward primer (SEQ ID NO: 4) and reverse primer (SEQ ID NO: 8), and the PCR result was used as a mega primer and reverse primer (SEQ ID NO: 8). The secondary PCR reaction was performed using No. 3).

실시예 2-1의 방법을 이용하여 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 1 내지 28번째 연속적인 아미노산으로 구성된 펩타이드가 제거되고, 175번째 아미노산이 트롬빈 인식 부위로 치환된 재조합 카스파제 3 전구체인 Δ28/175TS caspase 3(서열번호 55)을 과발현하는 벡터의 제작을 완료하였다.By inserting the PCR final product into the pET21a expression vector using the method of Example 2-1, the peptide consisting of 1 to 28 consecutive amino acids is removed, and the 175th amino acid is a recombinant caspase 3 precursor substituted with a thrombin recognition site. Construction of the vector overexpressing Δ28 / 175TS caspase 3 (SEQ ID NO: 55) was completed.

<2-5> 28TS/175TS caspae-3(서열번호 53) 발현벡터<2-5> 28TS / 175TS caspae-3 (SEQ ID NO: 53) Expression Vector

카스파제 3의 28번째 및 175번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 79)로 치환된 재조합 카스파제 3 전구체인 28TS/175TS caspae-3의 발현벡터를 제작하였다.28TS /, a recombinant caspase 3 precursor, wherein a peptide consisting of six consecutive amino acids, including the 28th and 175th amino acids of caspase 3, is substituted with a peptide consisting of consecutive amino acids recognized and cleaved by thrombin (SEQ ID NO: 79) An expression vector of 175TS caspae-3 was constructed.

실시예 2-1의 28TS caspase 3을 과발현하는 벡터를 주형 DNA로 하여 20 μΜ 정방향 프라이머(서열번호 7) 및 역방향 프라이머(서열번호 3)를 이용하여 실시예 2-1의 방법으로 과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머(megaprimer)로 사용하고 정방향 프라이머(서열번호 1)을 사용하여 2차 PCR 반응을 수행하였다.In the same manner as in Example 2-1, using 20 μΜ forward primer (SEQ ID NO: 7) and reverse primer (SEQ ID NO: 3) using the vector overexpressing 28TS caspase 3 of Example 2-1 as template DNA After performing the first PCR reaction, the PCR reaction product was used as a megaprimer and a second PCR reaction was performed using a forward primer (SEQ ID NO: 1).

실시예 2-1의 방법을 이용하여 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 175번째 아미노산이 치환된 재조합 카스파제 3 전구체인 28TS/175TS caspase 3(서열번호 53)을 과발현하는 벡터의 제작을 완료하였다.Preparation of the vector overexpressing 28TS / 175TS caspase 3 (SEQ ID NO: 53), a recombinant caspase 3 precursor substituted with the 175th amino acid, was performed by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1. It was.

<2-6> <2-6> L168FL168F /28/ 28 TSTS /175/ 175 TSTS caspasecaspase 3(서열번호 56)의 발현벡터 Expression vector of 3 (SEQ ID NO: 56)

실시예 2-4의 28TS/175TS caspase 3(서열번호 53)을 과발현하는 벡터를 주형 DNA로 하여 20 μΜ 정방향 프라이머(서열번호 9: GCC TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) 및 역방향 프라이머(서열번호 10: TGT CTC AAT GCC ACA GTC GAA TTC TGT ACC ACG GCA)를 이용하여 Site Directed Mutagenesis 키트(Stratagene, USA)를 이용하여 제조사의 방법에 따라 하기 조건으로 PCR 반응을 수행하였다: PCR 반응조건: 초기변성 95℃ 5분, 변성 95℃ 30초, 어닐링 45℃ 1분, 신장 68℃ 12분, 17회 반복, 최종 신장 72℃ 10분, 반응 종결 및 보관 4℃. PCR 반응 생성물을 제한효소 Dpn I(New England Biolabs, UK)으로 처리하여 37℃에서 1시간동안 반응 후 pET21a 발현벡터에 삽입하여 대장균에 형질전환하고 DNA 염기서열을 시퀀싱하여 확인하여 L168F/28TS/175TS caspase 3(서열번호 56)을 과발현하는 발현벡터를 제작하였다.20 μΜ forward primer (SEQ ID NO: 9: GCC TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) and reverse primer using the vector overexpressing 28TS / 175TS caspase 3 (SEQ ID NO: 53) of Example 2-4 (SEQ ID NO: 10: TGT CTC AAT GCC ACA GTC GAA TTC TGT ACC ACG GCA) using a Site Directed Mutagenesis kit (Stratagene, USA) to perform a PCR reaction under the following conditions according to the manufacturer's method: PCR reaction conditions : Initial denaturation at 95 ° C for 5 minutes, denaturation at 95 ° C for 30 seconds, annealing at 45 ° C for 1 minute, extension at 68 ° C for 12 minutes, 17 repetitions, final extension at 72 ° C for 10 minutes, reaction termination and storage at 4 ° C. The PCR reaction product was treated with restriction enzyme Dpn I (New England Biolabs, UK) for 1 hour at 37 ° C, inserted into a pET21a expression vector, transformed into E. coli, and sequenced and confirmed by DNA sequence. L168F / 28TS / 175TS An expression vector overexpressing caspase 3 (SEQ ID NO: 56) was constructed.

<2-7> <2-7> L168WL168W /28/ 28 TSTS /175/ 175 TSTS caspasecaspase 3( 3 ( 서열번호 57SEQ ID NO: 57 )의 발현벡터Expression vector

실시예 2-4의 28TS/175TS caspase 3(서열번호 53)을 과발현하는 벡터를 주형 DNA로 하여 정방향 프라이머(서열번호 11: TGC CGT GGT ACA GAA TGG GAC TGT GGC CTG GTG CCG) 및 역방향 프라이머(서열번호 12: CAC CAG GCC ACA GTC CCA TTC TGT ACC ACG GCA GGC)를 사용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L168W/28TS/175TS caspase 3(서열번호 57)을 과발현하는 발현벡터를 제작하였다.Forward primer (SEQ ID NO: 11: TGC CGT GGT ACA GAA TGG GAC TGT GGC CTG GTG CCG) and reverse primer (SEQ ID NO: 11) using the vector overexpressing 28TS / 175TS caspase 3 (SEQ ID NO: 53) of Example 2-4 No. 12: An expression vector overexpressing L168W / 28TS / 175TS caspase 3 (SEQ ID NO: 57) using the Site Directed Mutagenesis method described in Examples 2-6 using CAC CAG GCC ACA GTC CCA TTC TGT ACC ACG GCA GGC) Produced.

<2-8> 28TS/D175A/180TI caspase 3(서열번호 58) 발현벡터<2-8> 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 58) expression vector

카스파제 3의 28번째 아미노산을 포함하며 6개의 연속적인 아미노산으로 구성된 펩타이드를 연속적인 아미노산으로 치환하고, 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산이 삽입된 재조합 카스파제 3 전구체인 28TS/D175A/180TI caspase 3의 발현벡터를 제작하였다.Substituting a peptide consisting of six consecutive amino acids of the 28th amino acid of caspase 3 with a contiguous amino acid, between the 180 and 181 amino acids inserted six consecutive amino acids recognized and cleaved by thrombin An expression vector of 28TS / D175A / 180TI caspase 3, a recombinant caspase 3 precursor, was prepared.

<2-8-1> 28<2-8-1> 28 TSTS /180/ 180 TITI caspasecaspase 3(서열번호 54) 발현벡터 3 (SEQ ID NO: 54) Expression Vector

실시예 2-1의 28TS caspase 3(서열번호 51)을 과발현하는 벡터를 주형 DNA로 하여 20 μΜ 정방향 프라이머(서열번호 1) 및 역방향 프라이머(서열번호 13: TAT TTT ATG ACA CGC CAT GTC GCT GCC GCG CGG CAC CAG ATC ATC AAC ACC ACT)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행 후, 상기 PCR 반응 산물을 메가프라이머로 사용하고 역방향 프라이머(서열번호 3)를 사용하여 2차 PCR 반응을 수행하였다.20 μΜ forward primer (SEQ ID NO: 1) and reverse primer (SEQ ID NO: 13: TAT TTT ATG ACA CGC CAT GTC GCT GCC GCG) using the vector overexpressing 28TS caspase 3 (SEQ ID NO: 51) of Example 2-1 as template DNA CGG CAC CAG ATC ATC ATC AAC ACC ACT) using the first PCR reaction in the same manner as in Example 2-1, using the PCR reaction product as a mega primer and the second primer using a reverse primer (SEQ ID NO: 3) PCR reactions were performed.

또 다른 방법으로는 정방향 프라이머(서열번호 14: ACA GAC AGT GGT GTT GAT GAT CTG GTG CCG CGC GGC AGC GAC ATG GCG TGT CAT AAA) 및 역방향 프라이머(서열번호 3)를 이용하여 실시예 2-1과 동일한 방법으로 1차 PCR 반응 수행하여 얻은 PCR 반응 산물을 메가프라이머로 사용하고 서열번호 1을 정방향 프라이머로 하여 2차 PCR 반응 수행하여 목표유전자를 제조하였다.Another method is the same as Example 2-1 using a forward primer (SEQ ID NO: 14: ACA GAC AGT GGT GTT GAT GAT CTG GTG CCG CGC GGC AGC GAC ATG GCG TGT CAT AAA) and reverse primer (SEQ ID NO: 3) The target gene was prepared by using a PCR reaction product obtained by performing a first PCR reaction as a megaprimer and performing a second PCR reaction using SEQ ID NO: 1 as a forward primer.

실시예 2-1의 방법을 이용하여 상기 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 28TS/180TI caspase 3(서열번호 54)의 제작을 완료하였다.Preparation of 28TS / 180TI caspase 3 (SEQ ID NO: 54) was completed by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1.

<2-8-2> 28<2-8-2> 28 TSTS /Of D175AD175A /180/ 180 TITI caspasecaspase 3(서열번호 58) 발현벡터 3 (SEQ ID NO: 58) Expression Vector

이어서, 상기 제작된 벡터를 주형으로 정방향 프라이머(서열번호 15: GAC TGT GGC ATT GAG ACA GCG AGT GGT GTT GAT GAT)와 역방향 프라이머(서열번호 16: CAG ATC ATC AAC ACC ACT CGC TGT CTC AAT GCC ACA)를 사용한 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 175번 아스파틱산(Aspartic acid)를 알라닌(Alanine)으로 치환함으로써 28TS/D175A/180TI caspase 3(서열번호 58)를 과발현하는 발현벡터를 제작하였다.Subsequently, the prepared vector was used as a template with a forward primer (SEQ ID NO: 15: GAC TGT GGC ATT GAG ACA GCG AGT GGT GTT GAT GAT) and a reverse primer (SEQ ID NO: 16: CAG ATC ATC AAC ACC ACT CGC TGT CTC AAT GCC ACA) An expression vector overexpressing 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 58) was prepared by substituting alanine for aspartic acid No. 175 using the Site Directed Mutagenesis method described in Example 2-6. It was.

<2-9> L168F/28TS/D175A/180TI caspase 3(서열번호 59)발현벡터<2-9> L168F / 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 59)

실시예 2-7의 28TS/D175A/180TI caspase 3(서열번호 58)를 과발현하는 벡터를 주형 DNA로 하고 정방향 프라이머(서열번호 17: TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) 및 역방향 프라이머(서열번호 18: CTC AAT GCC ACA GTC GAATTC TGT ACC ACG GCA)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L168F/28TS/D175A/180TI caspase 3(서열번호 59)를 과발현하는 발현벡터를 제작하였다.The vector overexpressing 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 58) of Example 2-7 was used as template DNA, and the forward primer (SEQ ID NO: 17: TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) and reverse primer ( SEQ ID NO: 18: Expression vector overexpressing L168F / 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 59) using the Site Directed Mutagenesis method described in Example 2-6 using CTC AAT GCC ACA GTC GAATTC TGT ACC ACG GCA) Was produced.

<2-10> <2-10> L168WL168W /28/ 28 TSTS /Of D175AD175A /180/ 180 TITI caspasecaspase 3(서열번호 60) 발현벡터 3 (SEQ ID NO: 60) Expression Vector

실시예 2-7의 28TS/D175A/180TI caspase 3(서열번호 58)를 과발현하는 벡터를 주형 DNA로 하고 정방향 프라이머(서열번호 19: TGC CGT GGT ACA GAA TGG GAC TGT GGC ATT GAG) 및 역방향 프라이머(서열번호 20: CTC AAT GCC ACA GTC CCA TTC TGT ACC ACG GCA)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L168W/28TS/D175A/180TI caspase 3(서열번호 60)를 과발현하는 발현벡터를 제작하였다.The vector overexpressing 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 58) of Example 2-7 was used as template DNA, and the forward primer (SEQ ID NO: 19: TGC CGT GGT ACA GAA TGG GAC TGT GGC ATT GAG) and reverse primer ( SEQ ID NO: 20 Overexpresses L168W / 28TS / D175A / 180TI caspase 3 (SEQ ID NO: 60) using the Site Directed Mutagenesis method described in Example 2-6 using CTC AAT GCC ACA GTC CCA TTC TGT ACC ACG GCA) Vectors were produced.

<2-11> <2-11> C163SC163S caspasecaspase 3(서열번호 61) 발현벡터 3 (SEQ ID NO: 61) Expression Vector

음성대조군으로 카스파제 3의 활성잔기인 163번째 아미노산이 시스테인에서 세린으로 치환된 재조합 카스파제 3 전구체인 C163S caspase 3(서열번호 61)을, pET28a-Cas3 벡터를 주형 DNA로 하고 Pan S & Berk BC의 방법(Circ Res . 100(2):213-9, 2007)에 따라 수득하였다.As a negative control, C163S caspase 3 (SEQ ID NO: 61), a recombinant caspase 3 precursor (SEQ ID NO: 61) in which the 163th amino acid, the active residue of caspase 3, was substituted with a serine in cysteine, was used as the template DNA, and the pan S & Berk BC was used as the template DNA. Method of Circ Res . 100 (2): 213-9, 2007).

<2-12> 23<2-12> 23 TSTS /198/ 198 TSTS Caspase 7(서열번호 64) 발현벡터 Caspase 7 (SEQ ID NO: 64) Expression Vector

카스파제 7의 23번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 81: EDSVDAKPDR; 아미노산 19번째에서 28번째까지)를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 82: EDLVPRGSDR)로 치환 및 175번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 83: GIQADSGPIN; 아미노산 194번째에서 203번째까지)를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드(서열번호 98: GLVPRGSPIN)로 치환된 재조합 카스파제 7 전구체인 23TS/198TS Caspase 7(서열번호 64)의 발현벡터를 제작하였다.Peptides consisting of 10 consecutive amino acids including the 23rd amino acid of Caspase 7 (SEQ ID NO: 81: EDSVDAKPDR; amino acids 19-28) are peptides consisting of consecutive amino acids that are recognized and cleaved by thrombin : A peptide consisting of consecutive amino acids that are substituted by EDLVPRGSDR and consisting of 10 consecutive amino acids including the 175th amino acid (SEQ ID NO: 83: GIQADSGPIN; amino acids 194 through 203) recognized and cleaved by thrombin No. 98: An expression vector of 23TS / 198TS Caspase 7 (SEQ ID NO: 64), which is a recombinant caspase 7 precursor substituted with GLVPRGSPIN), was prepared.

pET28a-Cas7 벡터를 주형 DNA로 하고, 실시예 2-1의 방법으로 정방향 프라이머(서열번호 21) 및 역방향 프라이머(서열번호 24: TCA GCA AAT GAA CTG GTG CCG CGC GGC AGC CCA GAC CGG TCC TCG)를 이용하여 수득한 PCR 산물을 메가프라이머로 사용하고, 상기 메가프라이머 및 역방향 프라이머(서열번호 23)를 사용하여, 23번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환한 재조합 카스파제 7 전구체인 23TS Caspase 7(서열번호 63)을 과발현하는 벡터를 제작하였다.Using the pET28a-Cas7 vector as the template DNA, the forward primer (SEQ ID NO: 21) and the reverse primer (SEQ ID NO: 24: TCA GCA AAT GAA CTG GTG CCG CGC GGC AGC CCA GAC CGG TCC TCG) were prepared by the method of Example 2-1. Using the PCR product obtained as a megaprimer, using the megaprimer and the reverse primer (SEQ ID NO: 23), a peptide consisting of ten consecutive amino acids including the 23rd amino acid is recognized by the thrombin A vector overexpressing 23TS Caspase 7 (SEQ ID NO: 63), a recombinant caspase 7 precursor substituted with a peptide consisting of amino acids, was prepared.

또 다른 방법으로는 정방향 프라이머(서열번호 25: GGA CCG GTC TGG GCT GCC GCG CGG CAC CAG TTC ATT TGC TGA) 및 역방향 프라이머(서열번호 23)를 이용하여 수득한 PCR 산물을 메가프라이머로 사용하고, 정방향 프라이머(서열번호 21) 및 상기 메가프라이머를 사용하여 23TS Caspase 7(서열번호 63)을 과발현하는 벡터를 제작하였다.In another method, a PCR product obtained by using a forward primer (SEQ ID NO: 25: GGA CCG GTC TGG GCT GCC GCG CGG CAC CAG TTC ATT TGC TGA) and a reverse primer (SEQ ID NO: 23) is used as a mega primer. A vector overexpressing 23TS Caspase 7 (SEQ ID NO: 63) was prepared using the primer (SEQ ID NO: 21) and the megaprimer.

이어서, 상기 23TS Caspase 7(서열번호 63)을 과발현하는 벡터를 주형으로 정방향 프라이머(서열번호 27: GTC ATT GAT GGG GCT GCC GCG CGG CAC CAG GCC ATC ATC AAG)와 역방향 프라이머(서열번호 23)를 이용하여 생성된 PCR 산물을 메가프라이머로 사용하고, 정방향 프라이머(서열번호 21) 및 상기 메가프라이머를 사용하여 카스파제 7의 198번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환된 재조합 카스파제 7(23TS/198TS Caspase 7)의 유전자를 제작하였다.Subsequently, a forward primer (SEQ ID NO: 27: GTC ATT GAT GGG GCT GCC GCG CGG CAC CAG GCC ATC ATC AAG) and a reverse primer (SEQ ID NO: 23) were used as a template to overexpress the 23TS Caspase 7 (SEQ ID NO: 63). Using the PCR product generated as a megaprimer, and using a forward primer (SEQ ID NO: 21) and the megaprimer, a peptide consisting of ten consecutive amino acids including the 198th amino acid of caspase 7 was recognized and cleaved by thrombin. A gene of recombinant caspase 7 (23TS / 198TS Caspase 7) substituted with a peptide consisting of consecutive amino acids was prepared.

또 다른 방법으로는 정방향 프라이머(서열번호 21) 및 역방향 프라이머(서열번호 26: CTT GAT GAT GGC CTG GTG CCG CGC GGC AGC CCC ATC AAT GAC ACA)를 이용하여 수득한 PCR 산물을 메가프라이머로 사용하고, 상기 메가프라이머 및 역방향 프라이머(서열번호 23)를 사용하여 재조합 카스파제 7(23TS/198TS Caspase 7)의 유전자를 제작하였다.In another method, a PCR product obtained using a forward primer (SEQ ID NO: 21) and a reverse primer (SEQ ID NO: 26: CTT GAT GAT GGC CTG GTG CCG CGC GGC AGC CCC ATC AAT GAC ACA) is used as a megaprimer, Genes of recombinant caspase 7 (23TS / 198TS Caspase 7) were prepared using the megaprimer and reverse primer (SEQ ID NO: 23).

아울러, 실시예 2-1의 방법을 이용하여 상기 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 23 TS /198 TS Caspase 7(서열번호 64)을 과발현하는 발현벡터를 제작하였다.In addition, by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1 23 TS / 198 TS Caspase An expression vector overexpressing 7 (SEQ ID NO: 64) was prepared .

<2-13> 198<2-13> 198 TSTS CaspaseCaspase 7(서열번호 97) 발현벡터 7 (SEQ ID NO: 97) Expression Vector

카스파제 7의 23번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환된 재조합 카스파제 7 전구체인 198TS Caspase 7(서열번호 97)의 발현벡터를 제작하였다.Expression of 198TS Caspase 7 (SEQ ID NO: 97), a recombinant caspase 7 precursor in which a peptide consisting of 10 consecutive amino acids including the 23rd amino acid of caspase 7 is substituted with a peptide consisting of consecutive amino acids recognized and cleaved by thrombin Vectors were produced.

pET28a-Cas7 벡터를 주형 DNA로 하고, 실시예 2-1의 방법으로 정방향 프라이머(서열번호 27)와 역방향 프라이머(서열번호 23)를 이용하여 생성된 PCR 산물을 메가프라이머로 사용하고, 정방향 프라이머(서열번호 21) 및 상기 메가프라이머를 사용하여 카스파제 7의 198번째 아미노산을 포함한 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환된 재조합 카스파제 7(198TS Caspase 7)의 유전자를 제작하였다.Using the pET28a-Cas7 vector as template DNA, the PCR product generated using the forward primer (SEQ ID NO: 27) and the reverse primer (SEQ ID NO: 23) by the method of Example 2-1 was used as a mega primer, and the forward primer ( SEQ ID NO: 21) and a recombinant caspase 7 (substituted with a peptide consisting of 10 consecutive amino acids including the 198th amino acid of caspase 7 with a peptide consisting of consecutive amino acids recognized and cleaved by thrombin using the megaprimer) 198TS Caspase 7) was constructed.

또 다른 방법으로는 정방향 프라이머(서열번호 21) 및 역방향 프라이머(서열번호 26)를 이용하여 수득한 PCR 산물을 메가프라이머로 사용하고, 상기 메가프라이머 및 역방향 프라이머(서열번호 23)를 사용하여 재조합 카스파제 7(198TS Caspase 7)의 유전자를 제작하였다.As another method, a PCR product obtained by using a forward primer (SEQ ID NO: 21) and a reverse primer (SEQ ID NO: 26) is used as a megaprimer, and the recombinant caspa using the mega primer and a reverse primer (SEQ ID NO: 23). A gene of the seventh (198TS Caspase 7) was constructed.

아울러, 실시예 2-1의 방법을 이용하여 상기 PCR 최종산물을 pET21a 발현벡터에 삽입함으로써 198 TS Caspase 7(서열번호 97)을 과발현하는 발현벡터를 제작하였다.In addition, by inserting the PCR final product into the pET21a expression vector using the method of Example 2-1 198 TS Overexpressing Caspase 7 (SEQ ID NO: 97) Expression vectors were prepared.

<2-14> L191F/23TS/198TS Caspase 7(서열번호 65) 발현벡터<2-14> L191F / 23TS / 198TS Caspase 7 (SEQ ID NO: 65) Expression Vector

실시예 2-12의 23TS/198TS Caspase 7(서열번호 64)을 과발현하는 발현벡터를 주형 DNA로 하여 정방향 프라이머(서열번호 28: TGC CGA GGG ACC GAG TTT GAT GAT GGC CTG GTG CCG)와 역방향 프라이머(서열번호 29: CAC CAG GCC ATC ATC AAA CTC GGT CCC TCG GCA AGC)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L191F /23 TS /198 TS Caspase 7(서열번호 65)을 과발현하는 발현벡터를 제작하였다.Overexpressing 23TS / 198TS Caspase 7 (SEQ ID NO: 64) of Examples 2-12 Using an expression vector as a template DNA, a forward primer (SEQ ID NO: 28: TGC CGA GGG ACC GAG TTT GAT GAT GGC CTG GTG CCG) and a reverse primer (SEQ ID NO: 29: CAC CAG GCC ATC ATC AAA CTC GGT CCC TCG GCA AGC) L191F / 23 TS / 198 TS by the Site Directed Mutagenesis method described in Example 2-6 An expression vector overexpressing Caspase 7 (SEQ ID NO: 65) was prepared.

<2-15> L191W/23TS/198TS Caspase 7(서열번호 66) 발현벡터<2-15> L191W / 23TS / 198TS Caspase 7 (SEQ ID NO: 66) Expression Vector

실시예 2-12의 23TS/198TS Caspase 7(서열번호 64)을 과발현하는 발현벡터를 주형 DNA로 하여 정방향 프라이머(서열번호 30: TGC CGA GGG ACC GAG TGG GAT GAT GGC CTG GTG CCG)와 역방향 프라이머(서열번호 31: CAC CAG GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L191W /23 TS /198 TS Caspase 7(서열번호 66)을 과발현하는 발현벡터를 제작하였다.Overexpressing 23TS / 198TS Caspase 7 (SEQ ID NO: 64) of Examples 2-12 Using an expression vector as a template DNA, a forward primer (SEQ ID NO: 30: TGC CGA GGG ACC GAG TGG GAT GAT GGC CTG GTG CCG) and a reverse primer (SEQ ID NO: 31: CAC CAG GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC) L191W / 23 TS / 198 TS by the Site Directed Mutagenesis method described in Example 2-6. An expression vector overexpressing Caspase 7 (SEQ ID NO: 66) was prepared.

<2-16> 23TS/D198A/203TI Caspase 7(서열번호 67) 발현벡터<2-16> 23TS / D198A / 203TI Caspase 7 (SEQ ID NO: 67) Expression Vector

카스파제 7의 23번째 아미노산을 포함하며 10개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환하고, 203번째 아미노산과 204번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 10개의 연속적인 아미노산이 삽입된 재조합 카스파제 7 전구체인 23TS/D198A/203TI Caspase 7의 발현벡터를 제작하였다.A peptide comprising 10 consecutive amino acids of caspase 7 and consisting of 10 consecutive amino acids is replaced with a peptide consisting of consecutive amino acids that are recognized and cleaved by thrombin, and is recognized by thrombin between amino acids 203 and 204 An expression vector of 23TS / D198A / 203TI Caspase 7 , a recombinant caspase 7 precursor in which 10 consecutive amino acids were cut, was prepared .

실시예 2-12의 23TS Caspase 7(서열번호 63)을 과발현하는 벡터를 주형 DNA로하고 정방향 프라이머(서열번호 35: AGG ATT AGC ATC TGT GCT GCC GCG CGG CAC CAG GTC ATT GAT GGG CCC CGA)와 역방향 프라이머(서열번호 23)를 이용한 PCR 반응 산물을 메가프라이머(megaprimer)로 사용하고 정방향 프라이머(서열번호 21)을 사용하여 2차 PCR 반응을 수행하고, 실시예 2-1의 방법을 이용하여 최종 PCR 반응산물을 pET21a 벡터(Novagen, USA)에 삽입하여 23TS/203TI Caspase 7를 과발현하는 벡터를 제작하였다.A vector overexpressing 23TS Caspase 7 (SEQ ID NO: 63) of Examples 2-12 was used as template DNA and reversed with a forward primer (SEQ ID NO: 35: AGG ATT AGC ATC TGT GCT GCC GCG CGG CAC CAG GTC ATT GAT GGG CCC CGA) PCR reaction product using the primer (SEQ ID NO: 23) as a megaprimer (megaprimer), and performing a secondary PCR reaction using the forward primer (SEQ ID NO: 21), the final PCR using the method of Example 2-1 The reaction product was inserted into the pET21a vector (Novagen, USA) to prepare a vector that overexpresses 23TS / 203TI Caspase 7.

또 다른 방법으로는 정방향 프라이머(서열번호 21)와 역방향 프라이머(서열번호 34: GGG CCC ATC AAT GAC CTG GTG CCG CGC GGC AGC ACA GAT GCT AAT CCT CGA)를 이용한 PCR 반응 산물을 메가프라이머(megaprimer)로 사용하고 역방향 프라이머(서열번호 23)을 사용하여 2차 PCR 반응을 수행하고, 실시예 2-1의 방법을 이용하여 최종 PCR 반응산물을 pET21a 벡터(Novagen, USA)에 삽입하여 23TS/203TI Caspase 7를 과발현하는 벡터를 제작하였다.Alternatively, the PCR reaction product using a forward primer (SEQ ID NO: 21) and a reverse primer (SEQ ID NO: 34: GGG CCC ATC AAT GAC CTG GTG CCG CGC GGC AGC ACA GAT GCT AAT CCT CGA) is used as a megaprimer. Secondary PCR reaction using the reverse primer (SEQ ID NO: 23), and the final PCR reaction product was inserted into the pET21a vector (Novagen, USA) using the method of Example 2-1 to perform 23TS / 203TI Caspase 7 The vector overexpressed was produced.

상기 23TS/203TI Caspase 7를 과발현하는 벡터를 주형으로 정방향 프라이머(서열번호 32: GAT GGC ATC CAG GCC GAG TCG GGG CCC ATC AAT GAC)와 역방향 프라이머(서열번호 33: GTC ATT GAT GGG CCC CGA CTC GGC CTG GAT GCC ATC)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 198번 아스파틱산(Aspartic acid)을 알라닌으로 돌연변이 시킴으로써, 23 TS / D198A /203 TI Caspase 7(서열번호 67)을 과발현하는 발현벡터를 제작하였다.The vector overexpressing the 23TS / 203TI Caspase 7 was used as a template with a forward primer (SEQ ID NO: 32: GAT GGC ATC CAG GCC GAG TCG GGG CCC ATC AAT GAC) and a reverse primer (SEQ ID NO: 33: GTC ATT GAT GGG CCC CGA CTC GGC CTG 23 TS / D198A / 203 TI by mutating aspartic acid No. 198 to alanine using the Site Directed Mutagenesis method described in Example 2-6 using GAT GCC ATC) An expression vector overexpressing Caspase 7 (SEQ ID NO: 67) was prepared.

<2-17> L191F/23TS/D198A/203TI Caspase 7(서열번호 68) 발현벡터<2-17> L191F / 23TS / D198A / 203TI Caspase 7 (SEQ ID NO: 68) Expression Vector

실시예 2-14의 23TS/D198A/203TI Caspase 7(서열번호 67)을 과발현하는 발현벡터를 주형 DNA로 하여 정방향 프라이머(서열번호 36: TGC CGA GGG ACC GAG TTT GAT GAT GGC ATC CAG GCC)와 역방향 프라이머(서열번호 37: CTG GAT GCC ATC ATC AAA CTC GGT CCC TCG GCA AGC)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L191F /23 TS / D198A /203 TI Caspase 7(서열번호 68)를 과발현하는 발현벡터를 제작하였다. Reversed with forward primer (SEQ ID NO: 36: TGC CGA GGG ACC GAG TTT GAT GAT GGC ATC CAG GCC) using the expression vector overexpressing 23TS / D198A / 203TI Caspase 7 (SEQ ID NO: 67) of Example 2-14 as template DNA L191F / 23 TS / D198A / 203 TI using the Site Directed Mutagenesis method described in Examples 2-6 using a primer (SEQ ID NO: 37: CTG GAT GCC ATC ATC AAA CTC GGT CCC TCG GCA AGC) Caspase An expression vector overexpressing 7 (SEQ ID NO: 68) was prepared.

<2-18> L191W/23TS/D198A/203TI Caspase 7(서열번호 69) 발현벡터<2-18> L191W / 23TS / D198A / 203TI Caspase 7 (SEQ ID NO: 69) Expression Vector

실시예 2-14의 23TS/D198A/203TI Caspase 7(서열번호 67)을 과발현하는 발현벡터를 주형 DNA로 하여 정방향 프라이머(서열번호 38: TGC CGA GGG ACC GAG TGG GAT GAT GGC ATC CAG GCC)와 역방향 프라이머(서열번호 39: CTG GAT GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC)를 이용하여 실시예 2-6에 기술한 Site Directed Mutagenesis 방법으로 L191W /23 TS / D198A /203 TI Caspase 7(서열번호 69)를 과발현하는 발현벡터를 제작하였다. Reversed with forward primer (SEQ ID NO: 38: TGC CGA GGG ACC GAG TGG GAT GAT GGC ATC CAG GCC) using the expression vector overexpressing 23TS / D198A / 203TI Caspase 7 (SEQ ID NO: 67) of Example 2-14 as template DNA. L191W / 23 TS / D198A / 203 TI using the Site Directed Mutagenesis method described in Examples 2-6 using a primer (SEQ ID NO: 39: CTG GAT GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC) Caspase An expression vector overexpressing 7 (SEQ ID NO: 69) was prepared.

<< 실시예Example 3> 재조합  3> recombination 카스파제Caspase 전구체의 발현 Expression of precursors

하나한(Hanahan)이 기술한 방법(Hanahan D, DNA Cloning vol.1 109-135, IRS press 1985)에 의해 실시예 1 내지 2의 방법으로 수득한 재조합 카스파제 3의 전구체 및 재조합 카스파제 7의 전구체를 과발현하는 벡터들을 대장균에 형질전환 하였다.The precursor of recombinant caspase 3 and the recombinant caspase 7 obtained by the method of Examples 1 to 2 by the method described by Hanahan (Hanahan D, DNA Cloning vol. 1 109-135, IRS press 1985). E. coli were transformed with the vectors overexpressing the precursor.

구체적으로, CaCl2로 처리한 대장균 Rosetta DE3(Novagen, USA)에 실시예 1 내지 2의 벡터들을 열충격 방법으로 형질전환시킨 후, 앰피실린(ampicillin) 또는 카나마이신(kanamycin)이 포함된 배지에서 배양하여 상기 발현벡터가 형질전환되어 앰피실린 저항성 또는 카나마이신(kanamycin) 저항성을 나타내는 콜로니를 선별하였다. 상기 콜로니를 LB 배지(30 ㎍/㎖ 카나마이신 또는 50 ㎍/㎖ 앰피실린; sigma, USA)에 접종하고, 섭씨 37℃에서 16시간 배양한 후 적당량을 취하여 LB 배지(30 ㎍/㎖ 카나마이신 또는 50 ㎍/㎖ 앰피실린; sigma, USA)에 접종하였다. 배양액의 OD600이 0.7~0.9에 이르렀을 때 IPTG를 첨가(1 mM)하여 18℃에서 18 시간 동안 더 배양하여 재조합 유전자의 발현을 유도하였다.Specifically, E. coli Rosetta DE3 (Novagen, USA) treated with CaCl 2 transformed the vectors of Examples 1 and 2 by the thermal shock method, and then cultured in a medium containing ampicillin (kaniccin) or kanamycin (kanamycin) The expression vectors were transformed to select colonies showing ampicillin resistance or kanamycin resistance. The colonies were inoculated in LB medium (30 μg / ml kanamycin or 50 μg / ml ampicillin; sigma, USA), incubated at 37 ° C. for 16 hours, and then an appropriate amount was taken for LB medium (30 μg / ml kanamycin or 50 μg). / Ml ampicillin; sigma, USA). When the OD 600 of the culture reached 0.7-0.9, IPTG was added (1 mM) and further cultured at 18 ° C. for 18 hours to induce the expression of recombinant genes.

배양이 끝나고 배양액을 원심분리하여 침전된 균체를 25 ㎖ 세포 파쇄액(lysis buffer, 50 mM Tris pH 7.5, 200 mM NaCl, 5% 글리세롤)에 현탁하였고, 초음파 발생기로 파쇄하였다. 이를 원심분리하여 상층액을 탈론 수지(Talon resin, Clontech, USA)를 이용한 배치(batch) 방법으로 재조합 카스파제 전구체만을 정제하였다.After incubation, the culture medium was centrifuged and the precipitated cells were suspended in 25 ml of cell lysate (lysis buffer, 50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol) and crushed by an ultrasonic generator. The supernatant was centrifuged to purify only recombinant caspase precursors by a batch method using Talon resin (Clontech, USA).

배치방법을 이용한 단백질 정제를 상세히 기술하면 하기와 같다. 10 ㎖의 컬럼완충용액(50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol)으로 탈론 레진(1 ㎖)을 2번 씻어서 활성화시킨 후, 상기에서 분리한 상층액을 가하여 4℃에서 3시간 동안 천천히 진탕하여 단백질을 탈론 수지에 흡착시켰다. 수용액을 여과 제거한 후, 탈론 수지를 컬럼완충용액(10 ㎖)으로 두 번 씻어 준 후 10 ㎖의 용출 완충용액(elution buffer: 100 mM 이미다졸)을 가한 후 다시 1 시간 천천히 진탕(rocking)한 후 여과하여, 재조합 카스파제 전구체를 회수하였다. 5 ㎖의 용출 완충용액을 사용하여 한 번 더 반복해주어 수득량을 늘렸다. 상기 방법으로 정제된 단백질을 브래드포드 검출(Bradford assay)법을 통해 정량하였고, 15% SDS-PAGE 젤을 통해 단백질의 크기를 추정함으로써 재조합 카스파제 전구체 3의 과발현 및 정제 결과를 확인하였다.The protein purification using the batch method is described in detail as follows. 10 ml of column buffer solution (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol) was washed twice with activation of talon resin (1 ml), and the supernatant separated above was added thereto. The protein was adsorbed onto the Talon resin by shaking slowly at 4 ° C. for 3 hours. After the aqueous solution was filtered off, the Talon resin was washed twice with column buffer solution (10 mL), and then 10 mL of elution buffer (100 mM imidazole) was added, followed by slow rocking for 1 hour. Filtration recovered the recombinant caspase precursor. Repeat 5 more times with 5 ml of elution buffer to increase yield. Protein purified by the above method was quantified by the Bradford assay, and overexpression and purification of recombinant caspase precursor 3 was confirmed by estimating the size of the protein through a 15% SDS-PAGE gel.

그 결과, 도 3의 레인 4에 나타난 바와 같이 재조합 카스파제 3 전구체의 28번째 아미노산을 포함하는 6개의 아미노산으로 구성된 인식부위 및 175번째 아미노산을 포함하는 6개의 아미노산으로 구성된 인식부위를 트롬빈 인식부위로 치환함으로써 재조합 카스파제 3 전구체의 발현량이 높아지는 것을 확인하였다.As a result, as shown in lane 4 of FIG. 3, the recognition site consisting of six amino acids including the 28th amino acid of the recombinant caspase 3 precursor and the recognition site consisting of six amino acids including the 175th amino acid were referred to as thrombin recognition sites. By substitution, it was confirmed that the expression level of the recombinant caspase 3 precursor is increased.

<실시예 4> 정제된 재조합 카스파제 전구체의 활성화 측정Example 4 Determination of Activation of Purified Recombinant Caspase Precursor

실시예 2 및 3의 방법으로 수득한 트롬빈 기질부위서열이 치환된 재조합 카스파제 3의 전구체 또는 재조합 카스파제 7의 전구체를 활성화한 후 효소활성을 SDS-PAGE에서 측정하였다.After activating the precursor of recombinant caspase 3 or the precursor of recombinant caspase 7 substituted with the thrombin substrate sequence obtained by the method of Examples 2 and 3, the enzyme activity was measured in SDS-PAGE.

<4-1> 재조합 카스파제 3<4-1> Recombinant Caspase 3

정제된 단백질의 농도는 Bradford assay에 의하여 발색하는 정도를 흡광도 595 nm에서 측정하였다. 트롬빈은 Sulfo-NHS ester 그룹이 도입된 바이오틴(Pierce, USA)과 실온에서 반응시킨 후 탈염 컬럼(desalting column; Amersham Biosciences, USA)을 통해 미반응 바이오틴을 제거한 후 사용하였다.The concentration of the purified protein was measured by absorbance at 595 nm to the extent of color development by Bradford assay. Thrombin was used after removing unreacted biotin through a desalting column (Amersham Biosciences, USA) after reacting at room temperature with biotin (Pierce, USA) into which the Sulfo-NHS ester group was introduced.

실시예 2 및 3의 방법으로 수득한 재조합 카스파제 3 전구체(서열번호 53) 1 ㎎/㎖를 상기의 방법으로 바이오틴이 도입된 트롬빈을 이용하여 카스파제 200 ㎍ 당 트롬빈 2 NIH, 20 NIH 및 40 NIH 유닛이 되도록 섞어준 다음, 4℃와 18℃의 조건에서 활성화하였고, 4, 8 및 24 시간별로 트롬빈에 의해 활성화되는 정도를 SDS-PAGE 와 카스파제의 기질을 이용하여 확인하였다. 활성화 반응이 끝난 후 아비딘 수지(avidin resin)를 이용하여 상기 반응물에 아비딘 수지(avidin resin) 1 ㎖를 가하고 18℃에서 1 시간 동안 약하게 진탕한 후 여과함으로써 바이오틴이 도입된 트롬빈을 흡착 제거하였다. 상기 활성화반응이 제대로 수행되었는지 확인하기 위하여 시간별로 반응액을 회수하여 15% SDS-PAGE 젤을 통해 활성화 반응 결과를 확인하였다.1 mg / ml of the recombinant caspase 3 precursor (SEQ ID NO: 53) obtained by the method of Examples 2 and 3 was added to thrombin 2 NIH, 200 NIH and 40 μg of caspase using thrombin in which biotin was introduced by the above method. After mixing to the NIH unit, it was activated at the conditions of 4 ℃ and 18 ℃, the degree of activation by thrombin for 4, 8 and 24 hours was confirmed using the substrate of SDS-PAGE and caspase. After the activation reaction, 1 mL of avidin resin was added to the reaction product using an avidin resin, and then gently shaken at 18 ° C. for 1 hour, followed by filtration to remove the biotin-introduced thrombin. In order to confirm that the activation reaction was properly performed, the reaction solution was recovered by time and the activation reaction result was confirmed through 15% SDS-PAGE gel.

그 결과, 도 4에 나타난 바와 같이 본 발명의 재조합 카스파제 3 전구체(서열번호 53)는 18℃에서 8시간 또는 4℃에서 24시간 동안 트롬빈으로 가수분해하여 활성형 카스파제로 변환시키는 것이 적당한 것을 확인하였다. 또한, 상기 결과를 바탕으로 볼 때, 본 발명에서 트롬빈의 사용량은 반응 온도와 상관없이 40 NIH 유닛 이상이 바람직하였다.As a result, as shown in FIG. 4, the recombinant caspase 3 precursor (SEQ ID NO: 53) of the present invention was found to be suitable to be hydrolyzed to thrombin for 8 hours at 18 ° C. or 24 hours at 4 ° C. to convert it into active caspase. It was. In addition, based on the above results, the amount of thrombin used in the present invention is preferably at least 40 NIH units regardless of the reaction temperature.

<4-2> 재조합 카스파제 7<4-2> Recombinant Caspase 7

실시예 3과 동일한 방법으로 서열번호 97 및 서열번호 64의 재조합 카스파제 7의 전구체를 발현 및 정제하였고, 실시예 4-1과 동일한 방법으로 카스파제 200 ㎍ 당 트롬빈 40 NIH 유닛을 이용하여 활성화한 후 효소활성을 SDS-PAGE에서 측정하였다.The precursors of recombinant caspase 7 of SEQ ID NO: 97 and SEQ ID NO: 64 were expressed and purified in the same manner as in Example 3, and activated using thrombin 40 NIH units per 200 µg of caspase in the same manner as in Example 4-1. Post enzyme activity was measured on SDS-PAGE.

그 결과, 도 5에서 나타난 바와 같이 본 발명의 재조합 카스파제 7 전구체(서열번호 97 및 64)가 4℃ 또는 18℃에서 활성형 카스파제로 변환되는 것을 확인하였다.As a result, as shown in Figure 5 it was confirmed that the recombinant caspase 7 precursor (SEQ ID NOs: 97 and 64) of the present invention is converted to active caspase at 4 ℃ or 18 ℃.

<실시예 5> 활성형 카스파제의 효소반응속도Example 5 Enzyme Kinetics of Active Caspase

실시예 4의 방법으로 SDS-PAGE 젤 상에서 실시예 2 및 3의 방법으로 수득한 재조합 카스파제 3 전구체의 가수분해가 완료된 것을 확인한 후 기질을 사용하여 카스파제의 효소활성을 확인하였다.After confirming that the hydrolysis of the recombinant caspase 3 precursors obtained by the methods of Examples 2 and 3 was completed on the SDS-PAGE gel by the method of Example 4, the enzymatic activity of caspase was confirmed using a substrate.

카스파제 3의 기질로 널리 알려진 Ac-DEVD-pNA(pNA:para-nitroanilide; Anaspec, USA)를 사용하여 50 mM HEPES 완충액(pH 7.5, 50 mM KCl, 2 mM MgCl2, 1 mM EDTA, 10 mM DTT)에서 효소반응을 수행하였다.50 mM HEPES buffer (pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 1 mM EDTA, 10 mM) using Ac-DEVD-pNA (pNA: para-nitroanilide; Anaspec, USA), a well known substrate for caspase 3 Enzyme reaction was carried out in DTT).

효소동력학적 상수(parameters)인 Km과 kcat 값을 구하기 위하여 상기 완충용액으로 희석하여 준비한 다양한 농도의 기질 480 ㎕에 실시예 2 및 3의 방법으로 수득한 재조합 카스파제 전구체 및 실시예 4의 방법으로 수득한 활성형 카스파제 20 ㎕를 가한 후 UV/VIS 스펙트로포토미터(Beckman Coulter, USA)를 사용하여 405 ㎚에서 흡광도 변화를 측정함으로써 기질의 가수분해 반응속도를 측정하였다. 상기 측정값을 바탕으로 HYPER32.EXE, Version 1.0.0 (2003 for 32 bit Versions of MS Windows, Copyright J S Easterby, Freeware; //homepage.ntlworld.com/john.easterby/hyper32.html) 프로그램으로 효소동력학적 상수를 계산하였다.In order to obtain K m and k cat values, which are the enzyme kinetics parameters, the recombinant caspase precursors obtained by the method of Examples 2 and 3 and 480 μl of substrates of various concentrations prepared by diluting with the buffer solution were prepared. After adding 20 μl of the active caspase obtained by the method, the hydrolysis reaction rate of the substrate was measured by measuring the change in absorbance at 405 nm using a UV / VIS Spectrophotometer (Beckman Coulter, USA). Based on the measured values, enzyme power with HYPER32.EXE, Version 1.0.0 (2003 for 32 bit Versions of MS Windows, Copyright JS Easterby, Freeware; //homepage.ntlworld.com/john.easterby/hyper32.html) Calculation constants were calculated.

그 결과, 표 2에서 나타난 바와 같이 상기 재조합 카스파제 3 중, 175번째 아미노산을 포함한 6개의 연속적인 아미노산으로 구성된 펩타이드를 트롬빈에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환된 재조합 카스파제 3(Δ28TS/175TS, 28TS/175TS 또는 L168F/28TS/175TS) 보다 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산이 삽입된 재조합 카스파제 3(28TS/180TI, L168F/28TS/D175A/180TI 또는 L168W/28TS/D175A/180TI)의 kcat/Km(효소반응속도)가 더욱 높아 효소활성이 훨씬 우수한 것을 확인하였다.As a result, as shown in Table 2, the recombinant caspase 3 in which the peptide consisting of six consecutive amino acids including the 175th amino acid of the recombinant caspase 3 was replaced with a peptide consisting of consecutive amino acids recognized and cleaved by thrombin. Recombinant caspase 3 (28TS / 180TI, L168F) inserting six consecutive amino acids recognized and cleaved by thrombin between the 180th and 181th amino acids than (Δ28TS / 175TS, 28TS / 175TS or L168F / 28TS / 175TS) / 28TS / D175A / 180TI or L168W / 28TS / D175A / 180TI) higher k cat / K m (enzyme reaction rate) was confirmed that the enzyme activity is much better.

본 발명의 재조합 카스파제 3의 kcat/Km(효소반응속도)는 야생형 카스파제 3(서열번호 49)에 비해 낮으나, 트롬빈을 처리하기 전에는 활성을 나타내지 않는 비활성 형태로 대장균에서 발현되기 때문에, 카스파제 3의 대량생산이 가능할 수 있다.K cat / K m (enzyme reaction rate) of the recombinant caspase 3 of the present invention is lower than that of wild-type caspase 3 (SEQ ID NO: 49), but is expressed in E. coli in an inactive form that does not exhibit activity before thrombin treatment. Mass production of caspase 3 may be possible.

카스파제 전구체 및 활성형 카스파제의 효소동역학적 상수Enzyme Kinetic Constants of Caspase Precursor and Active Caspase 　 caspase 3caspase 3 K M(μM) K M (μM) k cat(min-1) k cat (min -1 ) k cat/K M(M-1min-1) k cat / K M (M -1 min -1 ) Wild type caspase 3 Wild type caspase 3 14.16±1.48 14.16 ± 1.48 432.40±56.00 432.40 ± 56.00 30.54 x 106 30.54 x 10 6 Δ28 caspase 3 Δ28 caspase 3 16.38±3.59 16.38 ± 3.59 203.38±34.97 203.38 ± 34.97 12.42 x 106 12.42 x 10 6 Δ28/175TS caspase 3Δ28 / 175TS caspase 3 93.20±33.23 93.20 ± 33.23 2.82±1.05 2.82 ± 1.05 0.03 x 106 0.03 x 10 6 Δ28/175TS caspase 3+ThrombinΔ28 / 175TS caspase 3 + Thrombin 41.13±1.90 41.13 ± 1.90 111.44±5.61 111.44 ± 5.61 2.71 x 106 2.71 x 10 6 28TS/175TS caspase 3 28TS / 175TS caspase 3 93.49±9.21 93.49 ± 9.21 1.39±0.12 1.39 ± 0.12 0.01 x 106 0.01 x 10 6 28TS/175TS caspase 3 +Thrombin 28TS / 175TS caspase 3 + Thrombin 27.81±5.73 27.81 ± 5.73 47.05±3.82 47.05 ± 3.82 1.69 x 106 1.69 x 10 6 28TS/180TI caspase 328TS / 180TI caspase 3 18.09±1.29 18.09 ± 1.29 75.44±2.00 75.44 ± 2.00 4.17 x 106 4.17 x 10 6 28TS/180TI caspase 3+Thrombin 28TS / 180TI caspase 3 + Thrombin 21.39±1.87 21.39 ± 1.87 105.99±7.90 105.99 ± 7.90 4.96 x 106 4.96 x 10 6 L168F/28TS/175TS caspase 3 L168F / 28TS / 175TS caspase 3 25.57±0.81 25.57 ± 0.81 1.43±0.02 1.43 ± 0.02 0.06 x 106 0.06 x 10 6 L168F/28TS/175TS caspase 3 + Thrombin L168F / 28TS / 175TS caspase 3 + Thrombin 66.02±1.03 66.02 ± 1.03 244.77±1.26 244.77 ± 1.26 3.71 x 106 3.71 x 10 6 L168F/28TS/D175A/180TI caspase 3 L168F / 28TS / D175A / 180TI caspase 3 9.99±0.61 9.99 ± 0.61 5.17±0.05 5.17 ± 0.05 0.52 x 106 0.52 x 10 6 L168F/28TS/D175A/180TI caspase 3 + Thrombin L168F / 28TS / D175A / 180TI caspase 3 + Thrombin 16.83±0.02 16.83 ± 0.02 170.16±10.47 170.16 ± 10.47 10.11 x 106 10.11 x 10 6 L168W/28TS/D175A/180TI caspase 3L168W / 28TS / D175A / 180TI caspase 3 5.73±1.16 5.73 ± 1.16 8.22±0.51 8.22 ± 0.51 1.43 x 106 1.43 x 10 6 L168W/28TS/D175A/180TI caspase 3 + ThrombinL168W / 28TS / D175A / 180TI caspase 3 + Thrombin 12.25±0.28 12.25 ± 0.28 293.29±30.70 293.29 ± 30.70 23.94 x 106 23.94 x 10 6

도 1은 본 발명의 재조합 카스파제 전구체가 트롬빈에 의해 활성형 카스파제 재조합 단백질로 되는 과정을 나타낸 도이다.1 is a diagram showing a process in which the recombinant caspase precursor of the present invention is activated caspase recombinant protein by thrombin.

도 2는 세포내에서 카스파제가 활성화되는 과정을 나타낸 도이다.2 is a diagram illustrating a process of activating caspase in cells.

도 3은 본 발명의 재조합 카스파제 전구체의 과발현 및 정제 결과를 나타낸 도이다:3 is a diagram showing the results of overexpression and purification of the recombinant caspase precursor of the present invention:

M: Size Marker;M: Size Marker;

1: caspase 3(1-277); 1: caspase 3 (1-277);

2: Δ28 caspase 3;2: Δ28 caspase 3;

3: Δ28/175TS caspase 3;3: Δ28 / 175TS caspase 3;

4: 28TS/175TS caspase 3;4: 28TS / 175TS caspase 3;

5: 28TS/D175A/180TI caspase 3; 및,5: 28TS / D175A / 180TI caspase 3; And,

6: C163S caspase 3(음성대조군).6: C163S caspase 3 (negative control).

도 4는 본 발명의 재조합 카스파제 전구체 정제 후 트롬빈 처리에 의해 카스파제 전구체에서 활성형 카스파제로 바뀌는 것을 시간별로 SDS-PAGE를 이용하여 관찰한 결과를 나타낸 도이다. 레인 1 내지 4는 4℃에서 반응한 것이고, 레인 5 내지 8은 18℃에서 반응한 것이다(M은 단백질 마커이며, 66 kDa에서 나오는 밴드는 트롬빈에서 유래한 것으로 추측된다.). 레인 1 및 5는 트롬빈과 반응하지 않은 카스파제 전구체이며, 레인 2 및 6은 카스파제 단백질 200 ㎍과 트롬빈 2 NIH 유닛이 반응한 것이며, 레인 3 및 7은 카스파제 단백질 200 ㎍과 트롬빈 20 NIH 유닛이 반응한 것이며, 레인 4 및 8은 카스파제 단백질 200 ㎍과 트롬빈 40 NIH 유닛이 반응한 것이다:Figure 4 is a diagram showing the results observed by using SDS-PAGE change the caspase precursor to the active caspase by thrombin treatment after purification of the recombinant caspase precursor of the present invention. Lanes 1 to 4 were reacted at 4 ° C., lanes 5 to 8 were reacted at 18 ° C. (M is a protein marker, and the band emerging at 66 kDa is presumably derived from thrombin). Lanes 1 and 5 are caspase precursors that did not react with thrombin, lanes 2 and 6 were reacted with 200 μg of caspase protein and thrombin 2 NIH units, lanes 3 and 7 with 200 μg caspase protein and thrombin 20 NIH units Lanes 4 and 8 were reacted with 200 μg of caspase protein and 40 thrombin units of thrombin:

a: 반응 4시간 후;a: after 4 hours of reaction;

b: 반응 8시간 후; 및,b: after 8 h of reaction; And,

c: 반응 24시간 후.c: after 24 hours of reaction.

도 5는 카스파제 7의 전구체를 대장균에서 발현 정제한 결과 및 이를 활성화한 결과를 나타낸 것이다:FIG. 5 shows the results of activating and expressing the caspase 7 precursor in E. coli:

M: Size Marker; M: Size Marker;

1: 198TS Cas7; 1: 198 TS Cas7;

2: 23TS/198TS Cas7; 2: 23TS / 198TS Cas7;

3: 198TS Cas7을 4℃에서 트롬빈 처리를 하여 활성화한 것; 3: activated by thrombin treatment of 198TS Cas7 at 4 ° C;

4: 198TS Cas7을 18℃에서 트롬빈 처리를 하여 활성화한 것; 4: 198TS Cas7 activated by thrombin treatment at 18 ° C .;

5: 23TS/198TS Cas7을 4℃에서 트롬빈 처리를 하여 활성화한 것; 및, 5: activated by thrombin treatment of 23TS / 198TS Cas7 at 4 ° C; And,

6: 23TS/198TS Cas7을 18℃에서 트롬빈 처리를 하여 활성화한 것. 6: Activated 23TS / 198TS Cas7 by thrombin treatment at 18 ° C.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for large scale preparation of procaspases, which can be artificially activated, in E. coli expression system and their activation <130> 8P-06-27 <160> 98 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for full length caspase-3 <400> 1 gggaattcca tatggagaac actgaaaact cag 33 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 for caspase-3 <400> 2 ccgctcgagt tagtgataaa aatagagttc 30 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 for caspase-3 (for pET21a vector) <400> 3 ccgctcgagg tgataaaaat agagttcttt tgt 33 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer for delta-28 caspase-3 <400> 4 gggaattcca tatgtctgga atatccctgg acaacagt 38 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer to prepare C-terminal megaprimer for 28TS caspase-3 <400> 5 actgaactgg tgccgcgcgg cagctccatt aaaaatttgg aaccaaag 48 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> reverse primer to prepare N-terminal megaprimer for 28TS caspase-3 <400> 6 aatggagctg ccgcgcggca ccagttcagt gttttcagtg ttctc 45 <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer to prepare C-terminal megaprimer for 175TS caspase-3 <400> 7 tgtggcctgg tgccgcgcgg cagcgttgat gatgacatgg cgtgtcat 48 <210> 8 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> reverse primer to prepare N-terminal megaprimer for 175TS caspase-3 <400> 8 atcaacgctg ccgcgcggca ccaggccaca gtccagttct gtaccacg 48 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For L168F mutation on 28TS/175TS caspase-3 <400> 9 gcctgccgtg gtacagaatt cgactgtggc attgag 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for L168F mutation on 28TS/175TS caspase-3 <400> 10 tgtctcaatg ccacagtcga attctgtacc acggca 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer for L168W mutation on 28TS/175TS caspase-3 <400> 11 tgccgtggta cagaatggga ctgtggcctg gtgccg 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for L168W mutation on 28TS/175TS caspase-3 <400> 12 caccaggcca cagtcccatt ctgtaccacg gcaggc 36 <210> 13 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 180TI caspase-3 <400> 13 tattttatga cacgccatgt cgctgccgcg cggcaccaga tcatcaacac cact 54 <210> 14 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 180TI caspase-3 <400> 14 acagacagtg gtgttgatga tctggtgccg cgcggcagcg acatggcgtg tcataaa 57 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for D175A mutation on 180TI caspase-3 <400> 15 gactgtggca ttgagacagc gagtggtgtt gatgat 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for D175A mutation on 180TI caspase-3 <400> 16 cagatcatca acaccactcg ctgtctcaat gccaca 36 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L168F mutation on 180TI caspase-3 <400> 17 tgccgtggta cagaattcga ctgtggcatt gag 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L168F mutation on 180TI caspase-3 <400> 18 ctcaatgcca cagtcgaatt ctgtaccacg gca 33 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L168W mutation on 180TI caspase-3 <400> 19 tgccgtggta cagaatggga ctgtggcatt gag 33 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L168W mutation on 180TI caspase-3 <400> 20 ctcaatgcca cagtcccatt ctgtaccacg gca 33 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer for wild type caspase-7 <400> 21 gggaattcca tatggcagat gagcagggct gtattgaag 39 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 for wild type caspase-7 <400> 22 ccgctcgagt tattgactga agtagagttc cttggt 36 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 for wild type caspase-7 <400> 23 ccgctcgagt tgactgaagt agagttcctt ggt 33 <210> 24 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 23TS caspase-7 <400> 24 tcagcaaatg aactggtgcc gcgcggcagc ccagaccggt cctcg 45 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 23TS caspase-7 <400> 25 ggaccggtct gggctgccgc gcggcaccag ttcatttgct ga 42 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 198TS caspase-7 <400> 26 cttgatgatg gcctggtgcc gcgcggcagc cccatcaatg acaca 45 <210> 27 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 198TS caspase-7 <400> 27 gtcattgatg gggctgccgc gcggcaccag gccatcatca ag 42 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For L191F mutation on 23TS/198TS caspase-7 <400> 28 tgccgaggga ccgagtttga tgatggcctg gtgccg 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191F mutation on 23TS/198TS caspase-7 <400> 29 caccaggcca tcatcaaact cggtccctcg gcaagc 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191W mutation on 23TS/198TS caspase-7 <400> 30 tgccgaggga ccgagtggga tgatggcctg gtgccg 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191W mutation on 23TS/198TS caspase-7 <400> 31 caccaggcca tcatcccact cggtccctcg gcaagc 36 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L198A mutation on 23TS/203TI caspase-7 <400> 32 gatggcatcc aggccgagtc ggggcccatc aatgac 36 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L198A mutation on 23TS/203TI caspase-7 <400> 33 gtcattgatg ggccccgact cggcctggat gccatc 36 <210> 34 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 203TI caspase-7 <400> 34 gggcccatca atgacctggt gccgcgcggc agcacagatg ctaatcctcg a 51 <210> 35 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 203TI caspase-7 <400> 35 aggattagca tctgtgctgc cgcgcggcac caggtcattg atgggccccg a 51 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191F mutation on 23TS/D198A/203TI caspase-7 <400> 36 tgccgaggga ccgagtttga tgatggcatc caggcc 36 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191F mutation on 23TS/D198A/203TI caspase-7 <400> 37 ctggatgcca tcatcaaact cggtccctcg gcaagc 36 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191W mutation on 23TS/D198A/203TI caspase-7 <400> 38 tgccgaggga ccgagtggga tgatggcatc caggcc 36 <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191W mutation on 23TS/D198A/203TI caspase-7 <400> 39 ctggatgcca tcatcccact cggtccctcg gcaagc 36 <210> 40 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-2 <400> 40 gggaattcca tatggcggcg ccgagcgcgg ggtctt 36 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-2 <400> 41 ccgctcgagt tatgtgggag ggtgtcctgg gaa 33 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-2 <400> 42 ccgctcgagt gtgggagggt gtcctgggaa 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-6 <400> 43 ctagctagca gctcggcctc ggggctccgc 30 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-6 <400> 44 cgcgtcgact taattagatt ttggaaagaa atg 33 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-6 <400> 45 cgcgtcgaca ttagattttg gaaagaaatg 30 <210> 46 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-9 <400> 46 ctagctagcg acgaagcgga tcggcggctc ctg 33 <210> 47 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-9 <400> 47 cgcgtcgact tatgatgttt taaagaaaag ttt 33 <210> 48 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-9 <400> 48 cgcgtcgact gatgttttaa agaaaagttt 30 <210> 49 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type Caspase-3 <400> 49 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 50 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> dalta28 Caspase-3 <400> 50 Ser Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met 1 5 10 15 Gly Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly 20 25 30 Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu 35 40 45 Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr 50 55 60 Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His 65 70 75 80 Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu 85 90 95 Gly Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr 100 105 110 Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys 115 120 125 Leu Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile 130 135 140 Glu Thr Asp Ser Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro 145 150 155 160 Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr 165 170 175 Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys 180 185 190 Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu 195 200 205 Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe 210 215 220 Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met 225 230 235 240 Leu Thr Lys Glu Leu Tyr Phe Tyr His 245 <210> 51 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> 28TS Caspase-3 <400> 51 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 52 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> 175TS Caspase-3 <400> 52 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ser Gly 20 25 30 Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys 35 40 45 Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser 50 55 60 Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg 65 70 75 80 Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu 85 90 95 Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg 100 105 110 Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile 115 120 125 Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe 130 135 140 Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile 145 150 155 160 Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val Pro Arg 165 170 175 Gly Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala 180 185 190 Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg 195 200 205 Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu 210 215 220 Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val 225 230 235 240 Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr 245 250 255 Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys 260 265 270 Glu Leu Tyr Phe Tyr His 275 <210> 53 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> 28TS/175TS Caspase-3 <400> 53 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val Pro Arg Gly 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 54 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> 28TS/180TI Caspase-3 <400> 54 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys 180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His 275 280 <210> 55 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> delta28/175TS Caspase-3 <400> 55 Ser Gly Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly 1 5 10 15 Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met 20 25 30 Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr 35 40 45 Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg 50 55 60 Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser 65 70 75 80 Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly 85 90 95 Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn 100 105 110 Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu 115 120 125 Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val 130 135 140 Pro Arg Gly Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val 145 150 155 160 Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser 165 170 175 Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala 180 185 190 Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr 195 200 205 Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp 210 215 220 Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu 225 230 235 240 Thr Lys Glu Leu Tyr Phe Tyr His 245 <210> 56 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> L168F/28TS/175TS Caspase-3 <400> 56 Met Glu Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Glu Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Phe Asp Cys Gly Leu Val Pro Arg Gly 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 57 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> L168W/28TS/175TS Caspase-3 <400> 57 Met Glu Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Glu Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Trp Asp Cys Gly Leu Val Pro Arg Gly 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 58 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> 28TS/D175A/180TI Caspase-3 <400> 58 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Ala Ser 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys 180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His 275 280 <210> 59 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> L168F/28TS/ D175A/180TI caspase 3 <400> 59 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Ala Ser 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys 180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His 275 280 <210> 60 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> L168W/28TS/ D175A/180TI caspase 3 <400> 60 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Trp Asp Cys Gly Ile Glu Thr Ala Ser 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys 180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly 195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser 210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val 260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His 275 280 <210> 61 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> C163S caspase-3 <400> 61 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Ser Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 62 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase 7 <400> 62 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 63 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 23TS Caspase-7 <400> 63 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 64 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 23TS/198TS Caspase-7 <400> 64 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 65 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> L191F/23TS/198TS Caspase-7 <400> 65 Asn Met Asn Phe Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys 1 5 10 15 Asn Phe Asp Lys Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys 20 25 30 Asp Ala Glu Ala Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val 35 40 45 Ile Val Tyr Asn Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys 50 55 60 Lys Ala Ser Glu Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile 65 70 75 80 Leu Leu Ser His Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val 85 90 95 Thr Pro Ile Lys Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys 100 105 110 Thr Leu Leu Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly 115 120 125 Thr Glu Phe Asp Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp 130 135 140 Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu 145 150 155 160 Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly 165 170 175 Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His 180 185 190 Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg 195 200 205 Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu 210 215 220 Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr 225 230 235 240 Phe Ser Gln <210> 66 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> L191W/23TS/198TS Caspase-7 <400> 66 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Trp Asp 180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 67 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> 23TS/D198A/203TI Caspase-7 <400> 67 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly 195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe 210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp 260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His 275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu 290 295 300 Tyr Phe Ser Gln 305 <210> 68 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> L191F/23TS/D198A/203TI Caspase-7 <400> 68 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Phe Asp 180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly 195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe 210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp 260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His 275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu 290 295 300 Tyr Phe Ser Gln 305 <210> 69 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> L191W/23TS/D198A/203TI Caspase-7 <400> 69 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Trp Asp 180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly 195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe 210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp 260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His 275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu 290 295 300 Tyr Phe Ser Gln 305 <210> 70 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-2 <400> 70 Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu 1 5 10 15 Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met 20 25 30 His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala 35 40 45 Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp 50 55 60 Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser 65 70 75 80 Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro 85 90 95 Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly 100 105 110 His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val 115 120 125 Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val 130 135 140 Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr 145 150 155 160 Val Glu His Ser Leu Asp Asn Lys Asp Gly Pro Leu Cys Leu Gln Val 165 170 175 Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr 180 185 190 Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val 195 200 205 His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val 210 215 220 Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val 225 230 235 240 His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln 245 250 255 Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val 260 265 270 Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly 275 280 285 Lys Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn 290 295 300 Cys Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys 305 310 315 320 Arg Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn 325 330 335 His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys 340 345 350 Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr 355 360 365 Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser 370 375 380 Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp 385 390 395 400 Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp 405 410 415 Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met 420 425 430 Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly 435 440 445 His Pro Pro Thr 450 <210> 71 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-6 <400> 71 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu 1 5 10 15 Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg Glu Met Phe Asp 20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu 35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg 50 55 60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp 65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu 85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp 100 105 110 Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr 115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys 130 135 140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp 165 170 175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val 180 185 190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met 195 200 205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn 210 215 220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val 245 250 255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys 260 265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe 275 280 285 Phe Pro Lys Ser Asn 290 <210> 72 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-9 <400> 72 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu 1 5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg 20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser 35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr 50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser 130 135 140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg 180 185 190 Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr 195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His 210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly 275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu 290 295 300 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405 410 415 <210> 73 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Caspase-2 cleavage site <400> 73 Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu 1 5 10 15 Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met 20 25 30 His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala 35 40 45 Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp 50 55 60 Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser 65 70 75 80 Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro 85 90 95 Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly 100 105 110 His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val 115 120 125 Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val 130 135 140 Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr 145 150 155 160 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Cys Leu Gln Val 165 170 175 Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr 180 185 190 Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val 195 200 205 His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val 210 215 220 Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val 225 230 235 240 His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln 245 250 255 Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val 260 265 270 Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly 275 280 285 Lys Leu Leu Gln Leu Gln Glu Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Asp Gln Gln Asp Gly Lys Asn 325 330 335 His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys 340 345 350 Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr 355 360 365 Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser 370 375 380 Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp 385 390 395 400 Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp 405 410 415 Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met 420 425 430 Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly 435 440 445 His Pro Pro Thr 450 <210> 74 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Caspase-3 cleavage site <400> 74 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu 1 5 10 15 Glu Pro Lys Ile Ile His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile 35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg 50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn 65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile 85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser 100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe 115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg 130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa 165 170 175 Xaa Xaa Xaa Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp 180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn 195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys 210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu 260 265 270 Leu Tyr Phe Tyr His 275 <210> 75 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Caspase-6 cleavage site <400> 75 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu 1 5 10 15 Glu Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Met Phe Asp 20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu 35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg 50 55 60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp 65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu 85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp 100 105 110 Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr 115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys 130 135 140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp 165 170 175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val 180 185 190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met 195 200 205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn 210 215 220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val 245 250 255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys 260 265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe 275 280 285 Phe Pro Lys Ser Asn 290 <210> 76 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Caspase-7 cleavage site <400> 76 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 77 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 cleavage site <400> 77 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu 1 5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg 20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser 35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr 50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr 65 70 75 80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala 85 90 95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg 100 105 110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro 115 120 125 Val Asp Ile Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser 130 135 140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg 180 185 190 Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr 195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His 210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys 260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly 275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro 325 330 335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val 340 345 350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp 355 360 365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu 370 375 380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser 405 410 415 <210> 78 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> caspase-3 25-30th <400> 78 Glu Ser Met Asp Ser Gly 1 5 <210> 79 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> thrombin cleavage site <400> 79 Leu Val Pro Arg Gly Ser 1 5 <210> 80 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> caspase-3 172-177th <400> 80 Ile Glu Thr Asp Ser Gly 1 5 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 19-28th <400> 81 Glu Asp Ser Val Asp Ala Lys Pro Asp Arg 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 19-28th -> thrombin cleavage site <400> 82 Glu Asp Leu Val Pro Arg Gly Ser Asp Arg 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 194-203th <400> 83 Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-2 162-171 <400> 84 Glu His Ser Leu Asp Asn Lys Asp Gly Pro 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-2 297-306 <400> 85 Phe Gln Leu Phe Asp Asn Ala Asn Cys Pro 1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-2 319-328 <400> 86 Ala Cys Arg Gly Asp Glu Thr Asp Arg Gly 1 5 10 <210> 87 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-6 19-28 <400> 87 Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg 1 5 10 <210> 88 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 134-143 <400> 88 Gly Gly Phe Gly Asp Val Gly Ala Leu Glu 1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 301-310 <400> 89 Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser 1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 311-320 <400> 90 Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 1 5 10 <210> 91 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Enterokinase cleavage site <400> 91 Asp Asp Asp Asp Lys 1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> TEV cleavage site <400> 92 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 93 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Factor Xa cleavage site 1 <400> 93 Ile Glu Gly Arg 1 <210> 94 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Factor Xa cleavage site 2 <400> 94 Ile Asp Gly Arg 1 <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-3 23-32th <400> 95 Gly Ser Glu Ser Met Asp Ser Gly Ile Ser 1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-3 170-179th <400> 96 Cys Gly Ile Glu Thr Asp Ser Gly Val Asp 1 5 10 <210> 97 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 198TS Caspase-7 <400> 97 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser 1 5 10 15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val 20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile 35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe 50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys 65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala 85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn 100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu 115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His 130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp 180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn 195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser 210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His 260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile 275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln 290 295 300 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 194-203th -> thrombin cleavage site <400> 98 Gly Leu Val Pro Arg Gly Ser Pro Ile Asn 1 5 10 <110> Korea Research Institute of Bioscience and Biotechnology <120> Method for large scale preparation of procaspases, which can be          artificially activated, in E. coli expression system and their          activation <130> 8P-06-27 <160> 98 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for full length caspase-3 <400> 1 gggaattcca tatggagaac actgaaaact cag 33 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 for caspase-3 <400> 2 ccgctcgagt tagtgataaa aatagagttc 30 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 for caspase-3 (for pET21a vector) <400> 3 ccgctcgagg tgataaaaat agagttcttt tgt 33 <210> 4 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> forward primer for delta-28 caspase-3 <400> 4 gggaattcca tatgtctgga atatccctgg acaacagt 38 <210> 5 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer to prepare C-terminal megaprimer for 28TS          caspase-3 <400> 5 actgaactgg tgccgcgcgg cagctccatt aaaaatttgg aaccaaag 48 <210> 6 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> reverse primer to prepare N-terminal megaprimer for 28TS          caspase-3 <400> 6 aatggagctg ccgcgcggca ccagttcagt gttttcagtg ttctc 45 <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> forward primer to prepare C-terminal megaprimer for 175TS          caspase-3 <400> 7 tgtggcctgg tgccgcgcgg cagcgttgat gatgacatgg cgtgtcat 48 <210> 8 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> reverse primer to prepare N-terminal megaprimer for 175TS          caspase-3 <400> 8 atcaacgctg ccgcgcggca ccaggccaca gtccagttct gtaccacg 48 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For L168F mutation on 28TS / 175TS caspase-3 <400> 9 gcctgccgtg gtacagaatt cgactgtggc attgag 36 <210> 10 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for L168F mutation on 28TS / 175TS caspase-3 <400> 10 tgtctcaatg ccacagtcga attctgtacc acggca 36 <210> 11 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> forward primer for L168W mutation on 28TS / 175TS caspase-3 <400> 11 tgccgtggta cagaatggga ctgtggcctg gtgccg 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for L168W mutation on 28TS / 175TS caspase-3 <400> 12 caccaggcca cagtcccatt ctgtaccacg gcaggc 36 <210> 13 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 180TI          caspase-3 <400> 13 tattttatga cacgccatgt cgctgccgcg cggcaccaga tcatcaacac cact 54 <210> 14 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 180TI          caspase-3 <400> 14 acagacagtg gtgttgatga tctggtgccg cgcggcagcg acatggcgtg tcataaa 57 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for D175A mutation on 180TI caspase-3 <400> 15 gactgtggca ttgagacagc gagtggtgtt gatgat 36 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for D175A mutation on 180TI caspase-3 <400> 16 cagatcatca acaccactcg ctgtctcaat gccaca 36 <210> 17 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L168F mutation on 180TI caspase-3 <400> 17 tgccgtggta cagaattcga ctgtggcatt gag 33 <210> 18 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L168F mutation on 180TI caspase-3 <400> 18 ctcaatgcca cagtcgaatt ctgtaccacg gca 33 <210> 19 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L168W mutation on 180TI caspase-3 <400> 19 tgccgtggta cagaatggga ctgtggcatt gag 33 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L168W mutation on 180TI caspase-3 <400> 20 ctcaatgcca cagtcccatt ctgtaccacg gca 33 <210> 21 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> forward primer for wild type caspase-7 <400> 21 gggaattcca tatggcagat gagcagggct gtattgaag 39 <210> 22 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 for wild type caspase-7 <400> 22 ccgctcgagt tattgactga agtagagttc cttggt 36 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 for wild type caspase-7 <400> 23 ccgctcgagt tgactgaagt agagttcctt ggt 33 <210> 24 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 23TS          caspase-7 <400> 24 tcagcaaatg aactggtgcc gcgcggcagc ccagaccggt cctcg 45 <210> 25 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 23TS          caspase-7 <400> 25 ggaccggtct gggctgccgc gcggcaccag ttcatttgct ga 42 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 198TS          caspase-7 <400> 26 cttgatgatg gcctggtgcc gcgcggcagc cccatcaatg acaca 45 <210> 27 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 198TS          caspase-7 <400> 27 gtcattgatg gggctgccgc gcggcaccag gccatcatca ag 42 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For L191F mutation on 23TS / 198TS caspase-7 <400> 28 tgccgaggga ccgagtttga tgatggcctg gtgccg 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191F mutation on 23TS / 198TS caspase-7 <400> 29 caccaggcca tcatcaaact cggtccctcg gcaagc 36 <210> 30 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191W mutation on 23TS / 198TS caspase-7 <400> 30 tgccgaggga ccgagtggga tgatggcctg gtgccg 36 <210> 31 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191W mutation on 23TS / 198TS caspase-7 <400> 31 caccaggcca tcatcccact cggtccctcg gcaagc 36 <210> 32 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L198A mutation on 23TS / 203TI caspase-7 <400> 32 gatggcatcc aggccgagtc ggggcccatc aatgac 36 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L198A mutation on 23TS / 203TI caspase-7 <400> 33 gtcattgatg ggccccgact cggcctggat gccatc 36 <210> 34 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer to prepare N-terminal megaprimer for 203 TI          caspase-7 <400> 34 gggcccatca atgacctggt gccgcgcggc agcacagatg ctaatcctcg a 51 <210> 35 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Forward primer to prepare C-terminal megaprimer for 203 TI          caspase-7 <400> 35 aggattagca tctgtgctgc cgcgcggcac caggtcattg atgggccccg a 51 <210> 36 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191F mutation on 23TS / D198A / 203TI caspase-7 <400> 36 tgccgaggga ccgagtttga tgatggcatc caggcc 36 <210> 37 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191F mutation on 23TS / D198A / 203TI caspase-7 <400> 37 ctggatgcca tcatcaaact cggtccctcg gcaagc 36 <210> 38 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for L191W mutation on 23TS / D198A / 203TI caspase-7 <400> 38 tgccgaggga ccgagtggga tgatggcatc caggcc 36 <210> 39 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for L191W mutation on 23TS / D198A / 203TI caspase-7 <400> 39 ctggatgcca tcatcccact cggtccctcg gcaagc 36 <210> 40 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-2 <400> 40 gggaattcca tatggcggcg ccgagcgcgg ggtctt 36 <210> 41 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-2 <400> 41 ccgctcgagt tatgtgggag ggtgtcctgg gaa 33 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-2 <400> 42 ccgctcgagt gtgggagggt gtcctgggaa 30 <210> 43 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-6 <400> 43 ctagctagca gctcggcctc ggggctccgc 30 <210> 44 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-6 <400> 44 cgcgtcgact taattagatt ttggaaagaa atg 33 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-6 <400> 45 cgcgtcgaca ttagattttg gaaagaaatg 30 <210> 46 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer For wild type caspase-9 <400> 46 ctagctagcg acgaagcgga tcggcggctc ctg 33 <210> 47 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 1 For wild type caspase-9 <400> 47 cgcgtcgact tatgatgttt taaagaaaag ttt 33 <210> 48 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer 2 For wild type caspase-9 <400> 48 cgcgtcgact gatgttttaa agaaaagttt 30 <210> 49 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type Caspase-3 <400> 49 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser                 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 50 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> dalta28 Caspase-3 <400> 50 Ser Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met   1 5 10 15 Gly Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly              20 25 30 Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu          35 40 45 Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr      50 55 60 Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His  65 70 75 80 Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu                  85 90 95 Gly Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr             100 105 110 Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys         115 120 125 Leu Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile     130 135 140 Glu Thr Asp Ser Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro 145 150 155 160 Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr                 165 170 175 Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys             180 185 190 Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu         195 200 205 Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe     210 215 220 Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met 225 230 235 240 Leu Thr Lys Glu Leu Tyr Phe Tyr His                 245 <210> 51 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> 28TS Caspase-3 <400> 51 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser                 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 52 <211> 278 <212> PRT <213> Artificial Sequence <220> <223> 175TS Caspase-3 <400> 52 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ser Gly              20 25 30 Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys          35 40 45 Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser      50 55 60 Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg  65 70 75 80 Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu                  85 90 95 Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg             100 105 110 Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile         115 120 125 Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe     130 135 140 Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile 145 150 155 160 Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val Pro Arg                 165 170 175 Gly Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala             180 185 190 Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg         195 200 205 Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu     210 215 220 Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val 225 230 235 240 Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr                 245 250 255 Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys             260 265 270 Glu Leu Tyr Phe Tyr His         275 <210> 53 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> 28TS / 175TS Caspase-3 <400> 53 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val Pro Arg Gly                 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 54 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> 28TS / 180TI Caspase-3 <400> 54 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser                 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys             180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly         195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser     210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe                 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val             260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His         275 280 <210> 55 <211> 248 <212> PRT <213> Artificial Sequence <220> <223> delta28 / 175TS Caspase-3 <400> 55 Ser Gly Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly   1 5 10 15 Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met              20 25 30 Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr          35 40 45 Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg      50 55 60 Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser  65 70 75 80 Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly                  85 90 95 Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn             100 105 110 Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu         115 120 125 Phe Ile Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Leu Val     130 135 140 Pro Arg Gly Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val 145 150 155 160 Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser                 165 170 175 Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala             180 185 190 Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr         195 200 205 Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp     210 215 220 Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu 225 230 235 240 Thr Lys Glu Leu Tyr Phe Tyr His                 245 <210> 56 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> L168F / 28TS / 175TS Caspase-3 <400> 56 Met Glu Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Glu Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Phe Asp Cys Gly Leu Val Pro Arg Gly                 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 57 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> L168W / 28TS / 175TS Caspase-3 <400> 57 Met Glu Asn Thr Glu Asn Ser Val Ala Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Glu Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Trp Asp Cys Gly Leu Val Pro Arg Gly                 165 170 175 Ser Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 58 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> 28TS / D175A / 180TI Caspase-3 <400> 58 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Ala Ser                 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys             180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly         195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser     210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe                 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val             260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His         275 280 <210> 59 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> L168F / 28TS / D175A / 180TI caspase 3 <400> 59 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Ala Ser                 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys             180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly         195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser     210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe                 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val             260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His         275 280 <210> 60 <211> 283 <212> PRT <213> Artificial Sequence <220> <223> L168W / 28TS / D175A / 180TI caspase 3 <400> 60 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Leu Val Pro Arg Gly Ser Gly Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Trp Asp Cys Gly Ile Glu Thr Ala Ser                 165 170 175 Gly Val Asp Asp Leu Val Pro Arg Gly Ser Asp Met Ala Cys His Lys             180 185 190 Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly         195 200 205 Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser     210 215 220 Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His 225 230 235 240 Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe                 245 250 255 Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val             260 265 270 Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His         275 280 <210> 61 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> C163S caspase-3 <400> 61 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Ser Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser                 165 170 175 Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 62 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase 7 <400> 62 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 63 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 23TS Caspase-7 <400> 63 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 64 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 23TS / 198TS Caspase-7 <400> 64 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 65 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> L191F / 23TS / 198TS Caspase-7 <400> 65 Asn Met Asn Phe Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys   1 5 10 15 Asn Phe Asp Lys Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys              20 25 30 Asp Ala Glu Ala Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val          35 40 45 Ile Val Tyr Asn Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys      50 55 60 Lys Ala Ser Glu Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile  65 70 75 80 Leu Leu Ser His Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val                  85 90 95 Thr Pro Ile Lys Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys             100 105 110 Thr Leu Leu Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly         115 120 125 Thr Glu Phe Asp Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp     130 135 140 Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu 145 150 155 160 Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly                 165 170 175 Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His             180 185 190 Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg         195 200 205 Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu     210 215 220 Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr 225 230 235 240 Phe Ser Gln             <210> 66 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> L191W / 23TS / 198TS Caspase-7 <400> 66 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Trp Asp             180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 67 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> 23TS / D198A / 203TI Caspase-7 <400> 67 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly         195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe     210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu                 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp             260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His         275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu     290 295 300 Tyr Phe Ser Gln 305 <210> 68 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> L191F / 23TS / D198A / 203TI Caspase-7 <400> 68 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Phe Asp             180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly         195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe     210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu                 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp             260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His         275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu     290 295 300 Tyr Phe Ser Gln 305 <210> 69 <211> 308 <212> PRT <213> Artificial Sequence <220> <223> L191W / 23TS / D198A / 203TI Caspase-7 <400> 69 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Leu Val Pro Arg Gly Ser Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Trp Asp             180 185 190 Asp Gly Ile Gln Ala Ala Ser Pro Ile Asn Asp Leu Val Pro Arg Gly         195 200 205 Ser Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe     210 215 220 Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro 225 230 235 240 Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu                 245 250 255 His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp             260 265 270 Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His         275 280 285 Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu     290 295 300 Tyr Phe Ser Gln 305 <210> 70 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-2 <400> 70 Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu   1 5 10 15 Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met              20 25 30 His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala          35 40 45 Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp      50 55 60 Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser  65 70 75 80 Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro                  85 90 95 Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly             100 105 110 His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val         115 120 125 Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val     130 135 140 Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr 145 150 155 160 Val Glu His Ser Leu Asp Asn Lys Asp Gly Pro Leu Cys Leu Gln Val                 165 170 175 Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr             180 185 190 Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val         195 200 205 His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val     210 215 220 Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val 225 230 235 240 His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln                 245 250 255 Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val             260 265 270 Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly         275 280 285 Lys Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn     290 295 300 Cys Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys 305 310 315 320 Arg Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn                 325 330 335 His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys             340 345 350 Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr         355 360 365 Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser     370 375 380 Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp 385 390 395 400 Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp                 405 410 415 Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met             420 425 430 Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly         435 440 445 His Pro Pro Thr     450 <210> 71 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-6 <400> 71 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu   1 5 10 15 Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg Glu Met Phe Asp              20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu          35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg      50 55 60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp  65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu                  85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp             100 105 110 Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr         115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys     130 135 140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp                 165 170 175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val             180 185 190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met         195 200 205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn     210 215 220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val                 245 250 255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys             260 265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe         275 280 285 Phe Pro Lys Ser Asn     290 <210> 72 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> Homo sapiens wild type caspase-9 <400> 72 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu   1 5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg              20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser          35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr      50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr  65 70 75 80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala                  85 90 95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg             100 105 110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro         115 120 125 Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser     130 135 140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly                 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg             180 185 190 Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr         195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His     210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys                 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys             260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly         275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu     290 295 300 Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro                 325 330 335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val             340 345 350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp         355 360 365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu     370 375 380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser                 405 410 415 <210> 73 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> Caspase-2 cleavage site <400> 73 Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu   1 5 10 15 Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met              20 25 30 His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala          35 40 45 Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp      50 55 60 Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser  65 70 75 80 Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro                  85 90 95 Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly             100 105 110 His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val         115 120 125 Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val     130 135 140 Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr 145 150 155 160 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Cys Leu Gln Val                 165 170 175 Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr             180 185 190 Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val         195 200 205 His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val     210 215 220 Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val 225 230 235 240 His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln                 245 250 255 Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val             260 265 270 Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly         275 280 285 Lys Leu Leu Gln Leu Gln Glu Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa     290 295 300 Xaa Xaa Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Xaa Xaa 305 310 315 320 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Val Asp Gln Gln Asp Gly Lys Asn                 325 330 335 His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys             340 345 350 Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr         355 360 365 Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser     370 375 380 Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp 385 390 395 400 Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp                 405 410 415 Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met             420 425 430 Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly         435 440 445 His Pro Pro Thr     450 <210> 74 <211> 277 <212> PRT <213> Artificial Sequence <220> <223> Caspase-3 cleavage site <400> 74 Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu   1 5 10 15 Glu Pro Lys Ile Ile His Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa              20 25 30 Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile          35 40 45 Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg      50 55 60 Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn  65 70 75 80 Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile                  85 90 95 Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser             100 105 110 Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe         115 120 125 Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg     130 135 140 Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Thr Glu Leu Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa                 165 170 175 Xaa Xaa Xaa Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp             180 185 190 Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn         195 200 205 Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys     210 215 220 Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn 225 230 235 240 Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe                 245 250 255 His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu             260 265 270 Leu Tyr Phe Tyr His         275 <210> 75 <211> 293 <212> PRT <213> Artificial Sequence <220> <223> Caspase-6 cleavage site <400> 75 Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu   1 5 10 15 Glu Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Met Phe Asp              20 25 30 Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu          35 40 45 Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg      50 55 60 Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp  65 70 75 80 Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu                  85 90 95 Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp             100 105 110 Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr         115 120 125 Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys     130 135 140 Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile 145 150 155 160 Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp                 165 170 175 Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val             180 185 190 Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met         195 200 205 Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn     210 215 220 Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly 225 230 235 240 Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val                 245 250 255 Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys             260 265 270 Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe         275 280 285 Phe Pro Lys Ser Asn     290 <210> 76 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> Caspase-7 cleavage site <400> 76 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 77 <211> 416 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 cleavage site <400> 77 Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu   1 5 10 15 Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg              20 25 30 Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser          35 40 45 Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr      50 55 60 Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr  65 70 75 80 Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala                  85 90 95 Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg             100 105 110 Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro         115 120 125 Val Asp Ile Gly Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser     130 135 140 Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys 145 150 155 160 Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly                 165 170 175 Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg             180 185 190 Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr         195 200 205 Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His     210 215 220 Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln 225 230 235 240 Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys                 245 250 255 Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys             260 265 270 Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly         275 280 285 Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Xaa Xaa Xaa Xaa     290 295 300 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro                 325 330 335 Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val             340 345 350 Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp         355 360 365 Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu     370 375 380 Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met 385 390 395 400 Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser                 405 410 415 <210> 78 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> caspase-3 25-30th <400> 78 Glu Ser Met Asp Ser Gly   1 5 <210> 79 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> thrombin cleavage site <400> 79 Leu Val Pro Arg Gly Ser   1 5 <210> 80 <211> 6 <212> PRT <213> Artificial Sequence <220> Caspase-3 172-177th <400> 80 Ile Glu Thr Asp Ser Gly   1 5 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-7 19-28th <400> 81 Glu Asp Ser Val Asp Ala Lys Pro Asp Arg   1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 19-28th-> thrombin cleavage site <400> 82 Glu Asp Leu Val Pro Arg Gly Ser Asp Arg   1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-7 194-203th <400> 83 Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn   1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-2 162-171 <400> 84 Glu His Ser Leu Asp Asn Lys Asp Gly Pro   1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-2 297-306 <400> 85 Phe Gln Leu Phe Asp Asn Ala Asn Cys Pro   1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-2 319-328 <400> 86 Ala Cys Arg Gly Asp Glu Thr Asp Arg Gly   1 5 10 <210> 87 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-6 19-28 <400> 87 Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg   1 5 10 <210> 88 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 134-143 <400> 88 Gly Gly Phe Gly Asp Val Gly Ala Leu Glu   1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 301-310 <400> 89 Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser   1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-9 311-320 <400> 90 Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln   1 5 10 <210> 91 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Enterokinase cleavage site <400> 91 Asp Asp Asp Asp Lys   1 5 <210> 92 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> TEV cleavage site <400> 92 Glu Asn Leu Tyr Phe Gln Gly   1 5 <210> 93 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Factor Xa cleavage site 1 <400> 93 Ile Glu Gly Arg   One <210> 94 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Factor Xa cleavage site 2 <400> 94 Ile Asp Gly Arg   One <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-3 23-32th <400> 95 Gly Ser Glu Ser Met Asp Ser Gly Ile Ser   1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> Caspase-3 170-179th <400> 96 Cys Gly Ile Glu Thr Asp Ser Gly Val Asp   1 5 10 <210> 97 <211> 303 <212> PRT <213> Artificial Sequence <220> <223> 198TS Caspase-7 <400> 97 Met Ala Asp Glu Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser   1 5 10 15 Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val              20 25 30 Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile          35 40 45 Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe      50 55 60 Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys  65 70 75 80 Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala                  85 90 95 Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn             100 105 110 Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu         115 120 125 Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His     130 135 140 Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys 145 150 155 160 Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu                 165 170 175 Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp             180 185 190 Asp Gly Leu Val Pro Arg Gly Ser Pro Ile Asn Asp Thr Asp Ala Asn         195 200 205 Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser     210 215 220 Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp 225 230 235 240 Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu                 245 250 255 Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His             260 265 270 Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile         275 280 285 Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln     290 295 300 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> caspase-7 194-203th-> thrombin cleavage site <400> 98 Gly Leu Val Pro Arg Gly Ser Pro Ile Asn   1 5 10  

Claims (34)

카스파제(Caspase) 내에 존재하는 시스테인계 단백질 가수분해효소의 인식부위가 비시스테인계 단백질 가수분해효소(Non-Cysteine proteases)의 인식부위로 치환된 재조합 카스파제 전구체.Recombinant caspase precursor in which the recognition site of cysteine-based protease present in caspase is replaced with the recognition site of non-cysteine proteases. 제 1항에 있어서, 카스파제는 카스파제 2, 카스파제 3, 카스파제 6, 카스파제 7 및 카스파제 9로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.2. The recombinant caspase precursor according to claim 1, wherein the caspase is any one selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. 제 1항에 있어서, 비시스테인계 단백질 가수분해효소는 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드인 것을 특징으로 하는 재조합 카스파제 전구체.2. The recombinant caspase precursor according to claim 1, wherein the bicysteine protease is a peptide consisting of 4 to 10 consecutive amino acids. 제 1항에 있어서, 비시스테인계 단백질 가수분해효소 인식부위는 트롬빈 인식부위(Thrombin; 서열번호 79: LVPRGS), 엔테로키나제 인식부위(Enterokinase; 서열번호 91: DDDDK), TEV 인식부위(서열번호 92: ENLYFQG) 및 Factor Xa 인식부위 (서열번호 93: IEGR 또는 서열번호 94: IDGR)로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.The bicysteine protease recognition site of claim 1 is a thrombin recognition site (Thrombin; SEQ ID NO: 79: LVPRGS), enterokinase recognition site (Enterokinase; SEQ ID NO: 91: DDDDK), TEV recognition site (SEQ ID NO: 92 : ENLYFQG) and a Factor Xa recognition site (SEQ ID NO: 93: IEGR or SEQ ID NO: 94: IDGR) any one selected from the group consisting of a recombinant caspase precursor. 제 1항에 있어서, 비시스테인계 단백질 가수분해효소는 트롬빈, 엔테로키나제, TEV 및 Factor Xa로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.The recombinant caspase precursor according to claim 1, wherein the bicysteine-based proteolytic enzyme is any one selected from the group consisting of thrombin, enterokinase, TEV, and Factor Xa. 제 1항에 있어서, 시스테인계 단백질 가수분해효소의 인식부위는 아스파틱산(aspartic acid)을 중심으로 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드인 것을 특징으로 하는 재조합 카스파제 전구체.[Claim 2] The recombinant caspase precursor according to claim 1, wherein the recognition site of the cysteine protease is a peptide composed of 4 to 10 consecutive amino acids centered on aspartic acid. 제 2항에 있어서, 카스파제 3은 23번째에서 32번째 아미노산(서열번호 95) 또는 170번째에서 179번째 아미노산까지(서열번호 96)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The method of claim 2, wherein caspase 3 is a continuous sequence in which the 23rd to 32nd amino acids (SEQ ID NO: 95) or the 170th to 179th amino acids (SEQ ID NO: 96) are recognized and cleaved by a bicysteine proteinase. Recombinant caspase precursor, characterized in that substituted with a peptide consisting of amino acids. 제 2항에 있어서, 카스파제 7은 19번째에서 28번째 아미노산(서열번호 81) 및 194번째에서 203번째 아미노산까지(서열번호 83)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The caspase 7 is a continuous protein wherein the 19th to 28th amino acid (SEQ ID NO: 81) and the 194th to 203rd amino acid (SEQ ID NO: 83) are recognized and cleaved by a bicysteine-based protease. Recombinant caspase precursor, characterized in that substituted with a peptide consisting of amino acids. 제 2항에 있어서, 카스파제 2는 162번째에서 171번째 아미노산(서열번호 84), 297번째에서 306번째 아미노산(서열번호 85) 또는 319번째에서 328번째 아미노산까지(서열번호 86)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The method of claim 2, wherein caspase 2 is the bicysteine group of amino acids 162 to 171 (SEQ ID NO: 84), 297 to 306 amino acids (SEQ ID NO: 85), or 319 to 328 amino acids (SEQ ID NO: 86). Recombinant caspase precursor, characterized in that substituted by a peptide consisting of a continuous amino acid that is recognized and cleaved by a proteolytic enzyme. 제 2항에 있어서, 카스파제 6은 19번째에서 28번째 아미노산까지(서열번호 87)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.3. The recombination according to claim 2, wherein caspase 6 is replaced with a peptide consisting of consecutive amino acids which are recognized by the bicysteine proteolytic enzymes from 19th to 28th amino acids (SEQ ID NO: 87). Caspase precursors. 제 2항에 있어서, 카스파제 9는 134번째에서 143번째 아미노산(서열번호 88), 301번째에서 310번째 아미노산(서열번호 89) 또는 311번째에서 320번째 아미노산까지(서열번호 90)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The caspase 9 is a bicysteine-based amino acid of 134 th to 143 th amino acids (SEQ ID NO: 88), 301 th to 310 th amino acids (SEQ ID NO: 89), or 311 th to 320 th amino acids (SEQ ID NO: 90). Recombinant caspase precursor, characterized in that substituted by a peptide consisting of a continuous amino acid that is recognized and cleaved by a proteolytic enzyme. 카스파제 3의 28번째 아미노산을 포함하는 6개의 연속적인 아미노산으로 구성된 펩타이드(서열번호 78: ESMDSG; 아미노산 25번째에서 30번째까지)를 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산(서열번호 79: LVPRGS)으로 치환하고, 180번째 아미노산과 181번째 아미노산 사이에 트롬빈에 의해 인식되어 절단되는 6개의 연속적인 아미노산(서열번호 79: LVPRGS)이 삽입된 재조합 카스파제 3 전구체.Peptides consisting of six consecutive amino acids comprising the 28th amino acid of Caspase 3 (SEQ ID NO: 78: ESMDSG; amino acids 25-30) are recognized by the thrombin and 6 consecutive amino acids (SEQ ID NO: 79 : Recombinant caspase 3 precursor substituted with LVPRGS and inserted with six consecutive amino acids (SEQ ID NO: 79: LVPRGS) that are recognized and cleaved by thrombin between the 180 and 181 amino acids. 카스파제 내에 존재하는, 적어도 하나 이상의 시스테인계 단백질 가수분해효소의 인식부위 내의 하나 이상의 아미노산 잔기가 상기 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산으로 치환되고, 상기 시스테인계 단백질 가수분해효소의 인식부위의 주변에 비시스테인계 단백질 가수분해효소 인식부위가 삽입된 재조합 카스파제 전구체.At least one amino acid residue in the recognition site of at least one cysteine proteolytic enzyme present in the caspase is replaced with an amino acid not recognized by the cysteine proteolytic enzyme, and the cysteine based proteolytic enzyme is recognized. A recombinant caspase precursor in which a bicysteine-based protease recognition site is inserted around a site. 제 13항에 있어서, 카스파제는 카스파제 2, 카스파제 3, 카스파제 6, 카스파제 7 및 카스파제 9로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.14. The recombinant caspase precursor according to claim 13, wherein the caspase is any one selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. 제 13항에 있어서, 비시스테인계 단백질 가수분해효소는 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드인 것을 특징으로 하는 재조합 카스파제 전구체.14. The recombinant caspase precursor according to claim 13, wherein the bicysteine protease is a peptide consisting of 4 to 10 consecutive amino acids. 제 13항에 있어서, 비시스테인계 단백질 가수분해효소 인식부위는 트롬빈 인식부위(Thrombin; 서열번호 79: LVPRGS), 엔테로키나제 인식부위(Enterokinase; 서열번호 91: DDDDK), TEV 인식부위(서열번호 92: ENLYFQG) 및 Factor Xa 인식부위(서열번호 93: IEGR 또는 서열번호 94: IDGR)로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.The bicysteine protease recognition site of claim 13 is a thrombin recognition site (Thrombin; SEQ ID NO: 79: LVPRGS), an enterokinase recognition site (Enterokinase; SEQ ID NO: 91: DDDDK), a TEV recognition site (SEQ ID NO: 92). : ENLYFQG) and a Factor Xa recognition site (SEQ ID NO: 93: IEGR or SEQ ID NO: 94: IDGR) any one selected from the group consisting of a recombinant caspase precursor. 제 13항에 있어서, 비시스테인계 단백질 가수분해효소는 트롬빈, 엔테로키나제, TEV 및 Factor Xa로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 재조합 카스파제 전구체.The recombinant caspase precursor according to claim 13, wherein the bicysteine-based proteolytic enzyme is any one selected from the group consisting of thrombin, enterokinase, TEV, and Factor Xa. 제 13항에 있어서, 시스테인계 단백질 가수분해효소의 인식부위는 아스파틱산(aspartic acid)을 중심으로 4 내지 10개의 연속적인 아미노산으로 구성된 펩타이드인 것을 특징으로 하는 재조합 카스파제 전구체.14. The recombinant caspase precursor according to claim 13, wherein the recognition site of the cysteine protease is a peptide composed of 4 to 10 consecutive amino acids centered on aspartic acid. 제 14항에 있어서, 카스파제 3은 23번째에서 32번째 아미노산(서열번호 95) 또는 170번째에서 179번째 아미노산까지(서열번호 96)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The method of claim 14, wherein caspase 3 is a continuous sequence in which the 23rd to 32nd amino acid (SEQ ID NO: 95) or the 170th to 179th amino acid (SEQ ID NO: 96) is recognized and cleaved by a bicysteine protease Recombinant caspase precursor, characterized in that substituted with a peptide consisting of amino acids. 제 14항에 있어서, 카스파제 7은 19번째에서 28번째 아미노산(서열번호 81) 및 194번째에서 203번째 아미노산까지(서열번호 83)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.15. The caspase 7 sequence is characterized in that the 19th to 28th amino acid (SEQ ID NO: 81) and the 194th to 203rd amino acid (SEQ ID NO: 83) are consecutively recognized and cleaved by a bicysteine-based protease. Recombinant caspase precursor, characterized in that substituted with a peptide consisting of amino acids. 제 14항에 있어서, 카스파제 2는 162번째에서 171번째 아미노산(서열번호 84), 297번째에서 306번째 아미노산(서열번호 85) 또는 319번째에서 328번째 아미노산까지(서열번호 86)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The method according to claim 14, wherein caspase 2 is the bicysteine system of amino acids 162 to 171 (SEQ ID NO: 84), 297 to 306 amino acids (SEQ ID NO: 85), or 319 to 328 amino acids (SEQ ID NO: 86). Recombinant caspase precursor, characterized in that substituted by a peptide consisting of a continuous amino acid that is recognized and cleaved by a proteolytic enzyme. 제 14항에 있어서, 카스파제 6은 19번째에서 28번째 아미노산까지(서열번호 87)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.15. Recombinant according to claim 14, wherein caspase 6 is substituted with a peptide consisting of consecutive amino acids which are recognized from the 19th to 28th amino acids (SEQ ID NO: 87) and cleaved by a bicysteine protease. Caspase precursors. 제 14항에 있어서, 카스파제 9는 134번째에서 143번째 아미노산(서열번호 88), 301번째에서 310번째 아미노산(서열번호 89) 또는 311번째에서 320번째 아미노산까지(서열번호 90)가 비시스테인계 단백질 가수분해효소에 의해 인식되어 절단되는 연속적인 아미노산으로 구성된 펩타이드로 치환되는 것을 특징으로 하는 재조합 카스파제 전구체.The method according to claim 14, wherein caspase 9 is amino acid 134 to 143 (SEQ ID NO: 88), 301 to 310 amino acids (SEQ ID NO: 89) or 311 to 320 amino acids (SEQ ID NO: 90) is bicysteine-based Recombinant caspase precursor, characterized in that substituted by a peptide consisting of a continuous amino acid that is recognized and cleaved by a proteolytic enzyme. 제 13항에 있어서, 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산은 아스파틱산(Aspartic acid)을 제외한 아미노산인 것을 특징으로 하는 재조합 카스파제 전구체.15. The recombinant caspase precursor according to claim 13, wherein the amino acid which is not recognized by the cysteine protease is an amino acid except for aspartic acid. 제 13항에 있어서, 시스테인계 단백질 가수분해효소가 인식할 수 없는 아미노산은 페닐알라닌, 알라닌, 류신 및 트립토판으로 이루어진 군으로부터 선택되는 것을 특징으로 하는 재조합 카스파제 전구체.14. The recombinant caspase precursor according to claim 13, wherein the amino acid not recognized by the cysteine-based proteolytic enzyme is selected from the group consisting of phenylalanine, alanine, leucine and tryptophan. 제 13항에 있어서, 아미노산 잔기가 치환되지 않은 시스테인계 단백질 가수분해효소 인식부위는 비시스테인계 단백질 가수분해효소 인식부위로 치환될 수 있는 것을 특징으로 하는 재조합 카스파제 전구체.14. The recombinant caspase precursor according to claim 13, wherein the cysteine-based protease recognition site in which the amino acid residue is not substituted may be substituted with the bicysteine-based protease recognition site. 제 1항 내지 제 26항 중의 어느 한 항의 재조합 카스파제 전구체를 암호화하는 폴리뉴클레오티드.27. A polynucleotide encoding the recombinant caspase precursor of any one of claims 1 to 26. 제 27항의 폴리뉴클레오티드를 포함하는 발현벡터.An expression vector comprising the polynucleotide of claim 27. 제 28항의 발현벡터가 형질도입된 형질전환체.A transformant transfected with the expression vector of claim 28. 1) 제 1항 내지 제 26항 중의 어느 한 항의 재조합 카스파제 전구체를 암호화하는 폴리뉴클레오티드를 포함하는 발현벡터를 제조하는 단계;1) preparing an expression vector comprising a polynucleotide encoding the recombinant caspase precursor of any one of claims 1 to 26; 2) 상기 발현벡터를 숙주세포에 도입하여 형질전환체를 제조하는 단계; 및,2) preparing a transformant by introducing the expression vector into a host cell; And, 3) 상기 형질전환체를 배양하여 재조합 단백질의 발현을 유도하고 이를 수득하는 단계로 구성되는 재조합 카스파제 전구체의 제조방법.3) A method for producing a recombinant caspase precursor, comprising culturing the transformant to induce expression of and obtain a recombinant protein. 제 1항 내지 제 26항 중의 어느 한 항의 재조합 카스파제 전구체를 비시스테인계 단백질 가수분해효소로 처리하는 단계를 포함하는 상기 재조합 카스파제 전구체를 활성화하는 방법.27. A method of activating the recombinant caspase precursor, comprising treating the recombinant caspase precursor of any one of claims 1 to 26 with a bicysteine proteolytic enzyme. 제 31항에 있어서, 상기 비시스테인계 단백질 가수분해효소는 트롬빈, 엔테로키나제, TEV 및 Factor Xa로 이루어진 군으로부터 선택된 어느 하나인 것을 특징으로 하는 방법.32. The method of claim 31, wherein the bicysteine protease is any one selected from the group consisting of thrombin, enterokinase, TEV, and Factor Xa. 제 32항의 방법으로 활성화된 재조합 카스파제.A recombinant caspase activated by the method of claim 32. 1) 제 33항의 활성화된 재조합 카스파제에 카스파제 특이적 기질 및 후보 물질을 처리하는 단계;1) treating the activated recombinant caspase of claim 33 with a caspase specific substrate and a candidate substance; 2) 상기 활성화된 재조합 카스파제의 기질에 대한 효소 활성을 측정하는 단계; 및,2) measuring enzyme activity on the substrate of the activated recombinant caspase; And, 3) 후보 물질을 처리하지 않은 대조군과 비교하여 상기 활성화된 재조합 카스파제의 효소 활성을 감소시키거나 증진시킨 후보 물질을 선별하는 단계를 포함하는 카스파제 활성 저해제 또는 촉진제의 스크리닝 방법.3) A method for screening a caspase activity inhibitor or promoter comprising selecting a candidate substance that has reduced or enhanced the enzymatic activity of the activated recombinant caspase compared to a control that has not been treated with the candidate substance.
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