WO2009044988A1

WO2009044988A1 - Method for large scale preparation of procaspases, which can be artificially activated, in e. coli expression system and their activation

Info

Publication number: WO2009044988A1
Application number: PCT/KR2008/003589
Authority: WO
Inventors: Sang Jeon Chung; Hyo Jin Kang; Young-Mi Lee; Moonil Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-10-01
Filing date: 2008-06-24
Publication date: 2009-04-09
Also published as: KR100980315B1; KR20090033785A

Abstract

The present invention relates to a recombinant caspase and a method for preparing the same, more precisely the present invention relates to a recombinant caspase expression vector capable of being over-expressed without cytotoxicity because the auto-activation recognition site of caspase is replaced with a non-cysteine protease recognition site to nullify the auto-activation activity during the mass-expression in E. coli. Caspase mass- produced from the auto-activation prevented recombinant caspase precursor of the present invention can be effectively used for the development of a novel drug and for studies on biological functions of enzymes.

Description

[DESCRIPTION]

[invention Title]

METHOD FOR LARGE SCALE PREPARATION OF PROCASPASES, WHICH CAN BE ARTIFICIALLY ACTIVATED, IN E. COLI EXPRESSION SYSTEM AND THEIR ACTIVATION

[Technical Field]

The present invention relates to recombinant caspases and methods for preparing the same.

[Background Art]

Apoptosis is an active death mechanism for the elimination of unnecessary cells in which intracellular enzymes are actively involved. This mechanism is essential for the elimination of unnecessary cells during the development of each organ. It was reported that autoimmune disease was caused by apoptosis disorder (Miller LJ & Marx

J, Science 281:1301-1326, 1998) . And methods for inducing apoptosis of cancer cells in gene therapy for cancer treatment were reported (Zhang XM et al . , EMBO J 16:2271-

2281, 1997) .

Caspase is a major protease which activates a series of apoptosis-related proteins in the last phase of apoptosis signal transduction to fragment DNA and nucleus and condense chromosome to induce apoptosis. Caspase is assumed to be involved in autoimmune disease such as Parkinson's disease and Alzheimer's disease (Korean Patent Publication No: 2002-0092042), and has been an important target of studies on mechanism of apoptosis and development of a novel drug. Caspase is composed of three domains which are prodomain, large subunit and small subunit. It is expressed as inactive procaspase in cells. Once apoptosis signal is transmitted, inactive caspase is decomposed into three domains by autolysis and prodomain is eliminated. Then, two large subunits and two small subunits are combined to produce 4 subunits which turn into active caspase (see Figure 2) . The active site of caspase contains cysteine, which specifically recognizes aspartic acid in the amino acid sequence of a substrate protein, to digest peptide bond. 11 caspases have been identified so far and among them caspases 1, 4, 5 and 11 are known to be involved in cytokine activation inducing inflammation and others are known to play an important role 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) .

Since caspase was confirmed to be involved in degenerative central nerve system disease and immune disorder, it has been a target of studies about novel drug development and has been in increasing demand with the increase of its utility in the field of apoptosis-related studies. Therefore, it is necessary to mass-produce caspase as a recombinant protein. However, if such a protein involved in apoptosis as caspase is over-expressed in E. coli system by using a whole gene, a specific region will be hydrolyzed, owing to weak activity of procaspase, and auto-activated (or auto-maturated) , resulting in cytotoxicity. Besides, accurate mechanism of the enzyme has not been disclosed, yet, making mass-expression and purification difficult.

To mass-express and purify caspase, it is most widely tried to express each subunit respectively and then renaturate or refold insoluble protein. Otherwise, it is tried to express caspase at a low level and then purify it small amount at a time (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, the above methods require high costs and endeavor, compared with the method for mass- expressing the whole protein as a water-soluble form, so that it is not appropriate for application in industry. In addition, active caspase obtained after every effort is likely to lose its activity by autolysis when it is stored for long term .

Thus, the present inventors developed E. coli expression system capable of expressing caspase precursor in which an amino acid sequence supposed to be hydrolyzed by cysteine proteases including caspase during the activation of caspase was replaced with another amino acid sequence that is hydrolyzed by a specific protease of another family. And the present inventors further completed this invention by confirming that the caspase precursor expressed in the said system can be prevented from being auto-activated during the expression process, can be easily purified and stored, and retains equal enzyme activity to natural caspase when it turns into active caspase. Therefore, the method of the present invention can be effectively used for mass-production of caspase precursor .

[Disclosure] [Technical Problem]

It is an object of the present invention to provide a recombinant caspase precursor in which cysteine protease recognition site is replaced with a non-cysteine protease recognition site and a method for preparing the same. It is another object of the present invention to provide a recombinant caspase precursor in which an amino acid residue of a cysteine protease recognition site is replaced with another amino acid not recognized by the said cysteine protease and a non-cysteine protease recognition site is inserted near the cysteine protease recognition site and a method for preparing the same.

It is further an object of the present invention to provide a method for activating the said recombinant caspase precursor by non-cysteine protease and a recombinant caspase activated by the method.

It is also an object of the present invention to provide a method for screening a caspase activity inhibitor or activator by using the said activated recombinant caspase .

[Technical Solution]

To achieve the above objects, the present invention provides a recombinant caspase precursor in which cysteine protease recognition site is replaced with a non-cysteine protease recognition site.

The present invention also provides a recombinant caspase 3 precursor in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 is replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin, and β consecutive amino acids recognized and digested by thrombin is inserted in between the 180^th amino acid and the 181^st amino acid.

The present invention further provides a recombinant caspase precursor in which at least one of amino acid residues of a cysteine protease recognition site of caspase is replaced with another amino acid not recognized by the said cysteine protease and a non-cysteine protease recognition site is inserted near the cysteine protease recognition site.

The present invention also provides a polynucleotide encoding one of the said recombinant caspase precursors.

The present invention also provides an expression vector containing the said polynucleotide. The present invention also provides a transformant transfected with the said expression vector.

The present invention also provides a method for preparing a recombinant caspase precursor comprising the following steps: 1) preparing an expression vector containing a polynucleotide encoding one of the recombinant caspase precursors ;

2) preparing a transformant by introducing the expression vector into a host cell; and, 3) culturing the transformant to induce the expression of the recombinant protein and obtaining thereof, The present invention also provides a method for activating the recombinant caspase precursor containing the step of treating the recombinant caspase precursor with non-cysteine protease.

The present invention also provides a recombinant caspase activated by the above method.

The present invention also provides a method for screening a caspase activity inhibitor or activator comprising the following steps:

1) treating the activated recombinant caspase with a caspase-specific substrate and candidates;

2) measuring the enzyme activity of the activated recombinant caspase against the substrate; and, 3) selecting candidates confirmed to inhibit or activate the enzyme activity of the activated recombinant caspase by comparing the activity of the control group not treated with candidates.

In addition, the present invention provides a use of the activated recombinant caspase for the screening of a caspase activity inhibitor or enhancer.

[Advantageous Effect!

Caspase mass-produced from the auto-activation prevented recombinant caspase precursor of the present invention can be effectively used for the development of a novel drug and for studies on biological functions of enzymes .

[Description of Drawings]

The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:

Figure 1 is a diagram illustrating the conversion of a recombinant caspase precursor into an active recombinant caspase protein by thrombin.

Figure 2 is a diagram illustrating the activation of caspase in cells.

Figure 3 is a diagram illustrating the over- expression of the recombinant caspase precursor of the present invention and purification of the same: M: Size Marker;

1: caspase 3(1-277); 2: Δ28 caspase 3; 3: Δ28/175TS caspase 3; 4: 28TS/175TS caspase 3 ; 5: 28TS/D175A/180TI caspase 3; and, 6: C163S caspase 3 (negative control) .

Figure 4 is a diagram illustrating the results of SDS-PAGE observing the changes of the recombinant caspase precursor of the present invention into active caspase by the treatment of thrombin over the time. Lanes 1 - 4: reacted at 4 ^°C , Lanes 5 - 8: reacted at 18 ^°C (M is a protein marker, and the 66 kDa band is presumed to be originated from thrombin) . Lanes 1 and 5: caspase precursors not reacted with thrombin, Lanes 2 and 6: 200 βg of caspase protein reacted with 2 NIH units of thrombin, Lanes 3 and 7: 200 βg of caspase protein reacted with 20 NIH units of thrombin, Lanes 4 and 8: 200 βg of caspase protein reacted with 40 NIH units of thrombin: a: 4 hours after the reaction; b: 8 hours after the reaction; and, c: 24 hours after the reaction.

Figure 5 is a diagram illustrating the expression and purification of caspase 7 precursor in E. coli and the activation of the same:

M: Size Marker;

1: 198TS Cas7;

2: 23TS/198TS Cas7; 3: 198TS Cas7 activated by thrombin at 4 ^°C ; 4: 198TS Cas7 activated by thrombin at 18°C;

5: 23TS/198TS Cas7 activated by thrombin at 4 ^°C ; and,

6: 23TS/198TS Cas7 activated by thrombin at 18^°C.

[Best Mode]

Hereinafter, terms used in this invention are described.

"Cysteine proteases" indicate proteases including caspase which contain cysteine in their active sites and recognize specifically aspartic acid in the amino acid sequence of a substrate protein to cut the amino acid bond. "Non-cysteine proteases" indicate all the proteases except cysteine proteases. "Megaprimer method" is one of PCRs, which used to be hired to complete mutation by two-step PCR especially when one-time PCR is not enough to induce mutation because a target site is located in the middle of a protein. For example, when PCR is performed using a forward primer containing nucleotide of targeted mutation and a reverse primer containing nucleotide of C-terminal of the protein, DNA that does not contain information on N-terminal located upstream of the forward primer is obtained. This produced DNA is called a megaprimer. In next PCR, this megaprimer is used as a reverse primer and oligo DNA encoding N- terminal of a protein is used as a forward primer. Then, DNA encoding a full length of the protein its targeted area is successfully mutated is obtained. If primary PCR is performed with a reverse primer encoding a mutation and a reverse primer encoding N-terminal of a protein to produce a megaprimer, the next PCR will be performed with a reverse primer containing C-terminal information to produce DNA encoding a full length mutated protein. This process is called megaprimer method. "Expression vector" is a linear or circular DNA molecule composed of fragments encoding polypeptide operably linked to an additional fragment provided for the transcription of the expression vector. The additional fragment includes a promoter and a stop codon sequence. The expression vector also contains one or more replication origins, one or more selection markers and polyadenylation signal. Such expression vector is generally derived from plasmid or virus DNA or both.

Hereinafter, the present invention is described in detail .

The present invention provides a recombinant caspase precursor in which cysteine protease recognition site is replaced with a non-cysteine protease recognition site. In a preferred embodiment of the present invention, an expression vector expressing a recombinant caspase 3 precursor (28TS, Δ28TS, 175TS, 28TS/175TS, Δ28TS/175TS Caspase 3; Δ: 1^st - 28^th amino acids were eliminated) in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG: 25^th - 30^th amino acids) and/or a peptide comprising 6 consecutive amino acids including the 175^th amino acid (SEQ. ID. NO: 80: IETDSG; 172^nd - 177^th amino acids) was replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS) was constructed. E. coli was transfected with the expression vector to express the recombinant caspase 3 precursor. The expressed recombinant caspase 3 precursor was purified by adsorption column binding to hexa-his tag recognition site. As a result, it was confirmed that the expression of the recombinant caspase 3 precursor was increased by the substitution of the recognition site comprising 6 amino acids including the 28^th amino acid of the recombinant caspase 3 precursor and the recognition site comprising 6 amino acids including the 175^th amino acid of the recombinant caspase 3 precursor with the thrombin recognition site (see Figure 3) . To activate the purified recombinant caspase 3 precursor (SEQ. ID. NO: 53), thrombin was treated for 8 hours at 18^°C or for 24 hours at 4 ^°C (see Figure 4) . The preferable dosage of thrombin in this invention was at least 40 NIH units regardless of reaction temperature. In a preferred embodiment of the present invention, an expression vector expressing a recombinant caspase 7 precursor (23TS, 198TS, 23TS/198TS Caspase 7) in which a peptide comprising 10 consecutive amino acids including the 23^rd amino acid of caspase 7 (SEQ. ID. NO: 81: EDSVDAKPDR; 19^th - 28^th amino acids) was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 82: EDLVPRGSDR) and/or a peptide comprising 10 consecutive amino acids including the 175^th amino acid (SEQ. ID. NO: 83: GIQADSGPIN; 194^th - 203^rd amino acids) is replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 98: GLVPRGSPIN) was constructed. E. coli was transfected with the expression vector to express and purify the recombinant caspase 7 precursor. As a result, the recombinant caspase 7 precursor (SEQ. ID. NO: 97 or NO: 64) was changed into active caspase at 4 ^°C or at 18^°C (see Figure 5) . Enzyme kinetics of the active caspase 3 obtained above were measured. As a result, k_cat/K_m of the recombinant caspase 3 of the present invention was lower than the wild type caspase 3 (SEQ. ID. NO: 49) (see Table 2) . However, by replacing a peptide comprising 6 consecutive amino acids including aspartic acid, the caspase 3 auto-activation recognition site, with amino acid sequence of thrombin recognition site, the recombinant caspase 3 precursor of the present invention can be mass-produced in E. coll without auto-cleavage mediated auto-activation (see Figure 3) .

The caspase of the present invention is not changed into an active form when it is expressed because the auto- activation recognition site is replaced, so that it does not induce auto-activation mediated cytotoxicity in host cells and it can be stored for a long term because it stays as an inactive form after separation and purification. Besides, the caspase of the present invention can be easily activated by using a commercial protease by replacing the protease recognition site with another recognition site.

The caspase herein is selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. The cysteine protease recognition site herein can be a peptide composed of 4 - 10 consecutive amino acids including aspartic acid. The cysteine protease recognition site is as follows:

(1) 162^nd ammo acid - 171^st amino acid (SEQ. ID. NO: 84), 297^th ammo acid - 306^th amino acid (SEQ. ID. NO: 85) and 319^th amino acid - 328^th amino acid (SEQ. ID. NO: 86) of full length caspase 2 (SEQ. ID. NO: 73) ; (2) 23^rd amino acid - 32^nd amino acid (SEQ. ID. NO: 95) and 170^th amino acid - 179^th amino acid (SEQ. ID. NO: 96) of full length caspase 3 (SEQ. ID. NO: 74);

(3) 19^th amino acid - 28^th amino acid (SEQ. ID. NO: 87) of full length caspase 6 (SEQ. ID. NO: 75);

(4) 19^th amino acid - 28^th amino acid (SEQ. ID. NO: 81) and 194^th amino acid - 203^rd amino acid (SEQ. ID. NO: 83) of full length caspase 7 (SEQ. ID. NO: 76) ; and,

(5) 134^th amino acid - 143^rd amino acid (SEQ. ID. NO: 88), 301^st amino acid - 310^th amino acid (SEQ. ID. NO: 89) and 311^th amino acid - 320^th amino acid (SEQ. ID. NO: 90) of full length caspase 9 (SEQ. ID. NO: 77) .

The non-cysteine protease recognition site herein can be any site that is not recognized to be digested by cysteine proteases including caspase, and can be a peptide composed of 4 - 10 consecutive amino acids. Caspase can be specifically activated by introducing the commercial non- cysteine protease recognition site according to the method of the present invention. The non-cysteine protease recognition site can be exemplified by 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 Factor Xa recognition site (SEQ. ID. NO: 93: IEGR or SEQ. ID. NO: 94: IDGR), etc, but not always limited thereto and any non- cysteine protease recognition site known to those in the art can be used. In a preferred embodiment of the present invention, thrombin recognition site was used. The non- cysteine protease herein can be exemplified by thrombin, enterokinase, TEV and Factor Xa, etc, and in a preferred embodiment of the present invention, thrombin was used.

The present invention also provides a recombinant caspase 3 precursor in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG; 25^th amino acid - 30^th amino acid) is replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS), and 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS) is inserted in between the 180^th amino acid and the 181^st amino acid.

In a preferred embodiment of the present invention, an expression vector expressing a recombinant caspase 3 precursor 28TS/180TI (SEQ. ID. NO: 54) in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 was replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin, and 6 consecutive amino acids recognized and digested by thrombin was inserted in between the 180^th amino acid and the 181^st amino acid was constructed. E. coli was transfected with the expression vector to express and purify the recombinant caspase 3 precursor. The precursor was treated with thrombin to turn into an active enzyme form. Then, enzyme kinetics was measured. This enzyme demonstrated higher enzyme activity than the recombinant caspase 3 precursor 28TS/175TS (SEQ. ID. NO: 53) in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 and a peptide comprising 6 consecutive amino acids including the 175^th amino acid were replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (see Table 2) .

The caspase of the present invention is not changed into an active form when it is expressed because the auto- activation recognition site is replaced, so that it does not induce auto-activation mediated cytotoxicity in host cells and it can be stored for a long term because it stays as an inactive form after separation and purification. Besides, the caspase of the present invention can be easily activated by using a commercial protease by replacing the hydrolyzed recognition site with another recognition site.

In a preferred embodiment of the present invention, an expression vector of L168F/28TS/175TS caspase 3 (SEQ. ID. NO: 56) in which leucine, the 168^th amino acid of 28TS/175TS caspase 3 was replaced with phenylalanine, was constructed. E. coli was transfected with the expression vector to express and purify the recombinant caspase 3 precursor. The precursor was treated with thrombin to be changed into an active enzyme form. Then, enzyme kinetics was measured. As a result, this enzyme demonstrated higher enzyme activity than that of 28TS/175TS caspase 3 (SEQ. ID. NO: 53) (see Table 2) . In a preferred embodiment of the present invention, L168F/28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 59) expression vector and L168W/28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 60) expression vector were constructed by replacing leucine, the 168^th amino acid of 28TS/180TI caspase 3 (SEQ. ID. NO: 54) with phenylalanine or with tryptophan, and by replacing aspartic acid, the 175^th amino acid, with alanine. E. coli was transfected with the expression vectors to express and purify the recombinant caspase 3 precursor. The precursor was treated with thrombin to be changed into an active enzyme form. Then, enzyme kinetics was measured. As a result, this enzyme demonstrated higher enzyme activity than that of 28TS/180TI caspase 3 (SEQ. ID. NO: 54) (see Table 2). The caspase herein is selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9. The cysteine protease recognition site herein can be a peptide composed of 4 - 10 consecutive amino acids including aspartic acid. The cysteine protease recognition site is as follows:

(1) 162^nd amino acid - 171^st amino acid (SEQ. ID. NO: 84), 297^th amino acid - 306^th amino acid (SEQ. ID. NO: 85) and 319^th amino acid - 328^th amino acid (SEQ. ID. NO: 86) of full length caspase 2 (SEQ. ID. NO: 73); (2) 23^rd amino acid - 32^nd amino acid (SEQ. ID. NO: 95) and 170^th amino acid - 179^th amino acid (SEQ. ID. NO: 96) of full length caspase 3 (SEQ. ID. NO: 74);

(3) 19^th amino acid - 28^th amino acid (SEQ. ID. NO: 87) of full length caspase 6 (SEQ. ID. NO: 75); (4) 19^th amino acid - 28^th amino acid (SEQ. ID. NO: 81) and 194^th amino acid - 203^rd amino acid (SEQ. ID. NO: 83) of full length caspase 7 (SEQ. ID. NO: 76); and,

(5) 134^th amino acid - 143^rd amino acid (SEQ. ID. NO:

88), 301^st amino acid - 310^th amino acid (SEQ. ID. NO: 89) and 311^th amino acid - 320^th amino acid (SEQ. ID. NO: 90) of full length caspase 9 (SEQ. ID. NO: 77) .

The non-cysteine protease recognition site herein can be any site that is not recognized to be digested by cysteine proteases including caspase, and can be a peptide composed of 4 - 10 consecutive amino acids. Caspase can be specifically activated by introducing the non-cysteine protease recognition site according to the method of the present invention. The non-cysteine protease recognition site can be exemplified by 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 Factor Xa recognition site (SEQ. ID. NO: 93: IEGR or SEQ. ID. NO: 94 : IDGR) , etc, but not always limited thereto and any non- cysteine protease recognition site known to those in the art can be used. In a preferred embodiment of the present invention, thrombin recognition site was used. The non- cysteine protease herein can be exemplified by thrombin, enterokinase, TEV and Factor Xa, etc, and in a preferred embodiment of the present invention, thrombin was used.

The amino acid used to replace one or more amino acid residues in the cysteine protease recognition site can be any amino acid except aspartic acid that cannot be recognized by cysteine protease, which is exemplified by phenylalanine, alanine, leucine and tryptophan, but not always limited thereto.

In the present invention, a cysteine protease recognition site in which no amino acid residue is replaced can be substituted with a non-cysteine protease recognition site.

The replacement of a cysteine protease recognition site of caspase with a non-cysteine protease recognition site and the insertion of a non-cysteine protease recognition site around the cysteine protease recognition site can be achieved by megaprimer PCR. One of the primers of the primer set used for the megaprimer PCR, either a reverse primer or a forward primer, is supposed to contain a nucleotide sequence encoding a non-cysteine protease recognition site. So, any primer that can give a nucleotide sequence encoding a non-cysteine protease recognition site in its PCR product can be used.

In a preferred embodiment of the present invention, the primer set represented by SEQ. ID. NO: 5 and NO: 6 and/or the primer set represented by SEQ. ID. NO: 7 and NO: 8 was used to replace a peptide comprising 6 consecutive amino acids including the 28^th and/or the 175^th amino acid of caspase 3 (SEQ. ID. NO: 49) with a thrombin recognition site. And the primer set represented by SEQ. ID. NO: 24 and NO: 25 and/or the primer set represented by SEQ. ID. NO: 26 and NO: 27 was used to replace a peptide comprising 10 consecutive amino acids including the 23^rd amino acid and/or the 198^th amino acid of caspase 7 (SEQ. ID. NO: 62) with a thrombin recognition site.

The replacement of one or more amino acids of a cysteine protease recognition site with other amino acids which cannot be recognized by cysteine protease can be performed by site directed mutagenesis method. The kit for site directed mutagenesis has already been commercialized, so it is well understood by those in the art that revival of the present invention is not difficult.

The present invention also provides an expression vector containing the said polynucleotide. The polynucleotide encoding the recombinant caspase with the said substitution is cloned into restriction enzyme site of a backcone vector. The backcone vector herein is not limited and any vector that can transfect E. coli is acceptable and might be selected from the group consisting of pET21a, pET28a, pET/Rb, pGEX, pET-22b(+) and pGEX . The expression vector of the present invention is prepared by inserting a replaced caspase gene into pET21a vector, which can be expressed as a recombinant caspase precursor.

The present invention also provides a transformant transfected with the said expression vector.

The present invention also provides a method for preparing a recombinant caspase precursor comprising the following steps:

1) preparing an expression vector containing a polynucleotide encoding one of the recombinant caspase precursors;

2) preparing a transformant by introducing the expression vector into a host cell; and,

3) culturing the transformant to induce the expression of the recombinant protein and obtaining thereof. According to the method of the present invention, mass-production of a recombinant caspase precursor is facilitated (see Figures 3 and 5) . The recombinant caspase precursor can be activated simply by treating non-cysteine protease (see Figures 4, 5 and Table 2) . Therefore, the recombinant caspase precursor of the present invention is expected to meet the demand of natural caspase required for the development of a novel drug and for the studies on biological functions of enzymes.

The present invention also provides a method for activating the recombinant caspase precursor containing the step of treating the recombinant caspase precursor with non-cysteine protease.

According to the method of the present invention, mass-production of a recombinant caspase precursor is facilitated (see Figures 3 and 5) . The recombinant caspase precursor can be activated simply by treating non-cysteine protease (see Figures 4, 5 and Table 2) . Therefore, the recombinant caspase precursor of the present invention is expected to meet the demand of natural caspase required for the development of a novel drug and for the studies on biological functions of enzymes. In a preferred embodiment of the present invention, thrombin was treated to activate the recombinant caspase 3 precursor (SEQ. ID. NO: 53) at 18^°C for 8 hours or at 4 ^°C for 24 hours, which was confirmed to be a proper condition to convert the precursor into active caspase by hydrolysis (see Figure 4) . The preferable dosage of thrombin in this invention was at least 40 NIH units regardless of reaction temperature .

The non-cysteine protease herein is exemplified by thrombin, enterokinase, TEV and Factor Xa and in a preferred embodiment of the present invention, thrombin was used.

2) measuring the enzyme activity of the activated recombinant caspase against the substrate; and, 3) selecting candidates confirmed to inhibit or activate the enzyme activity of the activated recombinant caspase by comparing the activity with that of the control group not treated with candidates.

The caspase specific substrate of step 1) can be Ac- DEVD-pNA (pNA: para-nitroanilide) , Ac-DEVD-AFC (7-amino-4- trifluoromethylcoumarin) , etc. In a preferred embodiment of the present invention, Ac-DEVD-pNA was used.

The candidate of step 1) is exemplified by natural compounds, synthetic compounds, RNA, DNA, polypeptides, enzymes, proteins, ligands, antibodies, antigens, metabolites of bacteria or fungi and biomolecules, but not always limited thereto.

The enzyme activity of step 2) can be determined by measuring the capacity of the activated recombinant caspase to decompose a substrate by UV/VIS spectrometer.

[Mode for Invention]

Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Vector for over-expression of natural caspase precursor

An expression vector for a natural caspase precursor that can be activated by cysteine protease was constructed.

First, template DNAs of genes encoding 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 caspase 9 (SEQ. ID. NO: 72) were purchased from 21C Human Gene Bank, Genome Research Center (KRIBB, Korea) . Each primer set was designed to amplify whole genes of the 5 caspases with harboring restriction enzyme site shown in Table 1, which was constructed by Bioneer (KOREA) . The sequences of the primers designed are as shown in Table 1.

[Table l] Primer sets for the amplification of whole genes of caspases

1 βt of template DNA, 1 μϊ of a 20 μM forward primer and reverse primer 1 or reverse primer 2 for each gene, 2.5 βt of polymerase activating buffer, 2 μt of dNTP, 1 μi of Taq polymerase and 16.5 βi of sterilized distilled water were all mixed to give 25 μl of reaction mixture, followed by PCR as follows: predenaturation at 95^°C for 5 minutes, denaturation at 95^°C for 30 seconds, annealing at 55^°C for 1 minute, extension at 68^°C for 2 minutes, 25 cycles from denaturation to extension, and final extension at

72^°C for 10 minutes. Reaction was terminated at 4 ^°C and the reaction product was stored at 4 ^°C . The reverse primer was selected among reverse primer 1 and reverse primer 2 according to the presence or absence of stop codon. The PCR product was electrophoresed on 1% agarose gel. As a result, 800-1300 bp DNA fragments were confirmed.

The final PCR product was reacted with pET28a vector (Novagen, USA) in the presence of each restriction enzyme

(New England Biolabs, UK) presented in Table 1 at 37^°C for

2 hours, leading to ligation using DNA ligation kit (Takara, Japan) . Nucleotide sequence of the vector was confirmed by nucleotide sequencing. As a result, pET28a-Cas3, pET28a- Cas2, pET28a-Casβ, pET28a-Cas7 and pET28a-Cas9 vectors over-expressing natural caspase precursors were constructed.

Example 2: Expression vector for a replaced recombinant caspase precursor

An expression vector for a replaced recombinant caspase precursor which cannot be activated by cysteine protease but can be activated by thrombin was constructed.

<2-l> 28TS caspase 3 (SEQ. ID. NO: 51) expression vector

An expression vector expressing a recombinant caspase

3 precursor in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG: 25^th - 30^th amino acids) was replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS) was constructed.

1 μ0 of template DNA (pET28a-Cas3 vector) , 1 μl of a 20 μM 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), 2.5 μi of polymerase activating buffer, 2 μϊ of dNTP, 1 //0 of Taq polymerase and 16.5 μi of sterilized distilled water were all mixed to give 25 μJl of reaction mixture, followed by the first PCR as follows: predenaturation at 95°C for 5 minutes, denaturation at 95^°C for 30 seconds, annealing at 55^°C for 1 minute, extension at 68^°C for 2 minutes, 25 cycles from denaturation to extension, and final extension at 72^°C for 10 minutes. Reaction was terminated at 4 ^°C and the reaction product was stored at 4 ^°C . The PCR product was used as a megaprimer without purification after the reaction. Then, 1 μJl of template DNA, 1 μl of the megaprimer and 20 μM reverse primer (SEQ. ID. NO: 3), 2.5 μQ of polymerase activating buffer, 2 μ& of dNTP, 1 μi of Taq polymerase and 16.5 μi of sterilized distilled water were all mixed to give 25 μJt of reaction mixture, followed by the second PCR as follows to produce a target gene: predenaturation at 95^°C for 5 minutes, denaturation at 95^°C for 30 seconds, annealing at 55^°C for 1 minute, extension at 68^°C for 2 minutes, 25 cycles from denaturation to extension, and final extension at 72^°C for 10 minutes. Reaction was terminated at 4 ^°C and the reaction product was stored at 4 ^°C .

PCR was performed another way using a 20 μM 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) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using a forward primer (SEQ. ID. NO: 1) and the PCR product as a megaprimer.

The final PCR product and pET21a vector (Novagen, USA) were treated with restriction enzymes Ndel and Xhol as shown in Table 1 at 37°C for 2 hours, leading to ligation using DNA ligation kit, followed by DNA sequencing to confirm nucleotide sequence of the vector. The confirmed nucleotide sequence was compared with a nucleotide sequence of natural caspase gene by Blast ( //www . ncbi . nlm. nih . gov) . As a result, it was confirmed that there was a change in a polynucleotide sequence encoding the peptide comprising 6 consecutive amino acids including the 28^th amino acid. Finally, the vector over-expressing 28TS caspase 3 (SEQ. ID. NO: 51), the recombinant caspase 3 precursor in which the 28^th amino acid was replaced with thrombin recognition site was constructed. <2-2> Δ28 caspase 3 (SEQ. ID. NO: 50) expression vector

An expression vector expressing a recombinant caspase 3 precursor in which the region from the 1^st amino acid to the 28^th amino acid of caspase 3 was eliminated was constructed.

PCR was performed using a 20 μM 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) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using reverse primer (SEQ. ID. NO: 3) and the PCR product as a megaprimer .

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing Δ28 caspase 3 (SEQ. ID. NO: 50), the recombinant caspase 3 precursor in which the region from the 1^st amino acid to the 28^th amino acid of caspase 3 was eliminated was constructed.

<2-3> 175TS caspase 3 (SEQ. ID. NO: 52) expression vector

An expression vector expressing 175TS caspase 3, the recombinant caspase 3 precursor in which a peptide comprising 6 consecutive amino acids including the 175^th amino acid of caspase 3 (SEQ. ID. NO: 80: IETDSG: 172^nd - 177^th amino acids) was replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS) was constructed . PCR was performed using a 20 μM 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) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using reverse primer (SEQ. ID. NO: 3) and the PCR product as a megaprimer .

PCR was performed another way using a 20 μM 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) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using a forward primer (SEQ. ID. NO: 1) and the PCR product as a megaprimer.

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing 175TS caspase 3 (SEQ. ID. NO: 52), the recombinant caspase 3 precursor in which the 175^th amino acid was replaced with thrombin recognition site was constructed. <2-4> Δ28/175TS caspase 3 (SEQ. ID. NO: 55) expression vector

An expression vector expressing a recombinant caspase 3 precursor in which a peptide comprising consecutive amino acids ranging from the first amino acid to the 28^th amino acid of caspase 3 was eliminated and a peptide comprising 6 consecutive amino acids including the 175^th amino acid of caspase 3 was replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79) was constructed.

PCR was performed using a 20 μM forward primer (SEQ. ID. NO: 4) and reverse primer (SEQ. ID. NO: 8) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using reverse primer (SEQ. ID. NO: 3) and the PCR product as a megaprimer .

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing Δ28/175TS caspase 3 (SEQ. ID. NO: 55), the recombinant caspase 3 precursor in which a peptide comprising consecutive amino acids ranging from the first amino acid to the 28^th amino acid of caspase 3 was eliminated and the 175^th amino acid was replaced with thrombin recognition site was constructed. <2-5> 28TS/175TS caspae-3 (SEQ. ID. NO: 53) expression vector

An expression vector expressing 28TS/175TS caspae-3, the recombinant caspase 3 precursor in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 and a peptide comprising 6 consecutive amino acids including the 175^th amino acid of caspase 3 were replaced with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79) was constructed.

PCR was performed with a 20 μM forward primer (SEQ. ID. NO: 7) and reverse primer (SEQ. ID. NO: 3) using the 28TS caspase 3 over-expressing vector of Example <2-l> as a template. The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using a forward primer (SEQ. ID. NO: 1) and the PCR product as a megaprimer.

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing 28TS/175TS caspase 3 (SEQ. ID. NO: 53), the recombinant caspase 3 precursor in which the 28^th and the 175^th amino acid was replaced with thrombin recognition site was constructed. <2-6> L168F/28TS/175TS caspase 3 (SEQ. ID. NO: 56) expression vector

PCR was performed using the 28TS/175TS caspase 3 (SEQ. ID. NO: 53) over-expressing vector prepared in Example <2- 4> as a template with a 20 μM forward primer (SEQ. ID. NO: 9: GCC TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) and reverse primer (SEQ. ID. NO: 10: TGT CTC AAT GCC ACA GTC GAA TTC TGT ACC ACG GCA) by using site directed mutagenesis kit (Stratagene, USA) according to the manufacturer's instruction as follows: predenaturation 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 cycles from denaturation to extension, and final extension at 72^°C for 10 minutes. Reaction was terminated at 4 ^°C and the reaction product was stored at 4 ^°C . The PCR product was treated with Dpnl (New England Biolabs, UK), followed by reaction at 37^°C for 1 hour, which was then inserted into pET21a expression vector. E. coli was transfected with the vector, followed by nucleotide sequencing. As a result, an expression vector over- expressing L168F/28TS/175TS caspase 3 (SEQ. ID. NO: 56) was constructed.

<2-7> L168W/28TS/175TS caspase 3 (SEQ. ID. NO: 57) expression vector

An expression vector over-expressing L168W/28TS/175TS caspase 3 (SEQ. ID. NO: 57) was constructed by using the vector over-expressing 28TS/175TS caspase 3 (SEQ. ID. NO: 53) prepared in Example <2-4> as a template with a forward primer (SEQ. ID. NO: 11: TGC CGT GGT ACA GAA TGG GAC TGT GGC CTG GTG CCG) and reverse primer (SEQ. ID. NO: 12: CAC CAG GCC ACA GTC CCA TTC TGT ACC ACG GCA GGC) by the site directed mutagenesis method described in Example <2-6>.

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

An expression vector expressing 28TS/D175A/180TI caspae-3, the recombinant caspase 3 precursor in which a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 was replaced with another peptide comprising consecutive amino acids and 6 consecutive amino acids recognized and digested by thrombin were inserted in between the 180^th amino acid and the 181^st amino acid of caspase 3 was constructed.

<2-8-l> 28TS/180TI caspase 3 (SEQ. ID. NO: 54) expression vector

PCR was performed with a 20 μM forward primer (SEQ. ID. NO: 1) and reverse primer (SEQ. ID. NO: 13: TAT TTT ATG ACA CGC CAT GTC GCT GCC GCG CGG CAC CAG ATC ATC AAC ACC

ACT) using the vector over-expressing 28TS caspase 3 (SEQ.

ID. NO: 51) of Example <2-l> as a template. The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using reverse primer (SEQ. ID. NO: 3) and the PCR product as a megaprimer.

PCR was performed another way using a 20 μM 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 a reverse primer (SEQ. ID. NO: 3) . The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using a forward primer (SEQ. ID. NO: 1) and the PCR product as megaprimer.

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing 28TS/180TI caspase 3 (SEQ. ID. NO: 54) was constructed.

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

An expression vector over-expressing 28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 58) was constructed by substituting the 175^th amino acid (aspartic acid) with alanine by the site directed mutagenesis method described in Example <2-6> by using the above constructed vector 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) .

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

An expression vector over-expressing L168F/28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 59) was constructed by the site directed mutagenesis method described in Example <2-6> by using the vector over- expressing 28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 58) of Example <2-7> as a template with a forward primer (SEQ. ID. NO: 17: TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) and a reverse primer (SEQ. ID. NO: 18: CTC AAT GCC ACA GTC GAATTC TGT ACC ACG GCA) .

<2-10> L168W/28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 60) expression vector An expression vector over-expressing L168W/28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 60) was constructed by the site directed mutagenesis method described in Example <2-6> by using the vector over- expressing 28TS/D175A/180TI caspase 3 (SEQ. ID. NO: 58) as a template with a forward primer (SEQ. ID. NO: 19: TGC CGT GGT ACA GAA TGG GAC TGT GGC ATT GAG) and a reverse primer (SEQ. ID. NO: 20: CTC AAT GCC ACA GTC CCA TTC TGT ACC ACG GCA) .

<2-ll> C163S caspase 3 (SEQ. ID. NO: 61) expression vector

The negative control was prepared by the method of

Pan S & Berk BC {Circ Res. 100 (2 ) : 213-9, 2007) using pET28a-Cas3 vector as a template. As a result, an expression vector expressing C163S caspase 3 (SEQ. ID. NO: 61), the recombinant caspase 3 precursor in which the 163^rd amino acid (cysteine), an active residue of caspase 3, was replaced with serine was constructed.

<2-12> 23TS/198TS Caspase 7 (SEQ. ID. NO: 64) expression vector

An expression vector expressing 23TS/198TS Caspase 7

(SEQ. ID. NO: 64), the recombinant caspase 7 precursor in which a peptide comprising 10 consecutive amino acids including the 23^rd amino acid of caspase 7 (SEQ. ID. NO: 81: EDSVDAKPDR: 19^th - 28^th amino acids) was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 82: EDLVPRGSDR) and a peptide comprising 10 consecutive amino acids including the 175^th amino acid of caspase 7 (SEQ. ID. NO: 83: GIQADSGPIN: 194^th - 203^rd amino acids) was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 98: GLVPRGSPIN), was constructed.

PCR was performed with a forward primer (SEQ. ID. NO: 21) and a reverse primer (SEQ. ID. NO: 24: TCA GCA AAT GAA CTG GTG CCG CGC GGC AGC CCA GAC CGG TCC TCG) using pET28a- Cas7 vector as a template. The first PCR was performed by the same manner as described in Example <2-l> and then the second PCR was performed using a reverse primer (SEQ. ID. NO: 23) and the PCR product as megaprimer. As a result, an expression vector over-expressing 23TS Caspase 7 (SEQ. ID. NO: 63), the recombinant caspase 7 precursor in which a peptide comprising 10 consecutive amino acids including the 23^rd amino acid of caspase 7 was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin was constructed.

An expression vector over-expressing 23TS Caspase 7 (SEQ. ID. NO: 63) was constructed by using the PCR product obtained from another PCR 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) as megaprimer and a forward primer (SEQ. ID. NO: 21) .

Then, a recombinant caspase 7 (23TS/198TS Caspase 7) gene in which a peptide comprising 10 consecutive amino acids including the 198^th amino acid of caspase 7 was replaced with a peptide comprising consecutive amino acids recognized and digested by thrombin was constructed by using megaprimer obtained from PCR using the vector over- expressing 23TS caspase 7 (SEQ. ID. NO: 63) as a template with 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) .

A recombinant caspase 7 (23TS/198TS Caspase 7) gene was constructed by using the PCR product obtained from another PCR 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) as megaprimer and a reverse primer (SEQ. ID. NO: 23) .

The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing 28TS/198TS caspase 7 (SEQ. ID. NO: 64) was constructed.

<2-13> 198TS Caspase 7 (SEQ. ID. NO: 97) expression vector An expression vector expressing 198TS Caspase 7 (SEQ.

ID. NO: 97), the recombinant caspase 7 precursor in which a peptide comprising 10 consecutive amino acids including the

23^rd amino acid of caspase 7 was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin was constructed. Then, a recombinant caspase 7 (198TS Caspase 7) gene in which a peptide comprising 10 consecutive amino acids including the 198^th amino acid of caspase 7 was replaced with a peptide comprising consecutive amino acids recognized and digested by thrombin was constructed by the same manner as described in Example <2-l> by using megaprimer obtained from PCR using the vector over- expressing pET28a-Cas7 as a template with a forward primer (SEQ. ID. NO: 27) and a reverse primer (SEQ. ID. NO: 23) . A recombinant caspase 7 (198TS Caspase 7) gene was constructed by using the PCR product obtained from another PCR using a forward primer (SEQ. ID. NO: 21) and a reverse primer (SEQ. ID. NO: 26) as megaprimer and a reverse primer (SEQ. ID. NO: 23) . The final PCR product was introduced into pET21a expression vector according to the method of Example <2-l> and as a result, an expression vector over-expressing 198TS caspase 7 (SEQ. ID. NO: 97) was constructed.

<2-14> L191F/23TS/198TS Caspase 7 (SEQ. ID. NO: 65) expression vector

An expression vector over-expressing L191F/23TS/198TS Caspase 7 (SEQ. ID. NO: 65) was constructed by using the vector over-expressing 23TS/198TS Caspase 7 (SEQ. ID. NO: 64) prepared in Example <2-12> as a template with 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) by the site directed mutagenesis method described in Example <2-6>,

<2-15> L191W/23TS/198TS Caspase 7 (SEQ. ID. NO: 66) expression vector

An expression vector over-expressing L191W/23TS/198TS Caspase 7 (SEQ. ID. NO: 66) was constructed by using the vector over-expressing 23TS/198TS Caspase 7 (SEQ. ID. NO: 64) prepared in Example <2-12> as a template with 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) by the site directed mutagenesis method described in Example <2-6>.

<2-16> 23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 67) expression vector

An expression vector expressing 23TS/D198A/203TI Caspase 7, the recombinant caspase 7 precursor in which a peptide comprising 10 consecutive amino acids including the 23^rd amino acid of caspase 7 was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin and 10 consecutive amino acids recognized and digested by thrombin were inserted in between the 203^rd amino acid and the 204^th amino acid of caspase 7 was constructed.

PCR was performed 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) and a reverse primer (SEQ. ID. NO: 23) using the vector over-expressing 23TS caspase 7 (SEQ. ID. NO: 63) of Example <2-12> as a template. Then, the second PCR was performed using a forward primer (SEQ. ID. NO: 21) and the PCR product as megaprimer. The final PCR product was introduced into pET21a expression vector (Novagen, USA) according to the method of Example <2-l> and as a result, an expression vector over-expressing 23TS/203TI Caspase 7 was constructed.

PCR was performed another way 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) . Then, the second PCR was performed using a reverse primer (SEQ. ID. NO: 23) and the PCR product as megaprimer. The final PCR product was introduced into pET21a expression vector (Novagen, USA) according to the method of Example <2-l> and as a result, an expression vector over-expressing 23TS/203TI Caspase 7 was constructed.

An expression vector over-expressing 23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 67) was constructed using the vector over-expressing 23TS/203TI Caspase 7 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 GAT GCC ATC) by replacing the 19^th amino acid (aspartic acid) with alanine by the site directed mutagenesis method described in Example <2-6>.

<2-17> L191F/23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 68) expression vector An expression vector over-expressing L191F/23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 68) was constructed using the expression vector over-expressing 23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 67) of Example <2-14> as a template with a forward primer (SEQ. ID. NO: 36: TGC CGA GGG ACC GAG TTT GAT GAT GGC ATC CAG GCC) and a reverse primer (SEQ. ID. NO: 37: CTG GAT GCC ATC ATC AAA CTC GGT CCC TCG GCA AGC) by the site directed mutagenesis method described in Example <2-6>.

<2-18> L191W/23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 69) expression vector

An expression vector over-expressing L191W/23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 69) was constructed using the expression vector over-expressing 23TS/D198A/203TI Caspase 7 (SEQ. ID. NO: 67) of Example <2-14> as a template with a forward primer (SEQ. ID. NO: 38: TGC CGA GGG ACC GAG TGG GAT GAT GGC ATC CAG GCC) and a reverse primer (SEQ. ID. NO: 39: CTG GAT GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC) by the site directed mutagenesis method described in Example <2-β>.

Example 3: Recombinant caspase precursor expression

E. coli was transfected with the vectors over- expressing recombinant caspase 3 precursors and recombinant caspase 7 precursors obtained in Examples 1 and 2 by the method of Hanahan (Hanahan D, DNA Cloning vol .1 109-135, IRS press 1985) .

Particularly, E. coli Rosetta DE3 (Novagen, USA) treated with CaCl₂ was transfected with the vectors of Examples 1 - 2 by heat shock method, followed by culture in media containing ampicillin or kanamycin. Colonies having resistance against ampicillin or kanamycin were selected. The colonies were inoculated in LB media (containing 30 /Jg/ ι\ύ kanamycin or 50 βg/mt ampicillin; Sigma, USA) , followed by culture at 37^°C for 16 hours. Some of the culture solution was inoculated in LB media (containing 30

kanamycin or 50

ampicillin; Sigma, USA) . When ODδoo of the culture solution reached 0.7 - 0.9, IPTG was added thereto, followed by further culture for 18 hours at 18^°C to induce the expression of the recombinant gene. Upon completion of the culture, the culture solution was centrifuged and the precipitated cells were resuspended in 25 mf of lysis buffer (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol), followed by cell lysis by ultrasonicator . The lysate was centrifuged. From the supernatant, the recombinant caspase precursor was purified by batch method using talon resin (Clontech, USA) .

Protein purification by batch method is described in detail hereinafter. Talon resin (1 mi) was washed twice with 10 ill? of column buffer (50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 0.05% β-mercaptoethanol ) to activate it. The supernatant was added thereto, followed by shaking at 4 ^°C for 3 hours to absorb proteins onto talon resin. The aqueous solution was eliminated by filtering. Then, talon resin was washed twice with column buffer (10 ill?)_/ to which 10 Hi-C of elution buffer (100 mM imidazole) was added, followed by rocking for one hour. After filtering, the recombinant caspase precursor was recovered. The above process was repeated again by using 5 ill<? of elution buffer to increase yield. The protein purified by the above process was quantified by Bradford assay and the protein size was measured by 15% SDS-PAGE gel electrophoresis to confirm the over-expression and purification of the recombinant caspase precursor 3. As a result, as shown in Figure 3, lane 4, the recombinant caspase 3 precursor expression was increased by replacing the recognition site comprising β consecutive amino acids including the 28^th amino acid of a recombinant caspase 3 and another recognition site comprising 6 consecutive amino acids including the 175^th amino acid of a recombinant caspase 3 with the thrombin recognition site.

Example 4: Measurement of level of actication of the purified recombinant caspase precursor activity After activating the recombinant caspase 3 precursor or the recombinant caspase 7 precursor with substitution with thrombin recognition site, prepared in Examples 2 and 3, their enzyme activities were measured by SDS-PAGE.

<4-l> Recombinant caspase 3

To measure the concentration of the purified protein, Bradford assay was performed and OD₅₉₅ was measured. Thrombin was reacted with Sulfo-NHS ester group conjugated biotin (Pierce, USA) at room temperature, which proceeded to desalting column (Amersham Biosciences, USA) to eliminate non-reacted biotin.

1 nig/mC of the recombinant caspase 3 precursor (SEQ. ID. NO: 53) prepared by the method of Example 2 or 3 was mixed with biotin conjugated thrombin (2 NIH, 20 NIH and 40 NIH units per 200 βg of caspase), followed by activation at 4^°C or 18°C. Level of activation was measured by SDS-PAGE and using caspase substrate respectively 4, 8 and 24 hours after the treatment. Upon completion of the activation, 1 mi of avidin resin was added to the reactant, followed by slow rocking at 18^°C for 1 hour, by which biotin conjugated thrombin was absorbed and eliminated. To confirm whether the above activation was successfully performed, the reaction solution was recovered at each hour and the activity was measured by 15% SDS-PAGE. As a result, as shown in Figure 4, the recombinant caspase 3 precursor of the present invention (SEQ. ID. NO: 53) was converted into an active caspase by thrombin mediated hydrolysis at 18^°C for 8 hours or at 4 ^°C for 24 hours. And it was also confirmed from the above result that the preferable dosage of thrombin was at least 40 NIH units regardless of reaction temperature.

<4-2> Recombinant caspase 7

Recombinant caspase 7 precursors (SEQ. ID. NO: 97 and NO: 64) were expressed and purified by the same manner as described in Example 3. The precursors were activated by using 40 NIH units of thrombin per 200 βg of caspase by the same manner as described in Example <4-l>. Then, the enzyme activity was measured by SDS-PAGE. As a result, as shown in Figure 5, the recombinant caspase 7 precursors of the present invention (SEQ. ID. NO: 97 and NO: 64) were converted into active caspases at 4 ^°C or at 18 ^°C .

Example 5: Enzyme kinetics of active caspase

After confirming the completion of hydrolysis of the recombinant caspase 3 precursors prepared by the method of Example 2 or 3 on SDS-PAGE gel according to the method of Example 4, the enzyme activity of caspase was measured by using a substrate.

Enzyme reaction was performed in 50 mM HEPES buffer (pH 7.5, 50 mM KCl, 2 mM MgCl₂, 1 mM EDTA, 10 mM DTT) using Ac-DEVD-pNA (pNA: para-nitroanilide; Anaspec, USA) , known as a substrate for caspase 3. To calculate K_n, and k_cat, the enzyme kinetic parameters, 20 βi of the active caspase prepared in Example 4 and the recombinant caspase precursors prepared in Examples 2 and 3 were added to 480 jΛ of substrates at different concentrations, diluted with the buffer, followed by measuring OD_40S by UV/VIS spectrometer (Beckman Coulter, USA) to measure the hydrolysis kinetics of the substrate. Based on the obtained values, the enzyme kinetic parameters were calculated using HYPER32.EXE, Version 1.0.0 (2003 for 32 bit Versions of MS Windows, Copyright J S Easterby, Freeware; //homepage . ntlworid. com/John. easterby/hyper32. html ) .

As a result, as shown in Table 2, k_cat/K_m (enzyme kinetics) of the recombinant caspase 3 in which 6 consecutive amino acids recognized and digested by thrombin were inserted in between the 180^th amino acid and the 181^st amino acid of caspase 3 (28TS/180TI, L168F/28TS/D175A/180TI or Ll 68W/28TS/D175A/180TI ) was significantly higher than that of the recombinant caspase 3 in which a peptide comprising 6 consecutive amino acids including the 175^th amino acid of caspase 3 was replaced with another peptide comprising consecutive amino acids recognized and digested by thrombin (Δ28TS/175TS, 28TS/175TS or L168F/28TS/175TS) .

K_cat/Km (enzyme kinetics) of the recombinant caspase 3 of the present invention is lower than that of wild type caspase (SEQ. ID. NO: 49), but it is expressed as an inactive form in E. coli, suggesting that it does not exhibit enzyme activity before being treated with thrombin, so that the caspase 3 can be mass-produced in E. coli.

[Table 2]

Enzyme kinetic parameters of caspase precursors and active caspases

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims,

Claims

[CLAIMS]

[Claim l]

A recombinant caspase precursor in which a cysteine protease recognition site of caspase is replaced with a non-cysteine protease recognition site.

[Claim 2]

The recombinant caspase precursor according to claim 1, wherein the caspase is selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9.

[Claim 3]

The recombinant caspase precursor according to claim 1, wherein the non-cysteine protease is the peptide composed of 4 - 10 consecutive amino acids.

[Claim 4]

The recombinant caspase precursor according to claim 1, wherein the non-cysteine protease recognition site is selected from the group consisting of 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 Factor Xa recognition site (SEQ. ID. NO: 93: IEGR or SEQ. ID. NO: 94: IDGR) .

[Claim 5]

The recombinant caspase precursor according to claim 1, wherein the non-cysteine protease is selected from the group consisting of thrombin, enterokinase, TEV and Factor Xa.

[Claim 6] The recombinant caspase precursor according to claim

1, wherein the cysteine protease recognition site is the peptide composed of 4 - 10 consecutive amino acids including aspartic acid in the middle.

[Claim 7]

The recombinant caspase precursor according to claim

2, wherein the caspase 3 has the replacement of the site ranging from the 23^rd amino acid to 32^nd amino acid (SEQ. ID. NO: 95) or from the 170^th amino acid to 179^th amino acid (SEQ. ID. NO: 96) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease.

[Claim 8] The recombinant caspase precursor according to claim 2, wherein the caspase 7 has the replacement of the site ranging from the 19^th amino acid to 28^th amino acid (SEQ. ID. NO: 81) or from the 194^th amino acid to 203^rd amino acid (SEQ. ID. NO: 83) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease .

[Claim 9]

The recombinant caspase precursor according to claim 2, wherein the caspase 2 has the replacement of the site ranging from the 162^nd amino acid to 171^st amino acid (SEQ. ID. NO: 84), from the 297^th amino acid to 306^th amino acid (SEQ. ID. NO: 85) or from the 319^th amino acid to 328^th amino acid (SEQ. ID. NO: 86) with a peptide comprising consecutive amino acids recognized and digested by non- cysteine protease.

[Claim 10]

The recombinant caspase precursor according to claim 2, wherein the caspase 6 has the replacement of the site ranging from the 19^th amino acid to 28^th amino acid (SEQ. ID. NO: 87) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease.

[Claim 11] The recombinant caspase precursor according to claim 2, wherein the caspase 9 has the replacement of the site ranging from the 134^th amino acid to 143^rd amino acid (SEQ. ID. NO: 88), from the 301^st amino acid to 310^th amino acid (SEQ. ID. NO: 89) or from the 311^th amino acid to 320^th amino acid (SEQ. ID. NO: 90) with a peptide comprising consecutive amino acids recognized and digested by non- cysteine protease.

[Claim 12]

A recombinant caspase 3 precursor prepared by replacing a peptide comprising 6 consecutive amino acids including the 28^th amino acid of caspase 3 (SEQ. ID. NO:

78: ESMDSG; 25^th - 30^th amino acids) with another peptide comprising 6 consecutive amino acids recognized and digested by thrombin (SEQ. ID. NO: 79: LVPRGS) and 6 consecutive amino acids recognized and digested by thrombin

(SEQ. ID. NO: 79: LVPRGS) are inserted in between the 180^th amino acid and the 181^st amino acid.

[Claim 13]

A recombinant caspase precursor prepared by replacing one or more amino acid residues of a cysteine protease recognition site of caspase with amino acids which cannot be recognized by cysteine protease and inserting a non- cysteine protease recognition site around the cysteine protease recognition site.

[Claim 14] The recombinant caspase precursor according to claim 13, wherein the caspase is selected from the group consisting of caspase 2, caspase 3, caspase 6, caspase 7 and caspase 9.

[Claim 15]

The recombinant caspase precursor according to claim 13, wherein the non-cysteine protease is the peptide composed of 4 - 10 consecutive amino acids.

[Claim 16]

The recombinant caspase precursor according to claim 13, wherein the non-cysteine protease recognition site is selected from the group consisting of 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 Factor Xa recognition site (SEQ. ID. NO: 93: IEGR or SEQ. ID. NO: 94: IDGR) .

[Claim 17] The recombinant caspase precursor according to claim 13, wherein the non-cysteine protease is selected from the group consisting of thrombin, enterokinase, TEV and Factor Xa.

[Claim 18]

The recombinant caspase precursor according to claim

13, wherein the cysteine protease recognition site is the peptide composed of 4 - 10 consecutive amino acids including aspartic acid in the middle.

[Claim 19]

The recombinant caspase precursor according to claim

14, wherein the caspase 3 has the replacement of the site ranging from the 23^rd amino acid to 32^nd amino acid (SEQ. ID.

NO: 95) or from the 170^th amino acid to 179^th amino acid (SEQ. ID. NO: 96) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease .

[Claim 20]

The recombinant caspase precursor according to claim 14, wherein the caspase 7 has the replacement of the site ranging from the 19^th amino acid to 28^th amino acid (SEQ. ID. NO: 81) or from the 194^th amino acid to 203^rd amino acid (SEQ. ID. NO: 83) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease .

[Claim 21]

The recombinant caspase precursor according to claim 14, wherein the caspase 2 has the replacement of the site ranging from the 162^nd amino acid to 171^st amino acid (SEQ. ID. NO: 84), from the 297^th amino acid to 306^th amino acid (SEQ. ID. NO: 85) or from the 319^th amino acid to 328^th amino acid (SEQ. ID. NO: 86) with a peptide comprising consecutive amino acids recognized and digested by non- cysteine protease.

[Claim 22]

The recombinant caspase precursor according to claim 14, wherein the caspase 6 has the replacement of the site ranging from the 19^th amino acid to 28^th amino acid (SEQ. ID. NO: 87) with a peptide comprising consecutive amino acids recognized and digested by non-cysteine protease.

[Claim 23]

The recombinant caspase precursor according to claim 14, wherein the caspase 9 has the replacement of the site ranging from the 134^th amino acid to 143^rd amino acid (SEQ. ID. NO: 88), from the 301^st amino acid to 310^th amino acid (SEQ. ID. NO: 89) or from the 311^th amino acid to 320^th amino acid (SEQ. ID. NO: 90) with a peptide comprising consecutive amino acids recognized and digested by non- cysteine protease.

[Claim 24]

The recombinant caspase precursor according to claim 13, wherein the amino acid that cannot be recognized by cysteine protease is any amino acid except aspartic acid.

[Claim 25]

The recombinant caspase precursor according to claim 13, wherein the amino acid that cannot be recognized by cysteine protease is selected from the group consisting of phenylalanine, alanine, leucine and tryptophan.

[Claim 26]

The recombinant caspase precursor according to claim 13, wherein the cysteine protease recognition site without substitution of amino acid residues can be replaced with a non-cysteine protease recognition site.

[Claim 27] A polynucleotide encoding anyone of the recombinant caspase precursors of claim 1 - claim 26.

[Claim 28]

An expression vector containing the polynucleotide of claim 27.

[Claim 29]

A transformant transfected with the expression vector of claim 28.

[Claim 30]

A method for preparing a recombinant caspase precursor comprising the following steps:

1) preparing an expression vector containing a polynucleotide encoding one of the recombinant caspase precursors of claim 1 - claim 26;

3) culturing the transformant to induce the expression of the recombinant protein and obtaining thereof.

[Claim 31]

A method for activating the recombinant caspase precursors containing the step of treating anyone of the recombinant caspase precursors of claim 1 - claim 26 with non-cysteine protease.

[Claim 32]

The method according to claim 31, wherein the non- cysteine protease is selected from the group consisting of thrombin, enterokinase, TEV and Factor Xa.

[Claim 33]

A recombinant caspase activated by the method of claim 32.

[Claim 34]

A method for screening a caspase activity inhibitor or activator comprising the following steps: 1) treating the activated recombinant caspase of claim 33 with a caspase-specific substrate and candidates;

2) measuring the enzyme activity of the activated recombinant caspase against the substrate; and,

3) selecting candidates confirmed to inhibit or activate the enzyme activity of the activated recombinant caspase by comparing the activity with that of the control group not treated with candidates.

[Claim 35] A use of the activated recombinant caspase of claim 33 for the screening of a caspase activity inhibitor or enhancer.