WO2009044988A1 - 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 PDFInfo
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- WO2009044988A1 WO2009044988A1 PCT/KR2008/003589 KR2008003589W WO2009044988A1 WO 2009044988 A1 WO2009044988 A1 WO 2009044988A1 KR 2008003589 W KR2008003589 W KR 2008003589W WO 2009044988 A1 WO2009044988 A1 WO 2009044988A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
Definitions
- the present invention relates to recombinant caspases and methods for preparing the same.
- 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
- 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.
- 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.
- 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) .
- caspase 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.
- 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 .
- 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 ⁇ 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 ;
- 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:
- the present invention provides a use of the activated recombinant caspase for the screening of a caspase activity inhibitor or enhancer.
- 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 .
- 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;
- caspase 3 (1-277); 2: ⁇ 28 caspase 3; 3: ⁇ 28/175TS 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 ⁇ g of caspase protein reacted with 2 NIH units of thrombin
- Lanes 3 and 7 200 ⁇ g of caspase protein reacted with 20 NIH units of thrombin
- Lanes 4 and 8 200 ⁇ g 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:
- 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.
- DNA that does not contain information on N-terminal located upstream of the forward primer is obtained.
- This produced DNA is called a megaprimer.
- 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.
- pression 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.
- the present invention provides a recombinant caspase precursor in which cysteine protease recognition site is replaced with a non-cysteine protease recognition site.
- an expression vector expressing a recombinant caspase 3 precursor (28TS, ⁇ 28TS, 175TS, 28TS/175TS, ⁇ 28TS/175TS Caspase 3; ⁇ : 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.
- ESMDSG 25 th - 30 th amino acids
- 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.
- 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) .
- 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.
- 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.
- 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) .
- the caspase 3 auto-activation recognition site 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.
- 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:
- 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.
- 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.
- 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.
- 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.
- 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.
- 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) .
- 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.
- 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:
- 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.
- 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.
- cysteine protease recognition site in which no amino acid residue is replaced can be substituted with a non-cysteine protease recognition site.
- 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.
- 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.
- the present invention also provides a polynucleotide encoding one of the said recombinant caspase precursors.
- 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.
- 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.
- 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:
- 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.
- the present invention also provides a recombinant caspase activated by the above method.
- 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.
- thrombin was treated to activate the recombinant caspase 3 precursor (SEQ. ID.
- the preferable dosage of thrombin in this invention was at least 40 NIH units regardless of reaction temperature .
- 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.
- the present invention also provides a method for screening a caspase activity inhibitor or activator comprising the following steps:
- the caspase specific substrate of step 1) can be Ac- DEVD-pNA (pNA: para-nitroanilide) , Ac-DEVD-AFC (7-amino-4- trifluoromethylcoumarin) , etc.
- 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.
- the present invention provides a use of the activated recombinant caspase for the screening of a caspase activity inhibitor or enhancer.
- Example 1 Vector for over-expression of natural caspase precursor
- 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.
- 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.
- Example 2 Expression vector for a replaced recombinant caspase precursor
- 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 ⁇ Jl of template DNA, 1 ⁇ l of the megaprimer and 20 ⁇ M reverse primer (SEQ. ID.
- PCR was performed another way using a 20 ⁇ M 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) .
- Blast //www . ncbi . nlm. nih . gov
- PCR was performed using a 20 ⁇ M 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 ⁇ 28 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.
- PCR was performed using a 20 ⁇ M 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 ⁇ M 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.
- 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> ⁇ 28/175TS caspase 3 (SEQ. ID. NO: 55) expression vector
- PCR was performed using a 20 ⁇ M 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 ⁇ 28/175TS 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.
- PCR was performed with a 20 ⁇ M 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.
- 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 ⁇ M forward primer (SEQ. ID. NO: 9: GCC TGC CGT GGT ACA GAA TTC GAC TGT GGC ATT GAG) and reverse primer (SEQ. ID.
- 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>.
- a forward primer SEQ. ID. NO: 11: TGC CGT GGT ACA GAA TGG GAC TGT GGC CTG GTG CCG
- reverse primer SEQ. ID. NO: 12: CAC CAG GCC ACA GTC CCA TTC TGT ACC ACG GCA GGC
- PCR was performed with a 20 ⁇ M 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
- 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 ⁇ M forward primer (SEQ. ID. NO: 14: ACA GAC AGT GGT GTT GAT GAT CTG
- 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.
- 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) .
- 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) .
- 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) .
- the negative control was prepared by the method of
- 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. 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
- 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.
- an expression vector over-expressing 23TS Caspase 7 (SEQ. ID.
- 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) .
- 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.
- caspase 7 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) .
- SEQ. ID. NO: 27 forward primer
- SEQ. ID. NO: 23 reverse primer
- 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.
- An expression vector over-expressing L191F/23TS/198TS Caspase 7 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>,
- 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>.
- a forward primer (SEQ. ID. NO: 30: TGC CGA GGG ACC GAG TGG GAT GAT GGC CTG GTG CCG)
- a reverse primer SEQ. ID. NO: 31: CAC CAG GCC ATC ATC CCA CTC GGT CCC TCG GCA AGC) by the
- 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.
- 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) .
- 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>.
- a forward primer SEQ. ID. NO: 32: GAT GGC ATC CAG GCC GAG TCG GGG CCC ATC AAT GAC
- a reverse primer SEQ. ID. NO: 33: GTC ATT GAT GGG CCC CGA CTC GGC CTG GAT GCC ATC
- 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>.
- a forward primer (SEQ. ID. NO: 36: TGC CGA GGG ACC GAG TTT GAT GAT GGC ATC CAG GCC) and
- 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- ⁇ >.
- a forward primer SEQ. ID. NO: 38: TGC CGA GGG ACC GAG TGG GAT GAT GGC ATC CAG GCC
- a reverse primer SEQ. ID. NO: 39: CTG GAT GCC ATC ATC CCA CTC GGT CCC TCG G
- 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) .
- E. coli Rosetta DE3 (Novagen, USA) treated with CaCl 2 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/ ⁇ kanamycin or 50 ⁇ g/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) .
- OD ⁇ oo 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.
- 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) .
- 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% ⁇ -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.
- column buffer 50 mM Tris pH 7.5, 200 mM NaCl, 5% glycerol, 0.05% ⁇ -mercaptoethanol
- 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.
- lane 4 the recombinant caspase 3 precursor expression was increased by replacing the recognition site comprising ⁇ 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.
- 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 ⁇ g 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.
- biotin conjugated thrombin 2 NIH, 20 NIH and 40 NIH units per 200 ⁇ g of caspase
- the reaction solution was recovered at each hour and the activity was measured by 15% SDS-PAGE.
- the recombinant caspase 3 precursor of the present invention SEQ. ID. NO: 53
- thrombin mediated hydrolysis 18 ° C for 8 hours or at 4 ° C for 24 hours.
- the preferable dosage of thrombin was at least 40 NIH units regardless of reaction temperature.
- 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 ⁇ g 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 .
- Enzyme reaction was performed in 50 mM HEPES buffer (pH 7.5, 50 mM KCl, 2 mM MgCl 2 , 1 mM EDTA, 10 mM DTT) using Ac-DEVD-pNA (pNA: para-nitroanilide; Anaspec, USA) , known as a substrate for caspase 3.
- the enzyme kinetic parameters 20 ⁇ i of the active caspase prepared in Example 4 and the recombinant caspase precursors prepared in Examples 2 and 3 were added to 480 j ⁇ 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 ) .
- 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 ( ⁇ 28TS/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.
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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 28th 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 180th amino acid and the 181st 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: 1st - 28th amino acids were eliminated) in which a peptide comprising 6 consecutive amino acids including the 28th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG: 25th - 30th amino acids) and/or a peptide comprising 6 consecutive amino acids including the 175th amino acid (SEQ. ID. NO: 80: IETDSG; 172nd - 177th 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 28th amino acid of the recombinant caspase 3 precursor and the recognition site comprising 6 amino acids including the 175th 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 23rd amino acid of caspase 7 (SEQ. ID. NO: 81: EDSVDAKPDR; 19th - 28th 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 175th amino acid (SEQ. ID. NO: 83: GIQADSGPIN; 194th - 203rd 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, kcat/Km 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) 162nd ammo acid - 171st amino acid (SEQ. ID. NO: 84), 297th ammo acid - 306th amino acid (SEQ. ID. NO: 85) and 319th amino acid - 328th amino acid (SEQ. ID. NO: 86) of full length caspase 2 (SEQ. ID. NO: 73) ;
(2) 23rd amino acid - 32nd amino acid (SEQ. ID. NO: 95) and 170th amino acid - 179th amino acid (SEQ. ID. NO: 96) of full length caspase 3 (SEQ. ID. NO: 74);
(3) 19th amino acid - 28th amino acid (SEQ. ID. NO: 87) of full length caspase 6 (SEQ. ID. NO: 75);
(4) 19th amino acid - 28th amino acid (SEQ. ID. NO: 81) and 194th amino acid - 203rd amino acid (SEQ. ID. NO: 83) of full length caspase 7 (SEQ. ID. NO: 76) ; and,
(5) 134th amino acid - 143rd amino acid (SEQ. ID. NO: 88), 301st amino acid - 310th amino acid (SEQ. ID. NO: 89) and 311th amino acid - 320th 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 28th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG; 25th amino acid - 30th 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 180th amino acid and the 181st 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 28th 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 180th amino acid and the 181st 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 28th amino acid of caspase 3 and a peptide comprising 6 consecutive amino acids including the 175th 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.
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.
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 168th 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 168th amino acid of 28TS/180TI caspase 3 (SEQ. ID. NO: 54) with phenylalanine or with tryptophan, and by replacing aspartic acid, the 175th 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) 162nd amino acid - 171st amino acid (SEQ. ID. NO: 84), 297th amino acid - 306th amino acid (SEQ. ID. NO: 85) and 319th amino acid - 328th amino acid (SEQ. ID. NO: 86) of full length caspase 2 (SEQ. ID. NO: 73); (2) 23rd amino acid - 32nd amino acid (SEQ. ID. NO: 95) and 170th amino acid - 179th amino acid (SEQ. ID. NO: 96) of full length caspase 3 (SEQ. ID. NO: 74);
(3) 19th amino acid - 28th amino acid (SEQ. ID. NO: 87) of full length caspase 6 (SEQ. ID. NO: 75); (4) 19th amino acid - 28th amino acid (SEQ. ID. NO: 81) and 194th amino acid - 203rd amino acid (SEQ. ID. NO: 83) of full length caspase 7 (SEQ. ID. NO: 76); and,
(5) 134th amino acid - 143rd amino acid (SEQ. ID. NO:
88), 301st amino acid - 310th amino acid (SEQ. ID. NO: 89) and 311th amino acid - 320th 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 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.
The present invention also provides a polynucleotide encoding one of the said recombinant caspase precursors.
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 28th and/or the 175th 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 23rd amino acid and/or the 198th 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.
The present invention also provides a recombinant caspase activated by the above method.
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.
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 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.
In addition, the present invention provides a use of the activated recombinant caspase for the screening of a caspase activity inhibitor or enhancer.
[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 28th amino acid of caspase 3 (SEQ. ID. NO: 78: ESMDSG: 25th - 30th 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 28th amino acid. Finally, the vector over-expressing 28TS caspase 3 (SEQ. ID. NO: 51), the recombinant caspase 3 precursor in which the 28th 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 1st amino acid to the 28th 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 1st amino acid to the 28th 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 175th amino acid of caspase 3 (SEQ. ID. NO: 80: IETDSG: 172nd -
177th 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 175th 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 28th amino acid of caspase 3 was eliminated and a peptide comprising 6 consecutive amino acids including the 175th 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 28th amino acid of caspase 3 was eliminated and the 175th 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 28th amino acid of caspase 3 and a peptide comprising 6 consecutive amino acids including the 175th 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 28th and the 175th 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 28th 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 180th amino acid and the 181st 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 175th 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 163rd 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 23rd amino acid of caspase 7 (SEQ. ID. NO: 81: EDSVDAKPDR: 19th - 28th 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 175th amino acid of caspase 7 (SEQ. ID. NO: 83: GIQADSGPIN: 194th - 203rd 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 23rd 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 198th 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
23rd 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 198th 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 23rd 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 203rd amino acid and the 204th 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 19th 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 28th amino acid of a recombinant caspase 3 and another recognition site comprising 6 consecutive amino acids including the 175th 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 Kn, and kcat, 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 OD40S 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, kcat/Km (enzyme kinetics) of the recombinant caspase 3 in which 6 consecutive amino acids recognized and digested by thrombin were inserted in between the 180th amino acid and the 181st 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 175th 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) .
Kcat/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 23rd amino acid to 32nd amino acid (SEQ. ID. NO: 95) or from the 170th amino acid to 179th 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 19th amino acid to 28th amino acid (SEQ. ID. NO: 81) or from the 194th amino acid to 203rd 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 162nd amino acid to 171st amino acid (SEQ. ID. NO: 84), from the 297th amino acid to 306th amino acid (SEQ. ID. NO: 85) or from the 319th amino acid to 328th 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 19th amino acid to 28th 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 134th amino acid to 143rd amino acid (SEQ. ID. NO: 88), from the 301st amino acid to 310th amino acid (SEQ. ID. NO: 89) or from the 311th amino acid to 320th 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 28th amino acid of caspase 3 (SEQ. ID. NO:
78: ESMDSG; 25th - 30th 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 180th amino acid and the 181st 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 23rd amino acid to 32nd amino acid (SEQ. ID.
NO: 95) or from the 170th amino acid to 179th 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 19th amino acid to 28th amino acid (SEQ. ID. NO: 81) or from the 194th amino acid to 203rd 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 162nd amino acid to 171st amino acid (SEQ. ID. NO: 84), from the 297th amino acid to 306th amino acid (SEQ. ID. NO: 85) or from the 319th amino acid to 328th 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 19th amino acid to 28th 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 134th amino acid to 143rd amino acid (SEQ. ID. NO: 88), from the 301st amino acid to 310th amino acid (SEQ. ID. NO: 89) or from the 311th amino acid to 320th 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;
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.
[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.
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US6348572B1 (en) * | 1996-11-12 | 2002-02-19 | Merck Frosst Canada & Co. | Ligands for phosphatase binding assay |
US20020034812A1 (en) * | 1998-07-17 | 2002-03-21 | Craen Marc Van De | Caspase homologue |
US20020045205A1 (en) * | 1998-07-27 | 2002-04-18 | Heinrikson Robert Leroy | Method for autoactivation of procaspase 8 |
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US6348572B1 (en) * | 1996-11-12 | 2002-02-19 | Merck Frosst Canada & Co. | Ligands for phosphatase binding assay |
US20020034812A1 (en) * | 1998-07-17 | 2002-03-21 | Craen Marc Van De | Caspase homologue |
US20020045205A1 (en) * | 1998-07-27 | 2002-04-18 | Heinrikson Robert Leroy | Method for autoactivation of procaspase 8 |
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