KR100512916B1 - Caspase Assay System - Google Patents

Abstract

The present invention relates to an evaluation method capable of measuring the activity of caspase, and inserts DEVD, a recognition amino acid sequence of the caspase-2 / 3/7 enzyme, into a peridot, a variant of the inserted fluorescent protein (InsYFP). Recombinant fluorescent proteins whose fluorescence changed by the activity of caspase-2 / 3/7 enzyme were prepared and named DEVDins. The prepared biosensor, Debdins, was introduced into the CHO-K1 cell line, and the cell lines expressing the same were selected, followed by treatment with okadaic acid, a cell death inducer, at a concentration of 20 mM / ml. The intensity of the fluorescence was analyzed under confocal microscopy. As a result, a biosensor molecule showing a change in caspase activity was invented, and by introducing the biosensor into a cell line, a cell based assay system capable of performing caspase activity evaluation was established. In addition, this study paves the way for the development of model animals for the evaluation of caspase activity using transgenic animals.

Description

Caspase Assay System

In multicellular organisms, programmed cell death is an essential process for development and maintenance of homeostasis. In this process, the cells are fragmented, gene-like waves, and cell contractions, which accumulate in the cytoplasm and are removed by phagocytic activity in the form of small cysts. Therefore, unlike cell necrosis, it does not cause an inflammatory response, thereby performing an important function for normal development. However, excessive cell death causes tissue damage and degenerative brain diseases caused by ischemia, and cancer or autoimmune diseases may occur when gene death by cells is not performed properly (Raff M, Cell suicide for beginners. 1998, 396: 119-122; Jacobson MD, Weil M, Raff MC, Programmed cell death in animal development. Cell 1997, 88: 347-354). Recent studies have shown that the molecular mechanisms of the regulation of cell death are known and, in particular, that the cysteine protease caspase plays an essential role in the multi-step process of cell death (Wolf BB, Green DR, Suicidal tendencies: apoptotic cell death by caspase family proteinases.J Biol Chem 1999, 274: 20049-20052). Cell death is based on cascading information that activates lower-level enzymes as the activities of various proteins occur sequentially. For example, when a ligand that causes cell death, such as tumor necrosis factor, attaches to a receptor due to the development or development of the immune system, a command to cause cell death into the cell is transmitted. 8/10 receive signals from the receptor. Cytochrome C secretion from mitochondria by intracellular stress activates caspase-9 and activates cascade of lower levels. These lower cascades directly induce cell death by altering or removing proteins responsible for gene repair, cell cycle regulation, and cell death inhibition (Green DR, Apoptotic pathways: the roads to ruin. Cell 1998, 94: 695-698; Ashkenazi A, Dixit VM, Death receptors: signaling and modulation.Science 1998, 281: 1305-1308; Green DR, Reed JC, Mitochondria and apoptosis.Science 1998, 281: 1309-1312).

Caspase recognizes the amino acid sequence following aspartic acid and cleaves the protein.In experiments with protein suppressors, caspase-1, 4, 5, 13, etc., WEHD, caspase-2, 3, 7, DEXD, , And caspase-6, 8, 9, 10 are known to show enzymatic activity by recognizing I (/ V / L) EXD. In particular, the well known substrate protein poly (ADP-ribose) polymerase (PARP) is a 115 kDa nucleoprotein that is known to bind DNA by recognizing breaks in DNA strands. DNA disruption activates PARP to produce poly (ADP-ribose), and these ADP-ribose polymers alter various proteins, including PARP. As a result, it induces chromatin decondensation to aid DNA repair (Pieper AA, Verma A, Zhang J, Snyder SH, Poly (ADP-ribose) polymerase, nitric oxide and cell death.Trends Pharmacol Sci 1999, 20 : 171-181). However, when cell death occurs, the cascade cuts the PARP into two fragments of 24 kDa and 89 kDa. In later studies, this step was used as an important marker when the signal of the cell death was cascaded (Lazebnik YA, Kaufmann). SH, Desnoyers S, Poirier GG, Earnshaw WC, Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE.Nature 1994, 371: 346-347). Based on this fact, Xu et al. Used a fluorescence energy transfer phenomenon (FRET), which is generated by placing a DEVD amino acid sequence between a green fluorescence protein (GFP) and a blue fluorescence protein (BFP). The activity of Phase-3 (CPP32) was measured (Xu X, Gerard AL, Huang BC, Anderson DC, Payan DG, Luo Y, Detection of programmed cell death using fluorescence energy transfer.Nucleic Acids Res 1998, 26: 2034-2035 (Bioscience Clontech) has developed a method for measuring caspase-3 activity through the intracellular location of YFP by placing the DEVD amino acid sequence in the NES (nuclear export sequence) (BD biosciences clontech). , PR1Z499W). However, the low signal-to-noise ratio (S / N ratio) was pointed out when measuring caspase activity using FRET, and the expensive equipment was measured by measuring the result of secondary signal when using intracellular protein transfer. Not only must be used, but there have been difficulties in quantifying enzyme activity. Thus, there is a need for the development of an efficient and economical method for measuring the activity of caspase in cells.

 In the present invention, the caspase-2 / 3/7 recognition amino acid sequence is added to the fluorescent protein to prepare a caspase-3 sensor so that the enzyme activity is proportional to the change in fluorescence exposed to the fluorescent protein by the enzymatic activity. The activity of Phase-3 was measured and the level of activity was quantified.

In order to position the amino acid sequence within a single protein, Baird et al. Have reported that the insertion of an external protein at position Tyr-145 of GFP does not affect fluorescence (Baird GS, Zacharias DA, Tsien RY, Circular). permutation and receptor insertion within green fluorescent proteins.Proc Natl Acad Sci USA 1999, 96: 11241-11246).

In the present invention, a peridot that emits a stable and powerful fluorescence as compared to a conventional insertion type fluorescent protein was used as a variant fluorescent protein reported in the patent (10-2002-0012409) filed by NeuroGenex. It inserts the amino acid sequence of the insertion site of YGGSGAS instead of tyrosine, amino acid No. 145 from citrine, a yellow-shifted green fluorescent protein made by substituting five amino acids of S65G, V68L, Q69M, S72A, and T203Y of GFP, and a P192L mutation. Is an added variant protein. Based on this fact, the DEVD amino acid sequence was inserted into the peridot and named DEVDins. The prepared DEVDins were introduced into a CHO-K1 (Chinese hamster ovarian) cell line, and then cell lines expressing DEVDins were made and named CHO-K1-DEVDins. This cell line was treated with a drug that induces cell death, and the activity of caspase-2 / 3/7 was quantitatively measured by changing the intensity of fluorescence.

The present invention is to develop a biosensor capable of measuring the activity of caspase, suitable for high-speed screening systems such as drug screens and introduced into transgenic laboratory animals or for intracellular targeting to quantitatively analyze enzyme activity. It is to develop this useful biosensor.

To this end, the present invention was intended to confirm the maintenance of fluorescence even when an insertion fluorescent protein having an enhanced fluorescence intensity capable of inserting an external gene was introduced and a cleavage site of the cascade substrate was introduced into its external gene insertion site. .

In addition, three recognition sequences were known according to the type of the cascade in the recognition sequence of the cleavage site of the cascade. In the present invention, a recombinant insertion-type protein was prepared using the DEVD recognition sequence and introduced into the cell, followed by selection. It was intended to show that the activity of caspase-2 / 3/7 can be quantitatively measured by treating a drug that induces and changing the fluorescence intensity accordingly.

The present invention is composed of the following configurations.

In the present invention, in the inserted YFP, caspase-1 / 4/5/13 is WEHD, caspase-2 / 3/7 is DEXD, and caspase-6 / 8/9/10 is I (/ Provided is a recombinant fluorescent protein characterized by the insertion of a cascade recognition amino acid sequence distinguished by V / L) EXD.

In addition, by introducing the recombinant fluorescent protein provided by the present invention into the cell can be used for cell-level drug analysis using a variety of cell lines will be a natural fact for those skilled in the art, the production of transgenic animals using the same is also obvious. would.

Recombinant fluorescent protein according to the present invention is an example of practical application by applying the inserted fluorescent protein to the activity measurement and quantification of protease activity is expected to have a ripple effect in various fields such as screens and high-speed evaluation system of various drugs in the future. In particular, DEVDins is a biosensor that can be useful for measuring the activity of caspases in inducing cell death, screening drugs related to caspase activity, cell lines using the recombinant vector, and producing transgenic animals. It will be obvious.

It will be apparent to those skilled in the art that the biosensor provided by the present invention can be introduced into an animal cell to make a cell line and use it for evaluation of caspase activity. In addition, it is also natural to those who are involved in the field of biotechnology that can make an experimental disease model animal capable of evaluating caspase activity by making a transgenic animal using Devdines provided by the present invention.

Figure 1. Fluorescence image obtained by fluorescence microscopy after treatment of CHO-K1-DEVDins with cell death inducer okadaic acid at a concentration of 20 mM / ml

Figure 2. Figure quantified fluorescence image of Figure 1 under confocal microscope

<110> KIM, Kyung Jin <120> Caspase Assay System <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 795 <212> DNA <213> Homo sapiens & Aequorea victoria chimeric protein <220> <221> CDS (222) (1) .. (792) <223> peridot containing recognition sequence of caspase substrate <400> 1 atg gtg agc aag ggc gag gag ctg ttc acc ggg gtg gtg ccc atc ctg 48 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 gtc gag ctg gac ggc gac gta aac ggc cac aag ttc agc gtg tcc ggc 96 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 gag ggc gag ggc gat gcc acc tac ggc aag ctg acc ctg aag ttc atc 144 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 tgc acc acc ggc aag ctg ccc gtg ccc tgg ccc acc ctc gtg act acc 192 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 ttc ggc tac ggc ctg atg tgc ttc gcc cgc tac ccc gac cac atg aag 240 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 cag cac gac ttc ttc aag tcc gcc atg ccc gaa ggc tac gtc cag gag 288 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 cgc acc atc ttc ttc aag gac gac ggc aac tac aag acc cgc gcc gag 336 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 gtg aag ttc gag ggc gac acc ctg gtg aac cgc atc gag ctg aag ggc 384 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 atc gac ttc aag gag gac ggc aac atc ctg ggg cac aag ctg gag tac 432 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 aac tac ggt gga tcc gcc atc aag aat gaa gga aag aga aaa ggc gac 480 Asn Tyr Gly Gly Ser Ala Ile Lys Asn Glu Gly Lys Arg Lys Gly Asp 145 150 155 160 gag gtg gat gga aca gat gaa gtg gcc gct agc aac agc cac aac gtc 528 Glu Val Asp Gly Thr Asp Glu Val Ala Ala Ser Asn Ser His Asn Val 165 170 175 tat atc atg gcc gac aag cag aag aac ggc atc aag gtg aac ttc aag 576 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys 180 185 190 atc cgc cac aac atc gag gac ggc agc gtg cag ctc gcc gac cac tac 624 Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr 195 200 205 cag cag aac acc ccc atc ggc gac ggc ctc gtg ctg ctg ccc gac aac 672 Gln Gln Asn Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn 210 215 220 cac tac ctg agc tac cag tcc gcc ctg agc aaa gac ccc aac gag aag 720 His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys 225 230 235 240 cgc gat cac atg gtc ctg ctg gag ttc gtg acc gcc gcc ggg atc act 768 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 245 250 255 ctc ggc atg gac gag ctg tac aag taa 795 Leu Gly Met Asp Glu Leu Tyr Lys 260 <210> 2 <211> 264 <212> PRT <213> Homo sapiens & Aequorea victoria chimeric protein <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Phe Gly Tyr Gly Leu Met Cys Phe Ala Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Tyr Gly Gly Ser Ala Ile Lys Asn Glu Gly Lys Arg Lys Gly Asp 145 150 155 160 Glu Val Asp Gly Thr Asp Glu Val Ala Ala Ser Asn Ser His Asn Val 165 170 175 Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys 180 185 190 Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr 195 200 205 Gln Gln Asn Thr Pro Ile Gly Asp Gly Leu Val Leu Leu Pro Asp Asn 210 215 220 His Tyr Leu Ser Tyr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys 225 230 235 240 Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 245 250 255 Leu Gly Met Asp Glu Leu Tyr Lys 260

Claims (8)

delete delete delete delete A recombinant fluorescent protein for measuring intracellular caspase activity, having the amino acid sequence of SEQ ID NO: 2. The nucleic acid base sequence of SEQ ID NO: 1 encoding the recombinant fluorescent protein of claim 5. Method for measuring intracellular caspase activity using the recombinant fluorescent protein of claim 5. Method for measuring intracellular caspase activity using the nucleotide sequence of claim 6.