WO1998008938A1

WO1998008938A1 - Proteins having telomerase activity

Info

Publication number: WO1998008938A1
Application number: PCT/JP1997/002976
Authority: WO
Inventors: Shonen Yoshida
Original assignee: Shonen Yoshida
Priority date: 1996-08-28
Filing date: 1997-08-27
Publication date: 1998-03-05
Also published as: AU4030697A

Abstract

Proteins having a telomerase activity and being useful in screening telomerase inhibitors, developing diagnostic methods with the use of antibodies, etc. Conjugated proteins including proteins having a telomerase activity which have the following physicochemical properties: function and substrate specificity: catalyzing the elongation of the telomere DNA 3'-OH terminal of chromosomes of eukaryotes; molecular weight: ranging from about 40 to about 120 kD, in particular, about 110, 58 and/or 45kD in some cases, as determined by SDS-PAGE; inactivation: inactivated by treating with RNase; and characteristic: binding to mouse telomerase RNA.

Description

Description Protein with telomerase activity

Technical field

The present invention relates to a protein having telomerase activity. More specifically, the present invention relates to a protein having telomerase activity purified from a rat-derived cultured cell. Background art

Telomerase is known to be an enzyme that catalyzes the extension of the telomere terminal (terminal portion of a linear chromosome), and many studies have been made (Greider CWand Blackburn EH, (1987) Cell, 51 Morine GB (1989) Cell, 59.521-529). In eukaryotic cells, telomerase is an enzyme containing type I RNA complementary to the 5 'TT AGGG3' of the telomere DNA sequence, and elongates the single-stranded telomere DNA based on type II RNA. This is an enzyme known as a reverse transcriptase that forms a single strand of 5 '(TTAGGG) η3' at the 3 'end of linear DNA in eukaryotic cells. This is the primer for the telomerase reaction.

Telomerase activity is not detected in normal cells except for some cells such as hematopoietic stem cells, but strong telomerase activity can be detected in most cancer tissues.Thus, telomerase is involved in maintaining infinite growth of cancer cells. Conceivable.

Since telomerase activity is selectively detected in cancer cells as described above, it is worth paying attention as a target of anticancer drugs.

The telomerase enzyme protein has not been purified for a long time throughout the whole organism, but has recently been purified with tetrahymena telomerase and its cDNA has also been cloned (K. Collins et al., Cell, Vol. 81, p. .677-686 (1995)).

However, a protein having telomerase activity purified and isolated from mammals such as rats or humans has not yet been reported. Disclosure of the invention

An object of the present invention is to provide a novel protein having telomerase activity, particularly a protein having telomerase activity derived from a mammal. Another object of the present invention is to establish a method for isolating and purifying a protein having telomerase activity using the TRAP-SPA method, which is one of the methods for measuring telomerase activity.

In order to solve the above problems, the present inventors have diligently studied purification conditions for purifying a protein having telomerase activity from rat-derived hepatocytes as a raw material. As a result, the present inventor succeeded in obtaining a fraction having relatively high telomerase activity by performing a combination of ammonium sulfate precipitation and a specific column chromatography purification treatment. completed.

That is, the present invention provides a complex protein comprising a protein having telomerase activity having the following physicochemical properties:

Action and substrate specificity: catalyzes elongation of the telomere DNA 3 'OH end of eukaryotic chromosomes:

Molecular weight: about 40 to about 120 kD as determined by SDS-PAGE;

Inactivation: Inactivated by RNase treatment;

Properties: binds to mouse telomerase RNA;

I will provide a.

The molecular weight of the protein having the above telomerase activity, in particular, as determined by SDS-P AGE, about 110 kD, in _c yet another aspect from about 58 kD and Roh or about 45 kD, the invention A protein having telomerase activity, characterized by having the following physicochemical properties:

Action and substrate specificity: catalyzes elongation of the telomere DNA 3 'OH end of eukaryotic chromosomes;

Molecular weight: about 110 kD, about 58 kD or about 45 kD as determined by SDS-PAGE;

Inactivation: Inactivated by RNase treatment;

Properties: Binds mouse telomerase RNA; I will provide a.

The protein having telomerase activity according to the present invention can preferably be obtained by purification from rat hepatocytes.

The protein having telomerase activity according to the present invention is preferably purified by subjecting the cell extract to ammonium sulfate precipitation, gel filtration chromatography, cation exchange chromatography, and then gel filtration chromatography. Can be obtained by

One of the properties of the protein having telomerase activity of the present invention is that it is inactivated by RNase treatment.

This is because telomerase is a lipoprotein (complex of protein and RNA), and the expression of its activity requires the use of an RNA subunit (which plays the role of type I in extending the telomere sequence). to cause. That is, when telomerase is treated with RNAse, its RNA subunit is degraded, thereby losing type II upon elongation of the telomere sequence and inactivated. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the amount of ³ H-uptake in each fraction fractionated by gel filtration chromatography using a Sephacryl S-300 column.

FIG. 2 is a graph showing the amount of ³ H-uptake in each fraction fractionated by cation exchange chromatography using a HyLoad SP column.

FIG. 3 is a graph showing the amount of ³ H-uptake in each fraction fractionated by gel filtration chromatography using a Sephacryl S-400 column. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, examples of the method for preparing the protein having telomerase activity of the present invention will be described.

Telomerase is known to be active in gonads and cancer cells in human tissues, and it has also been reported that living rat hepatocytes have high telomerase activity in both quiescent and proliferative phases. . Therefore, the telomerase of the present invention An active protein can be prepared from a material having such telomerase activity (such as a cultured cell or tissue). For example, in the examples described later, as an example of the present invention, it was prepared from rat AH7974 cells.

The method for preparing the protein having telomerase activity of the present invention from the material having telomerase activity as described above is not limited to a specific method, but generally, inorganic salts (for example, ammonium sulfate, Salting out method using alkali metal sulfate, alkali metal halide, etc., solvent precipitation method using hydrophilic organic solvents (eg, alcohols such as ethanol or isopropyl alcohol, ketones such as acetone), ion exchange resins and various columns It includes the use of a suitable combination of adsorption and desorption methods by chromatography, gel filtration methods, ultrafiltration methods, and use of protein precipitants (eg, nucleic acids, tannins, etc.).

Further, the protein having telomerase activity thus obtained can be further purified by isoelectric focusing, dialysis, electrodialysis, electrophoresis, or the like. For example, a rat hepatocyte extract is salt-broken with 40-60% saturated ammonium sulfate, the precipitate is dissolved in a buffer, filtered, filtered, and then subjected to gel filtration chromatography (eg, Sephacryl S-300 column). Fractionate. The active fraction is fractionated by cation exchange chromatography (flyLoad SP column, etc.). The active fraction was salt-broken with 60% saturated ammonium sulfate, the precipitate was dissolved in a buffer, filtered, and further fractionated by gel-based chromatography (eg, Sephacryl S-400 column). I do. When the active fraction is collected and analyzed by 8% SDS-PAGE, three bands corresponding to about 110 kD, about 58 kD and about 45 kD respectively are shown.

Furthermore, one of the characteristics of the protein having telomerase activity of the present invention is that it binds to mouse telomerase RNA. This binding is measured by adding labeled mouse telomerase RNA to a gel containing a protein having telomerase activity, leaving it to stand for a certain period of time, and performing autoradiography as described in the Examples below. Can be.

Telomerase activity, a key step in the purification and isolation of telomerase Several measurement methods have been reported so far. For example, as for detection of telomerase activity in Tetrahymena, detection of telomerase by a single primer extension assay system has been reported. However, this method had problems with sensitivity, detection time, quantification, and the ability to process large samples. To solve these problems, a measurement method based on the polymerase chain reaction called TRAP (telomeric repeat amplification protocol) was developed (Kim NW et al., (1994) Science, 206, 2011-2015; Piatyszek MA et al., (1995) Meth. Cell Sci; 17, 1-15), improved sensitivity and detection time issues.

However, the TRAP method, because it still requires an analysis of the polyacrylamide gel electrophoresis and HP LC such as by ³² P- labeled reaction product or fluorescence-labeled reaction products, there is a limit to the number of samples that can be measured, ³² There were problems such as handling of P, long operation time, and delay in results due to the need to quantify the intensity of the detected band.

In order to solve the above-mentioned problems, a method called TRAP-SPA has recently been reported as a method for detecting and quantifying telomerase activity quickly and with high sensitivity. Nippon 8-17830).

The present invention provides a novel protein having telomerase activity. The method for measuring telomerase activity in the purification and isolation step is not particularly limited, and any of the above-described methods can be used. be able to. In the following examples, the TRAP-SPA method, which is the most preferable method in that telomerase activity can be measured quickly and with high sensitivity, is used.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples. Example

Example 1: Preparation of CHAPS extract

Six rats of AH7974 cells (volume; 40 to 5 OmL) were suspended in ice-cold phosphate buffer, centrifuged, and the cells were washed twice. Then, wash the cells with ice-cold wash buffer After washing with: (1 OMIM of H ees (p H 7. 5) ; 1. Mg of 5mM C 1 _2;; 1 OmM of KC 1 1 11 ^ 0 felling of trees in), centrifuged, further the precipitate Mr 10111 of. ^ 18 PS cell extraction buffer (1 OmM Tris-HC1 (pH 7.5); lmM MgC12;

PMSF; 5 mM 2-mercaptoethanol; 0.5% CHAPS; 10% glycerol). After leaving on ice for 30 minutes, the mixture was centrifuged at 15,000 rpm for 30 minutes to obtain a supernatant fraction. This fraction had a protein-concentration of 3 Omg / m1X3Om1. This cell extract was used for the following purification. Example 2: Ammonium sulfate precipitation

After adding 1/30 volume of 1 M Tris-HCI (pH 8.3) to the cell extract obtained in Example 1, a 40-60% saturated ammonium sulfate fraction was collected. Subsequently, this fraction was added to buffer D containing 20% glycerol.

is-HC1 (pH 7.5); 1 mM EDTA; 1 mM Na-bisulfate); 0.01% NP-40; 10% glycerol; ImM Benzamidine) 5-1 OmL Dissolve and filter this (0.45

did. The protein concentration of the resulting solution was 42.78 mg / m 1 x 7 ml. Example 3: Gel-based chromatography on Sephacryl S-300 column

The ammonium sulfate fraction obtained in Example 2 was fractionated on a 45 mm × 50 cm Sephacryl S-300 (Pharmacia) column equilibrated with the above buffer D. The obtained fraction was analyzed by the TRAP-SPA method according to the following procedure. TRAP-S PA method, in order to detect specific ³ H uptake Te Romeraze activity for each fraction and those treated with RN ase (RN ase (+) ) and untreated ones (RN ase ( —))). The RNase treatment was performed for each fraction sample.

To 1 was performed by adding 1 ^ 1 RNase (1 Omg / m 1) and incubating at 30 ° C for 10 minutes.

(TRAP—SPA method)

The TS primer (SEQ ID NO: 1) and CX primer (SEQ ID NO: 2) were The DNA was synthesized using a DNA synthesizer manufactured by the company. The CX and TS primers that had been biotinylated were synthesized by coupling the oligonucleotide to the 5 ′ end with a biotin LC biotin-ON ^τΜ phosphoramidite (Clontech). Primers were purified using an ABIOPC column (purification was performed according to the manufacturer's instructions), lyophilized, and resuspended in water treated with DEPC (getyl piocarbonate).

0.01 to 0.1 g of a biotinylated CX primer (Biot-CX) was trapped under a wax layer of a Hot-Start tube (GIBC0-BR).

Next, 20/1 eluate fraction (RN ase treated as or untreated ones), 2 Omm of T ris- HC 1 (p H 8. 3), 1. Mg C 1 2 of 5 mM, 63 mM KC1, 0.005% Tween 20, 1 mM EGTA, 50 M dATP and dCTP, respectively dTTP, 50 M dGTP, 2Ci

^{[Me- 3 H] TTP (Amersham} , 114 C i / mm o 1), 0. 1 g / μ. \ Of BSA, 2 U T aq polymerase, and, 0. 1 g or a predetermined amount of TS primer Was incubated for 30 minutes at room temperature on a wax barrier in a final reaction mixture containing 50.

The mixture is then heated at 90 ° C for 90 seconds to amplify the synthesized telomere oligonucleotide, followed by one cycle at 94 ° C for 30 seconds, 50 ° C for 30 seconds and 72 ° C for 45 seconds. The polymerase chain reaction was performed in 31 cycles. The reaction product (40I) was transferred to a 96-well plate (Wallac), and 501 microparticle fluorofluorospheres coated with streptavidin (1: 4 solution in 0.56 M EDTA) And incubated at 37 ° C. for 10 minutes to bind the biotinylated ³ H-labeled reaction product to streptavidin beads.

Plates were counted on a MicroBeta scintillation counter (Wallac).

FIG. 1 shows the relationship between each fraction and ³ H—uptake amount.

Fractions containing telomerase activity (elution volume: 315-375 mL; fraction numbers 21 to 25 in FIG. 1) were collected. The protein concentration of this fraction was 1.3 mg / m1 x 75 ml. Example 4: Cation exchange chromatography with HyLoad SP column

75 ml of the gel ill overactive fraction obtained in Example 3 was applied to a HiLoad SP column (Pharmacia; 16 mm x 1 cm) equilibrated with buffer D. Eluted. Each fraction was analyzed by the TRAP-SPA method in the same manner as in Example 3. FIG. 2 shows the relationship between each fraction and ³ H—uptake amount.

27.5 mL of a fraction eluted by about 0.1 to 0.3 M KC1 in which telomerase activity was observed (fraction numbers 43 to 47 in FIG. 2) was collected.

The protein contained in this fraction was subjected to ammonium sulfate precipitation (60% saturation). Subsequently, the precipitate was dissolved in 4 mL of buffer D containing 20% glycerol. The protein concentration at this point was 1.0 mg / ml. The sample was then filtered (0.45 //). Example 5: Gel permeation chromatography on Sephacryl S-400 column

Purify the HiLoad column purified sample obtained in Example 4 with buffer D +

The fraction was further fractionated on a 15 mm x 75 cm Sephacryl S-400 (Pharmacia) column equilibrated in the same manner as above. Each fraction was analyzed by the TRAP-SPA method in the same manner as in Example 3, and the relationship between each _c fraction and the amount of ³ H-uptake is shown in FIG.

3 ml of a fraction containing telomerase activity (fraction number 22 in FIG. 3) was collected. The protein concentration of this fraction was 0.289 mgZm1. Example 6: Analysis by SDS-PAGE

The fraction containing telomerase activity obtained in Example 5 (fraction number 22 in FIG. 3) was analyzed by SDS-PAGE using a gel concentration of 8 to 10%.

As a result, a plurality of bands were detected between about 40 and about 120 kD, and particularly, bands at about 110 kD and about 58 kD and about 45 kD were strongly detected. Example 7: Binding of Telomerase RNA to Subunits of Protein Having Telomerase Activity (1) Cloning of mouse telomerase RNA

Using mouse Liver Total RNA (CLONTECH, CATALOG # 64042-1) as a source, the mouse telomerase RNA gene was amplified by RT-PCR and subcloned into the pGEM-3Zf (1) vector Small site (pGEM 3 Z f ZmTR). The 5 'and 3' primers used were

5 '-GGG GTA TTT AAG GTC GAG GGC GGC -3'

5 '-TTG TGA GAA CCG AGT TCC GGG TGC -3'

(SEQ ID NOs: 3 and 4).

(2) Preparation of mouse telomerase RNA

B AMH (as T 7 by Ri sense strand RNA polymerase is synthesized, base Kuta one telomerase RN A gene has been inserted) 1 p GEM3 Z f / mTR cleaved with the in铸型, ³² P label The prepared telomerase RNA was prepared using Riboprobe Combination System-SP6 / T7 (Promega) according to the instructions for use. (Synthesized telomerase RNA contains bases GGG CGA derived from the vector sequence at the 5 'end and 3' end.) AUU CGA GCU CGG UAC CCG and AGG GGA UC are added respectively)

(3) The binding of telomerase RNA to the band separated by SDS-PAGE was analyzed based on the method of Collins et al. (Cell, 81, 677-686).

The fraction containing high telomerase activity (fraction number 22 in FIG. 3) was separated by 8% SDS-PAGE as in Example 6. For the purpose of unwinding the protein, the SDS gel was washed by shaking with a 50% aqueous methanol solution for 15 minutes and then with a 10% aqueous ethanol solution for 4 hours. Furthermore, the gel of 50mM T ris- acetate (ρΗ8 0.), 10: 11 was equilibrated with \ 1 ^ 2 1 _2, 10% glycerol, ^{32 P-labeled} telomerase RNA with YeastRNA (Sigma) was added Was. After standing for a while, the gel was washed and subjected to autoradiography.

As a result, a plurality of bands were detected between about 40 and about 120 kD, and in particular, bands at about 110 kD, about 58 kD, and about 45 kD were strongly detected. Industrial applicability The protein having telomerase activity of the present invention is a novel protein. The protein having telomerase activity of the present invention is useful for screening for telomerase inhibitors, developing diagnostic methods using antibodies, and the like.

Sequence list SEQ ID NO: 1

Array length: 1 8

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acids (synthetic DNA) Sequence characteristics:

More information: TS Primer

Array:

AATCCGTCGA GCAGAGTT SEQ ID NO: 2

Array length: 2 4

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acids (synthetic DNA) Sequence characteristics

More information: CX Primer

Array:

CCCTTACCCT TACCCTTACC CTAA SEQ ID NO: 3

Array length: 2 4

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear Sequence type: Other nucleic acids (synthetic DNA) Column K:

GGGGTATTTA AGGTCGAGGG CGGC SEQ ID NO: 4

Array length: 24

Sequence type: nucleic acid

Number of chains: single strand

Topology: linear

Sequence type: Other nucleic acid (synthetic DNA) Sequence:

TTGTGAGAAC CGAGTTCCGG GTGC

Claims

The scope of the claims

1. A complex protein comprising a protein having telomerase activity having the following physicochemical properties:

Action and substrate specificity: catalyzes the extension of the telomere DNA 3 'OH terminus of eukaryotic chromosomes;

Molecular weight: about 40 to about 120 kD as determined by SDS-PAGE;

Inactivation: Inactivated by RNase treatment;

Properties: Binds mouse telomerase RNA;

2. The composite protein according to claim 1, wherein the molecular weight of the protein having telomerase activity is about 110 kD, about 58 kD and / or about 45 kD as measured by SDS-PAGE.

3. A protein having telomerase activity, characterized by having the following physicochemical properties:

Inactivation: Inactivated by RNase treatment:

Properties: binds to mouse telomerase RNA;

4. The protein according to any one of claims 1 to 3, wherein the protein is derived from rat hepatocytes.

5. The cell extract can be obtained by subjecting it to ammonium sulfate precipitation, gel filtration chromatography, cation exchange chromatography, and then gel-based chromatography to purify the cell extract. 5. The protein according to any one of 1 to 4.