WO1998008938A1 - Proteines ayant une activite de telomerase - Google Patents

Proteines ayant une activite de telomerase Download PDF

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Publication number
WO1998008938A1
WO1998008938A1 PCT/JP1997/002976 JP9702976W WO9808938A1 WO 1998008938 A1 WO1998008938 A1 WO 1998008938A1 JP 9702976 W JP9702976 W JP 9702976W WO 9808938 A1 WO9808938 A1 WO 9808938A1
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Prior art keywords
protein
telomerase
telomerase activity
fraction
sds
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PCT/JP1997/002976
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English (en)
Japanese (ja)
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Shonen Yoshida
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Shonen Yoshida
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Priority to AU40306/97A priority Critical patent/AU4030697A/en
Publication of WO1998008938A1 publication Critical patent/WO1998008938A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)

Definitions

  • 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.
  • 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).
  • 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) ⁇ 3' 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.
  • telomerase activity is selectively detected in cancer cells as described above, it is worth paying attention as a target of anticancer drugs.
  • 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)).
  • 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.
  • the present invention provides a complex protein comprising a protein having telomerase activity having the following physicochemical properties:
  • Inactivation Inactivated by RNase treatment
  • 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;
  • Inactivation Inactivated by RNase treatment
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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 iso
  • the protein having telomerase activity thus obtained can be further purified by isoelectric focusing, dialysis, electrodialysis, electrophoresis, or the like.
  • 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.
  • 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.
  • TRAP telomeric repeat amplification protocol
  • 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.
  • 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.
  • the TRAP-SPA method which is the most preferable method in that telomerase activity can be measured quickly and with high sensitivity, is used.
  • Example 2 Ammonium sulfate precipitation
  • 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.
  • 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 ⁇ 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).
  • Biot-CX biotinylated CX primer
  • 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.
  • FIG. 1 shows the relationship between each fraction and 3 H—uptake amount.
  • Example 3 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.
  • Example 4 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.
  • 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%.
  • 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
  • telomere 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 ⁇ , 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)
  • telomerase RNA 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.
  • 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.
  • the gel of 50mM T ris- acetate ( ⁇ 8 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.
  • 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 type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA) Sequence characteristics:
  • Sequence type nucleic acid
  • Sequence type Other nucleic acids (synthetic DNA) Sequence characteristics
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type Other nucleic acid (synthetic DNA) Sequence:

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Abstract

L'invention concerne des protéines ayant une activité de télomérase et pouvant servir à cribler les inhibiteurs de télomérase, à développer des méthodes de diagnostic utilisant des anticorps, etc. L'invention concerne aussi des protéines conjuguées, y compris des protéines ayant une activité de télomérase qui possèdent les propriétés physico-chimiques suivantes: fonction et spécificité du substrat: catalyser l'élongation de la terminaison 3'-OH d'ADN télomère de chromosomes d'eucaryotes; masse moléculaire: comprise entre environ 40 et environ 120 kD, notamment environ 110, 58 et/ou 45 kD dans certains cas, déterminé selon SDS-PAGE; inactivation: inactivé par traitement à la RNase; caractéristique: lier l'ARN télomérase chez la souris.
PCT/JP1997/002976 1996-08-28 1997-08-27 Proteines ayant une activite de telomerase WO1998008938A1 (fr)

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AU40306/97A AU4030697A (en) 1996-08-28 1997-08-27 Proteins having telomerase activity

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JP8/226869 1996-08-28
JP22686996 1996-08-28

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5919656A (en) * 1996-11-15 1999-07-06 Amgen Canada Inc. Genes encoding telomerase protein 1
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US6475789B1 (en) 1996-10-01 2002-11-05 University Technology Corporation Human telomerase catalytic subunit: diagnostic and therapeutic methods
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US6617110B1 (en) 1996-10-01 2003-09-09 Geron Corporation Cells immortalized with telomerase reverse transcriptase for use in drug screening
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators
US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CELL, Vol. 81, (1995), COLLINS K. et al., "Purification of Tetrahymena Telomerase and Cloning of Genes Encoding the Two Protein Components of the Enzyme", p. 677-686. *
MOLECULAR CARCINOGENESIS Vol. 16, (May 1996), YOSHIMI N. et al., "Telomerase Activity of Normal Tissues and Neoplasms in Rat Colon Carcinogenesis Induced by Methylazoxymethanol Acetate and Its Difference from That of Human Colonic Tissues", p. 1-5. *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7560437B2 (en) 1996-10-01 2009-07-14 Geron Corporation Nucleic acid compositions for eliciting an immune response against telomerase reverse transcriptase
US7585622B1 (en) 1996-10-01 2009-09-08 Geron Corporation Increasing the proliferative capacity of cells using telomerase reverse transcriptase
US8222392B2 (en) 1996-10-01 2012-07-17 Geron Corporation Kit for detection of telomerase reverse transcriptase nucleic acids
US6261836B1 (en) 1996-10-01 2001-07-17 Geron Corporation Telomerase
US7285639B2 (en) 1996-10-01 2007-10-23 Geron Corporation Antibody to telomerase reverse transcriptase
US7517971B1 (en) 1996-10-01 2009-04-14 Geron Corporation Muteins of human telomerase reverse transcriptase lacking telomerase catalytic activity
US6617110B1 (en) 1996-10-01 2003-09-09 Geron Corporation Cells immortalized with telomerase reverse transcriptase for use in drug screening
US6808880B2 (en) 1996-10-01 2004-10-26 Geron Corporation Method for detecting polynucleotides encoding telomerase
US6927285B2 (en) 1996-10-01 2005-08-09 Geron Corporation Genes for human telomerase reverse transcriptase and telomerase variants
US7005262B2 (en) 1996-10-01 2006-02-28 Geron Corporation Methods for detecting nucleic acids encoding human telomerase reverse transcriptase
US7056513B2 (en) 1996-10-01 2006-06-06 Geron Corporation Telomerase
US7195911B2 (en) 1996-10-01 2007-03-27 Geron Corporation Mammalian cells that have increased proliferative capacity
US6475789B1 (en) 1996-10-01 2002-11-05 University Technology Corporation Human telomerase catalytic subunit: diagnostic and therapeutic methods
US6174703B1 (en) 1996-11-15 2001-01-16 Amgen Inc. Genes encoding telomerase protein 1
US7390891B1 (en) 1996-11-15 2008-06-24 Amgen Inc. Polynucleotides encoding a telomerase component TP2
US5919656A (en) * 1996-11-15 1999-07-06 Amgen Canada Inc. Genes encoding telomerase protein 1
US5981707A (en) * 1996-11-15 1999-11-09 Amgen Inc. Genes encoding telomerase protein 1
US7413864B2 (en) 1997-04-18 2008-08-19 Geron Corporation Treating cancer using a telomerase vaccine
US7622549B2 (en) 1997-04-18 2009-11-24 Geron Corporation Human telomerase reverse transcriptase polypeptides
US7750121B2 (en) 1997-04-18 2010-07-06 Geron Corporation Antibody to telomerase reverse transcriptive
US8236774B2 (en) 1997-04-18 2012-08-07 Geron Corporation Human telomerase catalytic subunit
US8709995B2 (en) 1997-04-18 2014-04-29 Geron Corporation Method for eliciting an immune response to human telomerase reverse transcriptase
US6610839B1 (en) 1997-08-14 2003-08-26 Geron Corporation Promoter for telomerase reverse transcriptase
US7199234B2 (en) 1997-08-14 2007-04-03 Geron Corporation Regulatory segments of the human gene for telomerase reverse transcriptase
US7378244B2 (en) 1997-10-01 2008-05-27 Geron Corporation Telomerase promoters sequences for screening telomerase modulators

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