MXPA99009005A - Purified telomerase - Google Patents

Abstract

This invention provides purified human telomerase and methods of purifying it. The methods involve the use of several sequential steps, including the use of a first matrix that binds molecules bearing negative charges, a matrix that binds molecules bearing positive charges, a second matrix that binds molecules bearing negative charges, an affinity purification step and a matrix that separates molecules according to their size.

Description

PURIFIED TELOMERASE Background of the Invention The American population is aging. The fastest growing segment of the population is people over 85 years of age, who by the year 2040 are expected to represent more than 30 million. This demographic wave is producing significant needs for medications for the treatment of age-related diseases and fosters interest in increasing the aging process and the diseases associated with it, including cancer. Organisms age, in part, because their cells have a limited capacity to continue dividing. When they reach that limit, the cells begin to age. The senescence of the cells continued at the ends of the chromosomes of a cell, the telomeres. With each cell division, the telomeres lose some DNA and become shorter. At some point this reduction becomes critical. The cells perceive this and stop the reproduction of chromosomes to avoid further loss. Consequently, the cell no longer has the capacity to divide. Not all cells become senescent. Unicellular organisms and certain mammalian cells have no fixed limit for cell division. Researchers have discovered that many of these cells contain a ribonucleoprotein enzyme called telomerase. Telomerase replaces the DNA that is usually lost from telomeres during cell division. (E.H. Blackburn, (1992) Annu., Rev. Biochem. 61: 113-29). Therefore, telomeres never shrink beyond critical length and cells never reach aging. It is of particular interest that the researchers found that the cells of many human cancers have telomerase. (C.B. Harley, Mutation Research 1991 256: 271-282). This helps explain why cancer cells continue to divide without getting old. It also suggests a powerful weapon in the battle against. Cancer: If you can inhibit the activity of telomerase in cancer cells, you can expect these cells to age and stop dividing. The development of methods to regulate the activity of telomerase requires sources of purified telomerase and, in particular, purified human telomerase. Purified telomerase would be useful to develop and test assays to measure telomerase activity, for example, to evaluate the assay and to use it as a standard in assaying. Assays for telomerase are useful for characterizing cancer cells or precancerous cells, because most of these cells express telomerase. Purified telomerase would be more useful than crude telomerase preparations to identify and test the regulators, inhibitors or activators of telomerase activity in assays in vi tro. In addition, purified telomerase would facilitate a complete biochemical analysis of the mechanism of the enzyme, which can provide insight to develop regulators based on the mechanism. Purified telomerase would also be useful in the preparation of antibodies against telomerase. These antibodies at a certain moment would be a special help as reagents to purify human telomerase and as an aid for the diagnosis and prognosis of cancer. Purified telomerase will also help to provide amino acid sequence information useful for cloning the various components of the ribonucleoprotein. Although there is a need for purified telomerase, the purification of the human enzyme involves technical challenges. Telomerase is a rare ribonucleoprotein that is expressed in human cells only in very small amounts. It is estimated that human cells known to express the highest levels of telomerase activity can only have almost one hundred molecules of the enzyme per cell. The fact is that telomerase is a complex, multi-component structure that also hinders its purification. further, human cells have comparatively very high levels of non-telomerase ribonucleoproteins. These other ribonucl-eoproteins may have chromatographic purification properties similar to telomerase ribonucleoprotein, which makes it difficult to purify telomerase from human cells. Thus, purified telomerase and in particular, purified human telomerase is necessary. SUMMARY OF THE INVENTION Human telomerase is purified at up to 60,000 times purity in crude cytoplasmic cell preparations. Two polypeptides that purify together with fractions containing telomerase activity are present in the purified fractions in approximate stoichiometric amounts with the AUN component of the isolated human telomerase. A polypeptide has amino acid sequences equivalent to nucleolin. The other peptide has amino acid sequences equivalent to the elongation factor 2 homolog. In one aspect, this invention provides methods for purifying telomerase. The steps included in the method depend on the level of purification that is desired. A method for purifying telomerase from an impure composition containing organic biomolecules, for example, a nuclear extract of 293 cells, for at l 60,000 times compared to crude extract (approximately 4% relative purity) comprises: (1) contacting telomerase with a first matrix that binds negatively charged molecules, for example, POROS 50 HQ, separating telomerase from other organic biomolecules that do not bind with the matrix and pooling telomerase; (2) contacting telomerase with a matrix that binds positively charged molecules, for example, POROS® Heparin 20 HE-1, separating telomerase from other organic biomolecules that do not bind to the matrix and pooling telomerase; (3) contacting telomerase with a second first matrix that binds negatively charged molecules, for example, SOURCE 15Q®, separating telomerase from other organic biomolecules that do not bind to the matrix and pooling telomerase; (4) contacting the telomerase with an affinity agent having specific affinity to telomerase, for example an oligonucleotide having the sequence 5 '-cgttcctctt cctgcggcct-3' (Oligo 14ab) (SEQ ID NO: 7), separating telomerase of other organic biomolecules that do not bind to the affinity agent and bind telomerase; and (5) separating telomerase from other organic biomolecules according to molecular size, shape or floating density, for example separating the molecules according to size in a size classification column of TosoHaas TSK-Gel * G5000PWXL and bringing together the telomerase Telomerase can be separated into at least 500,000 times purity by adding a second affinity step using anti -inuclein antibodies or anti-TMG antibodies. Telomerase can be purified at substantial purity (eg, at least 1,000,000 times by further separating it by gel electrophoresis.) The purification protocol can also include the step of contacting telomerase with an intermediate selectivity matrix, separating telomerase from other organic biomolecules that do not bind to the intermediate selectivity matrix and bind telomerase, preferably before the affinity step.Timeomerase can be separated at different levels of purity by altering, changing sequences, or eliminating any of the steps In the purification protocol, however, any protocol will include the contact of telomerase with an affinity agent.Connecting telomerase with a matrix that binds negatively charged or positively charged molecules is the next step or preferred steps (s) to be included in the protocol The invention also provides purified telomerase and in a more particu lar, telomerase with increase of at least 2000 times, at least 3000 times, at least 20,000 times, at least 60,000 times, at least 100,000 times, at least 500,000 times or at least 1,000,000 times relative purity in comparison with crude cell extracts of 293 cells. Telomerase can be animal, mammalian, and even more specifically human. The invention also provides telomerase made by the above purification steps, or in a recombined manner. In another aspect, this invention provides a recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 5 consecutive amino acids for a human telomerase protein component. In one embodiment, the recombinant polynucleotide further comprises an expression control sequence operably linked to the nucleotide sequence. In another aspect, this invention provides a polynucleotide probe or primer that specifically mixes with a polynucleotide that encodes a human telomerase protein component. In another aspect, this invention provides a recombinant cell comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding a mammalian telomerase protein component, wherein the cell produces the RNA and the protein components of the medullar enzyme of telomerase. In one embodiment, the recombinant cell further comprises a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding the component of telomerase RNA. This invention provides a method for making telomerase comprise by culture a recombinant cell of this invention. In another aspect this invention provides a composition comprising recombinant human telomerase made by the methods of this invention. In another aspect this invention provides methods for inducing an immunization response against telomerase comprising the inoculation of an animal with purified telomerase or with an immunogenic fragment thereof., as a protein component. This includes the induction of a humoral immunization response that leads to the production of antibodies, as well as an immunization response with cell mediation. In another aspect this invention also provides a polypeptide fragment of a human telomerase protein component that, when presented in an animal as an immunogen, generates a humoral or cell mediated immune response.

In another aspect, this invention provides a composition comprising an antibody or antibody fragment that specifically binds to a human telomerase protein component. Brief Description of the Drawings Fig. 1 describes a four-step protocol for purifying telomerase. Fig. 2 describes a five-step protocol for purifying telomerase. Fig. 3 describes a first six step protocol for purifying telomerase. Fig. 4 describes a second six-step protocol for purifying telomerase. Fig. 5 shows the results of an initiation elongation assay from a CHAPS S-100 extract of 293 cells (lines 1-5) and from active accumulation of POROS® 50 HQ anion exchange chromatography (lines 1-5) in the four-step purification procedure. Fig. 6 shows the results of an initiator elongation assay from a telomerase preparation of the active accumulation of POROS® 50 HQ anion exchange chromatography (lines 1-4) and the active accumulation of POROS anion exchange chromatography ® Heparin 20 HE-1 (lines 5-8) in the four-step purification procedure.

Fig. 7 shows the results of an initiation elongation assay from a telomerase preparation from active affinity separation accumulation of Oligo 5 (lines 1-4), the active accumulation of POROS® Heparin 20 HE chromatography. -1 adjusted to the salt concentration used for affinity separation (lines 5-7), and active storage POROS® Heparin 20 HE-1 (lines 8 and 9) in the four-step purification procedure. Fig. 8 shows the results of an initiation elongation assay from telomerase preparations from active accumulation of POROS® Heparin 20 HE-1 chromatography (lines 1-3) and active accumulation of Spermidine POROS® chromatography (lines 4-6) in the five-step purification process, and from Superóse® active accumulation in the first six-step test. DETAILED DESCRIPTION OF THE INVENTION I. Definitions Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly known to a person skilled in the art to whom this invention pertains. The following references provide knowledge of a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd Edition, 1994); The Cambridge Dictionary of Science and Technology (Walker De., 1988); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified. "Polynucleotide" refers to a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related structural variants that occur naturally and synthetic analogs that do not naturally occur thereof) linked by means of phosphodiester linkages, naturally occurring related structural variants and synthetic analogs that do not occur naturally from them. Thus, the term includes polymers of nucleotides wherein the nucleotides and the links thereto include synthetic analogs that do not naturally occur therefrom, such as, for example, and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-0-methyl ribonucleotides, peptide nucleic acids ("PNAs"), and the like. The polynucleotides can be synthesized, for example, using an automated DNA synthesizer. "Nucleic acid" typically refers to large polynucleotides. "Oligonucleotide" typically refers to short polynucleotides, which are generally not greater than about 50 nucleotides. It is understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes an RNA sequence (ie, A, U, G, C) wherein " U "replaces" T ". At present a conventional annotation is used to describe polynucleotide sequences; the left end of a single band polynucleotide sequence is the 5 'end; the left direction of a double band polynucleotide sequence is referred to as the 5 'direction. The direction of addition of 5 'to 3' of nucleotides for transcripts of nascent RNA is referred to as the direction of transcripts. The DNA band that has the same sequence as a mRNA is termed as the "coding band"; sequences in the DNA band that have the same sequence of an mRNA transcribed from DNA and that are located at the 5 'end of the RNA transcript are referred to as "ascending sequences"; sequences in the DNA band that have the same RNA sequence and that are 3 r end to 30 transcription encoding RNA are referred to as "descending sequences". "Recombinant polynucleotides" refers to a polynucleotide that has sequences that do not naturally join. An amplified or pooled recombinant polynucleotide can be included in a suitable vector, and the vector can be used to transform a suitable host cell. A host cell comprising the recombinant polynucleotide is termed as "recombinant host cell". The gene is then expressed in the recombinant host cell to produce, for example, a "recombinant polypeptide". A recombinant polynucleotide can also provide a non-coding function service (e.g., origin of self-reproduction, ribosome binding site, etc.). Suitable unicellular hosts include any of those that are routinely used to express mammalian or eukaryotic nucleic acids, including, for example, prokaryotes, such as E. coli; and eukaryotes, including for example, fungi, such as yeast; and mammalian cells, including insect cells (e.g., Sf9) and animal cells such as CHO, Rl.l, BW, LM, African Green Monkey Kidney cells (e.g., COS 1, COS 7, BSC 1, BSC 40 and BMT 10) and human culture cells. "Expression control sequence" refers to a nucleotide sequence in a polynucleotide that regulates the expression (transcription and / or translation) of a nucleotide sequence operably linked thereto. "Operably linked" refers to a functional relationship between two parties where the activity of one party (for example, the ability to regulate transcription) results in an action of the other party (for example, transcription of the sequence). Expression control sequences may include, for example and without limitation, promoter (e.g., inducible or constitutive) promoter sequences, enhancers, transcription terminators, a start codon (i.e., ATG), binding signals for introns, and stop codons. "Expression vector" refers to a vector comprising a recombinant polynucleotide comprising the expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be delivered by the host cell or in vitro expression system. Expression vectors include all those that are known in the art, such as cosmids, plasmids (e.g., single or contained in liposomes) and viruses that incorporate the recombinant polynucleotide. "Coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (ie, rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties that result from them. Thus, a gene encodes a protein if the transcription and translation of mRNA produced by a gene produces the protein in a cell or other biological system. Both the coding band, the nucleotide sequence that is identical to the mRNA sequence and that is generally provided in sequence listings, such as the non-coding band, used as a template for transcription, of a gene or cDNA can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of one and the other and that encode the same amino acid sequence. The nucleotide sequences encoding proteins and RNA may include introns. "Allelic variant" refers to any of two or more polymorphic forms of a gene that occupies the same genetic point. Allelic variations arise naturally through mutation, and can result in phenotypic polymorphism within populations. Mutations of genes can be silent (without change in the encoded polypeptide) or can encode polypeptides having altered amino acid sequences. Allelic variants may also be referred to as "allelic variants" to transcripts of mRNAs from genetic allelic variants, as well as the proteins encoded by them. "Hybridizes specifically to" or "specific hybridization" or "selectively hybridizes to", refers to the binding, duplication or hybridization of a nucleic acid molecule preferably to a particular nucleotide sequence under severe conditions when that sequence is present in DNA or Complex mixture RNA (for example, total cell). The term "severe conditions" refers to the conditions under which a probe will preferably mix its white secondary sequence, and to an extent less than, or not at all, other sequences. "Severe hybridization" and "severe hybridization washing conditions" in the context of nucleic acid hybridization experiments as southern and northern hybridizations depend on the sequence, and are different under different environmental parameters. An extensive guide for the hybridization of nucleic acids is found in Tijsen (1993) Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization wi th Nucleic Acid Probes (Laboratory Techniques of Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes) part I chapter 2"General Principles of Hybridization and Nucleic Acid Probe Assay Strategy", Elsevier, New York. In general, highly stringent hybridization and washing conditions are selected at about 5 ° C below the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) in which 50% of the target sequence is mixed for a probe with perfect coincidence. Very severe conditions are selected to equal the Tm for a particular probe. An example of severe hybridization conditions for hybridization of complementary nucleic acids having more than 100 complementary residues in a filter in a south or north spot is 50% formalin with 1 mg of heparin at 42 ° C, hybridization taking place during the night. An example of highly severe washing conditions is 0.15 M CaCl at 72 ° C for almost 15 minutes. An example of severe washing conditions is a 0.2X SSC wash at 65 ° C for 15 minutes (see Sambrook et al. For a description of SSC buffer). Often, a severe wash is preceded by a mild wash to remove the previous probe signal. An example of washing of average severity for a duplex of, for example, more than 100 nucleotides, is IX SSC at 45 ° C for 15 minutes. An example of low severity wash for duplex of eg, more than 100 nucleotides, is 4-6x SSC at 40 ° C for 15 minutes. In general, a signal to noise ratio of 2x (or greater) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. "Polypeptide" refers to a polymer composed of amino acid residues, naturally occurring related structural variants, and non-naturally occurring synthetic analogs thereof linked through peptide linkages, naturally occurring related structural variants, and synthetic analogs that do not naturally occur therefrom. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" typically refers to large polypeptides. The term "peptide" typically refers to short polypeptides. At present conventional annotation is used to represent polypeptide sequences; the left end of a polypeptide sequence is the aminotermin; the right end of a polypeptide sequence is the carboxyl-terminus. "Antibody" refers to a polypeptide substantially encoded by an immunoglobulin gene, or fragments thereof, that specifically bind and recognize an analyte (antigen). Immunoglobulin genes comprise genes from kappa, lambda, alpha, gamma, delta, epsilon and mu constant regions, as well as immunoglobulin variable region iriagenes. The antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. This includes, for example, Fab 'and F (ab)' 2 fragments. The term "antibody", as used herein, also comprises fragments of antibodies produced either by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. An antibody "binds specifically to" or "is in-reactive specifically with" a protein when the antibody functions in a binding reaction that determines the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated conditions of immunoassays, the specified antibodies bind in preference to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under these conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Labora tory Manual (Antibodies, Laboratory Manual), Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. "Immunoassay" refers to an assay that uses an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of a particular antibody to separate, target and / or label the analyte. "Complementary" refers to the topological compatibility or mutual correspondence of interaction surfaces of two polynucleotides. In this way, the two molecules can be described as complementary, and in addition, the contact surface characteristics complement each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is identical to the nucleotide sequence of the polynucleotide binder co-ingredient of the second polynucleotide. Thus, the polynucleotide whose sequence is 5 '-TATAC-3', is complementary to the polynucleotide whose sequence is 5'-GTATA-3 '. "Conservative substitution" refers to a substitution in a polypeptide of an amino acid for an amino acid with similar functionality. Each of the following six groups contains amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Triptofan (W). "Substantially pure" means that an objective species is the predominant species present (ie, on a molar basis, more abundant than any other individual organic biomolecular species in the composition), and a substantially purified fraction is a composition wherein the target species it comprises at least about 50% (on a molar basis) of all organic biomolecular species present. As usual, a substantially pure composition means that about 80% to 90% or more of the organic biomolecular species present in the composition is the purified species of interest. The target species is purified to obtain essential homogeneity (contaminating species can not be detected in the composition by conventional detection methods) if the composition essentially comprises a single organic biomolecular species, "organic biomolecule" termed an organic molecule of biological origin, by example, proteins, nucleic acids, carbohydrates or lipids. For the purpose of this definition, solvent species, small molecules (<) are not considered organic biomolecular species.500 Daltons), stabilizers (for example, BSA), and elemental ion species. "What happens naturally" when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be separated from a source of nature and that was not intentionally modified by man in the laboratory, occurs naturally. "Telomerase" or "telomerase ribonucleoprotein complex" refers to a ribonucleoprotein enzyme of eukaryotic origin identifiable by its ability to polymerize a DNA sequence of a eukaryotic telomere. Telomerase is also characterized by an RNA component that has complementary sequences with at least part of the telomeric repeat of the origin species and by one or more protein components. As used herein, "animal telomerase", "mammalian telomerase" and "human telomerase" refer to telomerases that can be found naturally in various animal, mammalian and human multicellular cells, respectively, or that have polypeptide components with the same amino acid sequences, and RNA components with the same nucleotide sequences. Human telomerase contains the RNA component, "hTR". (U.S. Patent, 5,583,016, Villeponteau et al.). The term "telomerase" comprises all allelic forms of telomerase, including wild-type and mutant forms. "Telomerase protein component" refers to a protein component of the medullary telomerase enzyme. "Medullary telomerase enzyme" refers to the joint assembly of telomerase components, both RNA and protein components, sufficient for telomerase activity in vi tro. "Protein associated with telomerase" refers to a protein that binds to the medullar enzyme of telomerase but that is not necessarily for the activity of telomerase in vitro. "Telomerase activity factor" refers to a protein that, when included with the medullar enzyme of telomerase, increases the activity of telomerase in vi tro. "Telomerase-related protein" refers, collectively, to the components of the telomerase protein, proteins associated with telomerase and telomerase activity factors.

"Telomerase activity" refers to the synthesis of telomeric DNA by telomerase. One test method for detecting telomerase activity is the TRAP assay. See Harley et al., International Application WO 95/13381. This assay measures the amount of radioactive nucleotides incorporated in the elongation products, polynucleotides, formed by the addition of nucleotides to a substrate or telomerase initiator. Incorporated radioactivity can be measured as a function of the intensity of a band on a Phosphorlmager ™ screen exposed to a gel where the radioactive products are separated. A test experiment and a control experiment can be visually compared using the Phosphorlmager ™ screens. See also TRAP-eze ™ telomerase assay kit (Oncor); and Morin, Cell (Cell) 59: 521-529 (1989). The "specific activity" of telomerase is the amount of telomerase activity according to the amount of protein in a unit volume. "Purified telomerase" refers to preparations of telomerase that have at least 2000 times increased relative purity. As used herein, a telomerase preparation has 2000 times increased relative purity if the specific activity of telomerase in the preparation is at least 2000 times greater than the telomerase specific activity of crude cytoplasmic extracts of 293 cells with of suspension, which are described hereinafter, and as measured by the primer elongation assay described hereinbelow in Example IA It is calculated that in a telomerase preparation having approximately 100,000 times increased relative purity, approximately 6% of the proteins are telomerase, while in the telomerase preparation having approximately 1,000,000 times increased relative purity, approximately 60% of the molecules are telomerase. . II. Methods for Purifying Telomerase This invention provides purified telomerase and the methods for preparing it. In particular, this invention is directed to purified mammalian or human telomerase and recombinant telomerase. This invention provides purified telomerase separated from any cell expressing telomerase, for example, normal crude cell extracts, cancer cells, immortalized cells, human and animal tissues, tumors or cells expressing telomerase recombinantly. A. Assays for Telomerase Activity In methods for purifying telomerase it is often useful to determine the presence or amount of telomerase or telomerase activity in a preparation. For this, several assays are available. As stated above, in order to determine the relative purity, the most preferred method for measuring the specific activity of telomerase is the primer elongation assay. This assay is described in Example I.A., below. Briefly, this assay measures the amount of radioactive nucleotides incorporated into polynucleotides synthesized in an initiator sequence. The amount incorporated is measured as a function of the intensity of a band on a phosphoimager screen exposed to a gel where the radioactive products are separated. A test experiment and a control experiment can be visually compared on phosphoimager screens. This assay is based on an assay described by G.B. Morin, (1989) Cell (Cell) 59: 521-529. Another test for telomerase activity is the dot spot assay is useful for routine screening because it has higher performance and hundreds of assays can be carried out in a single day with a large portion of the work done automatically. The results can be obtained the afternoon of the second day. Spot spot testing is more effective in comparing the activity of the samples to almost the same level of purity and less effective in different stages of purity. Therefore, it is not a preferred assay to determine the relative purity. A protocol for spot testing with spots is provided in Example I.B. Other assays include detecting the presence of the RNA component of telomerase. The sequence of the RNA component of telomerase for several species is known. A polynucleotide comprising the sequence for the RNA component of human telomerase ("hTR") was separated. Human genomic DNA hTR was cloned, sequenced and placed in reservoir. A lambda clone designated "28-1" contains a ~ 15 kb insert containing sequences of human telomerase RNA component genes. Clone 28-1 was deposited in the American Type Culture Collection in accordance with the Budapest Treaty and assigned the acquisition record number ATCC 75925. Plasmid pGRN33 contains an insert ~ 2.5 kb HindIII-Saci containing sequences from lambda clone 28-1 containing the hTR sequence. The plasmid pGRN33 was deposited in the American Type Culture Collection according to the Budapest Treaty and assigned the acquisition record number ATCC 75926. An hTR fragment of the ~ 2.4 kb SauIIIAl-HindlII fragment of clone 28-1 also contains the sequence of hTR. The sequence of the PstI fragment is given below in SQ ID NO: l. The nucleotides of hTR are indicated before the sequence indicated by the asterisks and numbered 1 to 451. The region of the template is underlined. 1 CTGCAGAGGAtAGAAAAAAGOCCCTCTGATACCT - AAGTTAGTTTCACCTTTAAAGAAGG GACGTCTCCTATCTTTTTTC7GGGAGACTATGGAGTTCAATCAAAGTGGAAATtTCTTCC -psti- 61 TCGGAAGTAAAGACGOUVAGCCTTTCCCGGACGTGCGGAAGGGCAACGTCCTTCCTCATG AGCCrrCATTTCTGCGTTTCGGAAAGGGCCTGCACGCCTrCCCGtTsCAGGAAGGAGTAC 121 GCCsGAAATGGAACTTtAA TTCCCsTTCCCCCCAACCAGCCCGCCCsAGAGAGTGA-TC CGGCCTTTACCTTGAAATTAAAGGGCAAGGGGGGTTGGTCGGGCGGGCTCTCTCACtGAG 181 TCACGAGAGCCGCGAGAGTCAGCTTGGCCAATCCGTGCGGTCGGCGGCCGCTCCCTTTAT AGTGCTCTCGGCGCTCTCAGTCGAACCGGTTAGGCACGCCAGCCGCCGGCGAGGGAAATA January 10 20 30 241 40 50 AAGCCGACTCGCCCGGCAGCGCACCaGGTTGCGGAGGGTGGGCCTGGGAGGGGTGGTGGC TTCGGCTGAGCGGGCCGTCGCGTGGCCCAACGCCTCCCACCCGGACCCTCCCCACCACCG 60 70 80 90 **************** *********************** 301 CAT TrrrGTCTAACCCTAACTGAGAAGGGCGTAGGCGCCGTGCTTTrGCTCCCCGCGCG GTA-? UU? C-AGATTG - GATTGACTCTTCCCG - ATCCGCGG - ACGAAAACGAGGGGCGCGC 100 110 120 130 140 ISO ************* + *************************** *** ++ *** + ***** 3ßi CGGTTTTTCTCGCTGACTTTCAGCGGGCGGAAAAGCCTCGGCCTGCCGCCGTC CACCGGT GACAAAAAGAGCGACTG-AAAG7CGCCCsCCTTTTCGGAGCCGGACGGCGGAAGGTGG - AA 160 170 180 190 200 210 421 (- tTCrAGAGCAAACA - ^^ AAATsT - AGCTGCTGGCCCGTTCGCCCCTCCCGGGGACCTGC GTAAGAT -tCG TTGTTTTTTACAGTCGACGACCGGGC-AAGCGGGGAGGGCCCC- ssACG HTR 220230240250 '260270481 GGCGGGTCGCCTGCC CAGCCCCCGAACCCCGCCTGGAGGCCGCGGTCGGCCCGGGGCTTC CCGCCCAGCGGACGGGTCGGGGGCTTGGGGCGGACCTCCGGCGCCAGCCGGGCCCCGAAG For the sequence of the telomerase RNA component of humans, mice, yeast, ciliates, see Feng et al. (1995) Science (Science) 269: 1236-41; Villeponteau et al., United States patent no. 5,583,016; WO96 / 01835 (Villeponteau et al.), WO 96/01604 (Andrews et al.), Blasco et al. (1995) Sci en 269: 1267-1270; Romero and Blackburn 280 290 300 310 320 330 541 340 3S0 TCCGGAGGCACCCACTGCCACCsCGAAGAGTTGssCTCTGTCAGCCGCGGGTCTCTCGGG AGGCCTCCsTGsGTGACGGTGsCGCTTCTCAACCCGAGACAGTCGGCGCCCAGAGAGCCC 360 370 380 390 601 410 420 GGCGAGGGCGAGGTTCAGGCCTrTCAGGCCGCAGGAAGAGGAACGGAGCGAGTCCCCGCG CCGCTCCCGCTCCAAGTCCGGAAAGTCCGGCGTCCTTCTCCTTGCCTCGCTCAGGGGCGC 400 430 440 450 661 721 CGCGGCGCGATtCCCTGAGCTGTGGGACGTGCACCCAGGACTCGGCTCACACATGCAGTT GCGCCGCGCTAAGsGACTCGACACCCTGCACGTGGGTCCTGAGCCGAGTGTGTACGTCAA CsCTTTCCTGTTGGTGGGGGGAACGCCGATCGTGCGCATCCsTCACCCCTCsCCGGCAGT GCGAAAGGACAACCACCCCCCTTGCGGCTAGCACGCGTAGGCAGTGGGGAGCGGCCGTCA 781 GGGGGCTTsTGAACCCCCAAACCTGACTGACTGGGCCAGTGTGCTGCAAATTGsCAGGAG CCCCCGAACACTTGGGGGTTTGGACTGACTGACCCGGTCACACGACGTTTAACCGTCCTC August 1 ACGTGAAGGC-ACCTCCAAAGtCGGCC-AAAATGAATGGsCAGTGAGCCGGGsTTGCCTGGA TsCACTTCCsTGGAGGTTTCAGCCGGTTTTACTTACCCsTCACTCsGCCCCAACGGACCT 901 GCCGTTCCTGCGTGGGTTCtCCCGTCT CCGCTTTTTGTTGCCTTTtATGGTTGTATTAC CGG-- AAGGACGCACC --- ^ - AGGGCAGAAGGCG - Ul -? ---- ^ 961 AACTTAGTTCCTGCT CTGCAG (SEQ ID NO: l) (S «" 7"IS A OR T) TTGAATCAAGGACGAGACGTC -PSTl- (1991) Cell (Cell) 67: 343-353; Lingner et al. (1994) Genes and Devel. (Genes and Development) 8: 1984-1988; and Singer and Gottschling (1994) Science (Science) 266: 404-409. PCR ("RT-PCR") of reverse transcription is a useful assay to determine the amount of telomerase RNA because it is very sensitive. A protocol for an RT-PCR assay is provided in Example I.C. See also, United States patent application 08 / 770,565, filed December 20, 1996. Other methods for specific detection of RNA may be used, for example, Northern Analysis. In Example I.D. a protocol for Northern is provided. The main limitation for using any of the hTR assays to detect telomerase is that the presence of hTR does not mean that there is presence of active telomerase. For example, normal somatic cells and some fractions of partially purified telomerase have significant amounts of hTR but no detectable telomerase activity. Another highly sensitive assay for telomerase activity is the TRAP assay, described in Harley et al., International publication WO 95/13381; Harley et al. International application PCT / US96 / 09669, filed on June 7, 1996 and Kim et al. (1994) Sci ence 266: 2011-2015. B. Purification Protocols 1. General Considerations This invention provides methods for preparing purified telomerase from an impure composition., that is, a composition containing telomerase and other contaminating organic biomolecules. Starting with an impure telomerase composition, a method that produces telomerase that is greater than 60,000 times pure includes the following steps: (1) providing an impure preparation containing telomerase, for example, a nuclear extract; (2) separating telomerase from other organic biomolecules with a first matrix that binds molecules that are negatively charged, for example, 50 HQ POROS®; (3) additionally purifying telomerase with a matrix that binds molecules that are positively charged, for example, Heparin 20 HE-1 POROS®; (4) further purify the telomerase with a second matrix that binds molecules that are negatively charged, for example, SOURCE 15Q®; (5) additionally purifying telomerase with an affinity agent having telomerase-specific affinity, for example oligonucleotide 14ab, having the sequence 5'-cgttcctctt-3 '(SEQ ID NO: 7); and (6) further purifying the telomerase from other organic biomolecules according to the molecular size, shape or floating density, for example separating the molecules according to size in a size classification column TosoHaas TSK-Gel * G5000PWXL. Additional optional steps may be included in the purification methods to produce preparations of telomerase with relatively higher purity. Telomerase can be further purified with a resin of intermediate selectivity (preferably a matrix containing spermidine) before affinity purification. See Fig. 2. The specific purification steps used and their sequence are performed at the discretion of the practitioner. However, the following guidance is provided. In general, it is preferred to start with the steps that have higher capacity and relatively lower selectivity, then with the steps that have intermediate capacity and / or selectivity, then with the steps that have low capacity and high selectivity. Matrices that bind molecules with positive or negative charges have high capacity and are useful as initial steps because telomerase is present in small amounts in cells and purification methods will typically start with large amounts of crude cell extract. Among these resins, purification is preferred first with anion exchange resins, then purification with resins that bind molecules carrying positive charges. The order can be reversed. The steps with selectivity and intermediate capacity are preferred after the steps with large capacity. These include matrices of intermediate selectivity and separation based on molecular size, shape or floating density. Although purification with intermediate selectivity resins is preferred first, the intermediate purification steps should not be limited in any particular order. In the four step purification procedure described above, the intermediate steps are not included. Specific affinity matrices have relatively low capacity, but high selectivity, and are subsequently preferred in the purification process when telomerase is present with few contaminating materials. This step can be a single purification step and is more useful when the increased relative purity of telomerase is at least 40 times. 2. Source of Telomerase The purification of telomerase begins with an impure source composition, such as a nuclear extract or crude cell extract, preferably rich in telomerase activity. Unicellular organisms without limits of number of cell divisions, such as Tetrahymena or yeast, elaborate telomerase and are good sources for cellular extract in the preparation of telomerase from these organisms. However, in multicellular organisms, especially mammals, not all cells make telomerase. Therefore, sources should be identified from the types of cells available. In mammals, germ cells and tissue, cancerous cells and tissue and immortalized cell lines are all sources of telomerase activity. In rodents and possibly in other non-primate mammals, some normal somatic tissues, for example, mouse liver, are sources of telomerase activity. Immortalized cell lines are in particular a useful source of crude telomerase preparations because they can be grown in culture and harvested in large quantities, thereby providing cellular extract for large-scale preparations of telomerase. In particular, in the preparation of purified human telomerase, 293 cells are preferred. The 293 cells originate from the human embryonic kidney transformed with DNA fragments of adenovirus type 5 (Graham et al., 1977 J. Gen. Virol.; 59-77). The cell line, which grows in single layer cultures, was adapted to grow in suspension Stillman and Gluzman, (1985) Mol. and Cell Bio. 5: 2051-2060). They can be obtained from the American Type Culture Collection (ATCC) (Registration of Acquisition No. ATCC CRL 1573). The crude nuclear extract can be prepared by separating the nucleus from the cytoplasm by a low speed rotation. In Example VI, Section A, a more detailed description is provided. The crude cell extract can be prepared normally. In general, the cells can be homogenized at approximately 4 ° C in buffers at physiological pH. In Example II a more detailed protocol is provided. The 293 cells grow as suspension cultures in 8-liter spinner bottles in Joklik's MEM, 5% newborn calf serum, 2 g / 1 NaHCO3, 1% non-essential amino acids, 1% glutamine, 1% penicillin / streptomycin at 37 °. The cultures are conserved in 0.6 x 106 cells / ml and duplicate every 24 hours. You can also hire a cell culture specialist to grow large batches of cells. The contractors include Analytical Biological Systems (Wilmington, DE), Cellex (Minneapolis, MN) and Berlex (South San Francisco, CA). Other cell types, particularly those that grow rapidly in suspension cultures (which facilitates large-scale cultures), are also useful for purifying human telomerase. Candidates include cell lines of B or T cell lineage, such as Namalwa lines (Burkitt's lymphoma), Daudi (Burkitt's lymphoma), Jurkat (acute T cell leukemia) and HUT 78 (cutaneous T-cell lymphoma). In addition, HeLa cells (cervical carcinoma) have telomerase activity. HeLa cell extracts can be obtained from the Computer Cell Culture Center (Mons, Belgium). Nuclear extracts of 293 cells are a preferred source of telomerase. The crude cell extract of the mammalian cells used in the methods of this invention may be cell extract or whole cytoplasmic extract. The amount of impure preparation necessary to purify telomerase depends, in part, on the abundance of telomerase in the cell, the amount of telomerase that is "lost in" each step and the last degree of purification and amount desired. Example III describes the use of 128 liters of suspension culture of 293 cells in a procedure that purified telomerase more than 3000 times. As purification progresses, telomerase becomes purer and more diluted. In this state, telomerase can be lost due to the adhesion of telomerase to tubes, lines, tips, etc. This loss can be minimized by the addition of detergent. In particular, the addition of up to about 0.1% Nonidet P-40 and about 1% Tween®-20 (non-ionic detergents) does not inhibit telomerase activity, and can be added to all chromatography buffers. 3. Matrices Binding Molecules with Negative Loading A preferred method for purifying telomerase from a crude cell extract comprises contacting telomerase with the first and second matrices that bind molecules that have negative charges, separating telomerase from other organic biomolecules that they do not join the matrix and put together the telomerase. Matrices that bind molecules with a negative charge are useful for purifying telomerase because of the pH between about 6 and about 9, the complex telomerase seems to have at least local negative charges. In a preferred embodiment of the invention,. The matrix is an anion exchange resin packed in a column. Anion exchange resins bind negatively charged molecules. A benefit of using anion exchange at this crude stage of preparation is the high binding capacity of these resins. To separate human telomerase, anion exchange resins that are characterized by functional groups of tertiary or quaternary amines provide the best results. 50 HQ of POROS® (PerSeptive Biosystems, Cambridge, Massachusetts) and SOURCE 15Q (Pharmacia, Uppsala, Sweden) are preferred anion exchange resins. Super Q-650M are also useful (TosoHaas, Montgomeryville, Pennsylvania), DEAE Sepharose® CL-6B (Pharmacia, Uppsala, Sweden) and Mono Q® (Pharmacia, Uppsala, Sweden). In washing fractions from the anion exchange resin, elution with salt step is preferred. Elution of linear salt gradients can also be used to wash the telomerase using preferably gradient volumes of less than the column volumes. 4. Matrices that link molecules with positive charge A preferred method for purifying telomerase comprising, as a second step between two purification steps with anion exchange, contacting telomerase with a matrix that binds molecules that have positive charges, separating telomerase from other organic biomolecules that do not bind to the column and gather the telomerase. In a preferred embodiment, the matrix comprises heparin.

In particular, Heparin 20 HE-1 POROS® (PerSeptive Biosystems, Cambridge, Massachusetts) is useful as a matrix at this stage. Other useful matrices that bind molecules with positive charges include SP Sepharose® CL-6B and Resource ™ (Pharmacia, Uppsala, Sweden). 5. Intermediate Purification Steps After purification with high capacity matrices and before purification of affinity matrix, telomerase can be further purified through one or more intermediate purification steps. In an intermediate purification step, the telomerase is contacted with hydroxylapatite, the telomerase is separated from other organic biomolecules that do not bind to the hydroxylapatite and binds the telomerase. Hydroxylapatite is a crystalline form of calcium hydroxylphosphate that has the ability to bind proteins according to their basic or acidic character. Its base of protein separation differs from that of ion exchange resins. The fractions can be washed with buffers or different composition. A preferred step of intermediate purification comprises contacting the telomerase with a matrix of intermediate selectivity; Separate telomerase from other organic biomolecules that do not bind to the intermediate selectivity matrix and bind telomerase. This step may be the fourth step of the purification of the five or six purification schemes outlined in Figures 2 and 3. The main purpose of this step is to further purify the telomerase preparation before it comes in contact with an agent of affinity. The kinds of matrices included in this step have, in general, few binding capacities but they can be more selective than the ion exchange resins, thus they provide greater purification. They bind telomerase through interactions that are more complex than charge, independent, and are not as specific as, for example, antibodies. Binding characteristics may include, for example, combinations of electrostatic attraction, hydrophobic / hydrophilic attraction, affinity for particular components, such as particular nucleic acids or amino acids in the protein, affinity for functional chemical groups in the molecules, and the variety of known compounds and used in the purification of proteins for the specific separation of molecules. In particular, and without limitation, this invention contemplates the use of the following intermediate selectivity matrices. In this step, matrices comprising a polyamine, such as spermidine or spermine, are preferred. These molecules contain primary and secondary hydrocarbon chains of amines to idst. In the binding of telomerase both the charge and the hydrophobic interactions can be understood. In this step matrices comprising polynucleotides are useful. In the case of human telomerase, poliguanilic acid retains telomerase activity. Without wishing to be bound by theory, it is believed that these matrices are useful because telomerase is an enzyme that synthesizes DNA and contains one half of RNA. Thus, it is presumed to have an influence that specifically binds polynucleotides. Matrices comprising divalent metal ions are also useful in this step. Metals can be chelated on solid supports through molecules such as iminodiacetic acid or nitrilotriacetic acid. Nickel is the most preferred metal and copper and zinc are also useful in the purification of human telomerase. Without wishing to limit them by theory, it is assumed that metals selectively interact with specific amino acid residues in proteins. In these interactions it is normal to include histidine residues. Depending on the immobilized metal, only proteins with sufficient local histidine densities will be retained by the column. Some interactions between metals and proteins can be so strong that the protein can not recover. In this way, each metal must be tested empirically to detect its usefulness in the purification of a certain protein. Both the binding and the release of telomerase must be efficient. Matrices that comprise proteinaceous substances with positive charge are also useful in this step of the purification method. As used herein, the proteinaceous substances comprise amino acids, polyamino acids, peptides and proteins. Two materials with positive charge, poly-L-lysine and histone, selectively retain human telomerase. Matrices comprising aminophenyl-boronic acid also retain human telomerase. Although not wishing to be bound by theory, the affinity of the protein for boronic acid is complicated but essentially comprises interaction between groups of diols on proteins with immobilized acid. In one embodiment of the invention, the telomerase is contacted sequentially with at least 2, at least 3 or at least 4 matrices of intermediate selectivity. Since it is not possible for proteins to act as telomerase in matrices that have different characteristics, the use of more than one matrix improves the purification level of this step. Sequences that produce high yields and purifications can be determined empirically, and matrices having higher capacity are initially preferred in the sequence. Some combinations of two matrices in the series are not preferred due to their similar modes of separation, for example, a divalent metal column followed by another, spermine followed by espemidine, poly-L-lysine followed by histone. Another step of intermediate purification in a method for purifying telomerase comprises separating telomerase from other organic biomolecules according to molecular size, shape or floating density and pooling telomerase. This may be the fifth step in the six-step procedure outlined in Figure 3. A preferred embodiment of this step comprises fractionating the telomerase preparation by gel filtration chromatography. The size classification of gel matrices that separate proteins in the size range of 200 kD to 2000 kD are most useful in the purification of human telomerase. Preferred matrices are HW65 (TosoHaas, Montgomeryville, Pennsylvania), Superóse® 6 (Pharmacia, Uppsala, Sweden) and in particular, TSK-Gel * G5000PW. Another embodiment of this step comprises gradient centrifugation of the telomerase in gradients of different compositions that produce separation of the molecules in the preparation. Preferred gradients are composed of Cs2S0 or glycerol. Another embodiment of this step comprises the use of gel electrophoresis, which separates the molecules based on their charge, size and shape. The gel compositions can vary greatly in this embodiment. A preferred gel, is a native gel composed of agarose, polyacrylamide or both, which runs under physiological conditions of buffer and pH resistance, which tend to preserve the native complex and the activity of human telomerase. 6. Affinity Matrix After the steps of high capacity purification and after the optional steps of intermediate purification, telomerase is further purified by contacting telomerase with an affinity agent having specific affinity to telomerase, separating telomerase from other organic biomolecules that do not bind to the affinity agent, and pooling telomerase from the affinity agent. The affinity agents in this step of the purification method are orders of magnitude more specific to bind telomerase over other organic biomolecules, than agents in other steps of the method. Affinity agents that have telomerase specific affinity comprise, for example, oligonucleotides that are complementary to the RNA component of telomerase, oligonucleotides (RNA or DNA) having an initiator sequence recognized by telomerase, antibodies that recognize telomerase epitopes. or telomerase-associated proteins, and compounds that inhibit telomerase. A preferred affinity agent is Oligo 14ab, an oligonucleotide whose sequence is given in Table 2. The anti-nucleolin antibody and the anti-TMG antibody are also useful. Oligonucleotides comprising a nucleotide sequence complementary to a telomerase RNA component sequence (eg, an "antisense sequence") are useful affinity agents. In one embodiment, the oligonucleotide binds to a recoverable label, for example, biotin. "Peptide nucleic acids", polymers having a support of amides and linked bases, are also useful as affinity agents. The label can be on one or both ends. After contacting telomerase with the labeled affinity agent, the affinity agent is contacted with a binding agent that binds to the recoverable label, for example, streptavidin or derivative agent. Preferably, the binder is bound to a matrix. Then, the molecules that did not bind to the recoverable affinity agent and therefore did not bind to the binding agent, are separated or removed from the mixture. Releasing telomerase from the affinity agent, telomerase is purified. The use of recoverable tags is well known in the art. The recoverable label and the binder can be any kind of binder and co-bound binder. In a preferred embodiment, the recoverable tag is biotin and the binding agent is NeutrAvidin ImmunoPure® (Pierce, Rockford, IL, catalog number 53157). In one embodiment, the recoverable label is an antigen and the binding agent is an antibody that binds the antigen. A general experimental procedure for testing nucleic acid affinity approaches is as follows: (i) biotin-labeled oligonucleotides are mixed under various conditions with partially purified telomerase; (ii) beads with bound NeutrAvidin are added to the mixture; (iii) the beads are separated from the mixture (the telomerase that stuck to the oligonucleotide was also stuck to the beads, so the activity is reduced from the mixture); (iv) the beads are washed to remove bound material (the telomerase is detached from the beads and the activity is recovered). Oligonucleotides complementary to a sequence of the human telomerase RNA component were tested for their capacity as affinity agents. Oligonucleotides useful in the methods of the invention are provided in Table 1. Each of the antisense oligos was tested in parallel with a non-specific control oligo. The reduction refers to the retention of telomerase by the oligo; Recovery refers to the subsequent release of telomerase from the oligo. Oligo 14ab is the preferred oligo for affinity purification. Oligo 14ab hybridizes to a region of hTR that is accessible in the holoenzyme. Oligo 5 is also a good oligonucleotide for affinity purification. Although it does not want the union by theory, Oligo 5 is a strong initiator without elaboration, so it may not act as an antisense ligand; can act as an initiating binder. When using Oligo 5 and washing with varying saline concentrations, the activity of telomerase is recovered in a set of fractions containing low levels of detectable protein (10 μg / ml), making it a very rich telomerase preparation. The production of telomerase activity was 19%. (See Example III).

Table 1: Complementary oligonucleotides (antisense) for human telomerase RNA. Name of Oligo Size (nt) Description Performance anti-P 31 hTR antisense Good reduction, direct, covers the inhibits the activity P3 22 hTR antisense plus a good reduction, initiator term recovery of telomerase Oligo 5 30 hTR antisense Good reduction, recovery of telomerase Oligo 13 30 hTR antisense Little reduction.

Oligo 14 30 hTR antisense Good reduction.

Oligo 14ab 20 hTR antisense for Good reduction, accessible region recovery of telomerase Table 2: Oligonucleotide sequences anti-P (SEQ ID NO: 2): 5 'BIOTIN - gcctacgcc ttctcagtta gggttagaca - a - 3' BIOTINA P3 (SEQ ID NO: 3): 5 'BIOTIN - cgcccttctc agttagggtt ag - 3' Oligo 5 (SEQ ID NO: 4): 5 'BIOTIN - gccgagtcct gggtgcacgt cccatagct c - 3' Oligo 13 (SEQ ID NO: 5): 5 'BIOTIN - gaacgggcca gcagctgaca ttttttgttt - 3' Oligo 14 (SEQ ID NO: 6): 5 'BIOTIN - gctcagaat gaacggtgga aggcggcagg - 3' Oligo 14ab (SEQ ID NO: 7): 5 'BIOTIN - cgttcctctt cctgcggcct - 3' En Another embodiment of this step, the affinity agent is an oligonucleotide with a primer sequence recognized by telomerase. The oligonucleotide is contacted with telomerase and dideoxy nucleotides under conditions for an initiator elongation reaction.

This results in the termination of the DNA synthesis chain by telomerase. Under these conditions, telomerase can block the finished chain starter. The initiator and the telomerase attached to it are separated. The oligonucleotide preferably includes sequences that are known to be efficient initiators in the primer elongation assay. This includes the sequence synthesized by telomerase as well as non-telomeric sequence primers such as M2 / TS. For example, telomerase synthesizes telomerase DNA sequences (TTAGGG) n at the 3 'end of single-stranded DNA (and RNA) primers. Thus, an oligonucleotide for separating human telomerase can have the sequence (TTAGGG) 3 (SEQ ID NO: 8). Sequences synthesized by other telomerases are identified, for example, in E.H. Blackburn, (1991) TIBS 16: 378-381. The above embodiment was tested using biotinylated oligonucleotides as primers, which were then recovered with NeutrAvidin beads. The controls were non-initiating oligos, such as (CCCTAA) 3 (SEQ ID NO: 9). Preferred oligonucleotides for affinity purification in this embodiment are M2 / TS and (TTAGGG) 3 (SEQ ID NO: 8). The sequence of useful oligonucleotides is given in Table 3.

TABLE 3 Name of Oligo Size (nt) Description Performance (TTAGGG) s 18 Telomeric initiator Good reduction, recovery of (SEC ID NO: 8) telomerase M2 / TS 18 Initiator no Good reduction, telomeric recovery of telomerase Biotin-SS- Telomeric initiator Light reduction, (TTAGGG) 3 divisible little recovery (SEQ ID NO: 8) Oligonucleotide Sequences: M2 / TS (SEQ ID NO: 10): 5'- BIOTIN - aatccgtcga gcagagtt - 3 'With the various primers used, the efficiency of the reduction correlated with the resistance of the initiator in an elongation assay of telomerase primer. The M2 / TS initiator showed the best ability to reduce and retain activity. Some of the telomerase activity was eluted with DTT from a reduction of biotinyl-SS- initiator (TTAGGG) 3 (SEQ ID NO: 8) (the DTT breaks the disulfide bond in the initiator, freeing it from the beads) . The production was only 1-5 $, but the purity was rather high (the lower protein concentration for detection by assay of standard proteins). In another embodiment of this step, the affinity agent is an antibody that specifically recognizes a telomerase epitope. Antibodies useful as affinity reagents include the antibodies against nucleolin and antibodies against the tri-methyloguanisine plug structure ("TMG") found in several small nuclear RNAs. A source of antibodies that can recognize human telomerase are antibodies that recognize telomerase from other organisms. These antibodies can react by crossing with telomerase by homology. The 80 kD and 95 kD primary sequences of tetrahimena Telomerase protein subunits were analyzed for regions of antigenicity and surface probability. From this analysis, two peptides were designed for those of 80 kD and one for those of 95 kD. These peptides have the following sequences: GP 80A: CRKKTMFRYLS VTNKQKWDQT KKKRKEN (SEQ ID NO: 11) - 80 kD GP 80B protein: CHISEPKERV YKILGKKYPK TEEE (SEQ ID NO: 12) 80 kD GP 95A protein: DNNLCILALL RFLLSLERFN IL (ID SEC NO: 13) - 95 kD protein These peptides were used to increase the antibodies. When Tetrahimena protein sequences are chosen for this purpose, it is very important to select the surface probability because it is very possible that antibodies against external forms of telomerase immunoprecipitate the activity of telomerase from other organisms. In another embodiment, the antibodies are augmented against fusion proteins that contain a portion of a telomerase polypeptide component and prepared in, for example, an E. coli system. In another embodiment, the antibodies are identified from antibodies that recognize epitopes in enzymes that are functionally related to telomerase, for example, DNA replication enzymes and reverse transcripts. In another embodiment, the antibodies are augmented against peptides of the human telomerase protein sequence. In another embodiment of this step, the affinity agent is an inhibitor of telomerase activity that binds to telomerase. Telomerase inhibitors and methods of assaying for them are described in Prowse et al., United States patent application 08 / 288,501, filed August 10, 1994. When they adhere to a recoverable label, these compounds provide a hook through which they separate telomerase. After molecules that do not bind to the affinity agent are removed, the bound telomerase is released therefrom. When the affinity agent is an oligonucleotide, ends of ionic strength (e.g., elevated salt (approximately 500 mM NaCl) or low (zero)), exposure to high concentrations of nucleotides, and the use of divisible oligos (oligos) are attempted. of disulfide that appear in Table 3) as efforts to release the bound enzyme. Washing a column of Oligo 5 with saline solutions of increasing ionic strength liberates telomerase. When affinity column matrix of M2 / TS was boiled with bound telomerase, and the released material was analyzed in an SDS gel impregnated in silver, very few bands appeared (less than 10). Although in this experiment the telomerase proteins were rather below the limit of detection, this result indicated that very few of the other proteins bound to the affinity matrix. Thus, the specificity of this affinity reduction was high. Adding a second affinity step significantly increases the purity of telomerase. For example, if an affinity step of an oligo 14ab is followed by purification of affinity with antinucleolin or anti-TMG, the telomerase is purified at about 500,000 times. 7. Purification Based on Molecular Size, Shape or Floating Density In a preferred method of the invention, the affinity purification step is followed by a step that includes separating telomerase from other organic biomolecules according to molecular size, shape or the floating density. Matrices that separate molecules according to size, such as gel exclusion chromatography, are especially useful at this stage. A preferred matrix is the size classification column TosoHaas TSK-Gel * G5000PWXL. Telomerase in these fractions was purified at more than 60,000 times relative purity. The fractions containing telomerase activity in this step were separated by SDS-PAGE. These contained approximately 50 visible protein bands. The separation of the bands from the gel results results in a substantially pure protein.

Purified telomerase is useful to produce antibodies against the protein. It is also useful as a control in assays to detect telomerase in a sample. Recombinant telomerase, produced in cells, is useful for immortalizing those cells. Immortalization is useful in the preparation of self-replicating cell lines. III. Telomerase Related Proteins After purifying telomerase using the protocol presented in Fig. 4, the proteins in fractions containing telomerase activity were examined by gel electrophoresis. Two polypeptides were identified in the purified fractions that were co-purified with telomerase activity and found in approximate stoichiometric amounts with the RNA component of telomerase. One protein, designated bl20, had an apparent molecular mass of 120 kD in SDS-PAGE. Another protein, designated yl05, had an apparent molecular mass of 105 kD on SDS-PAGE. The proteins were further characterized. The proteins were fragmented with protase and the amino acid sequence was determined by mass spectrometry with nano-eletrospray (nanoelectroroidal). This method is described in M. Wilm et al., (1996) "Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectometry" (Sequence of the femtomole of polyacrylamide gel proteins by means of mass spectrometry with nano- eletrospray (nanoelectroroidal)), Nature 379: 466-469. The proteins were further characterized. The proteins were fragmented with protase, and the amino acid sequence of the fragments was determined by mass spectrometry of nanoelectrospray (nanoelectrorored). This method is described in M. Wilm et al., (1996) "Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray spectrometry" (Sequence of femtomole proteins of polyacrylamide gels by means of nanoelectrospray spectrometry), Nature 379: 466-469. The amino acid sequences of peptides derived from bl20 / yl05 were compared to known amino acid sequences in the Genbank sequence database. It was identified that the bl20 peptide fragments have identical amino acid sequences to the elongation factor 2 homologue sequences ("HF2E"). See N. Nomura et al., "Prediction of the coding sequences of unidentified human genes I. The coding sequences of 40 new gene-s (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human ommature myeloid cell line KG -1"(Prediction of the coding sequences of unidentified human genes I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of cDNA clones from random samples from myeloid ommature cell line KG -1), (1994) DNA Res. (Japan) 1: 27-35. GENBANK acquisition record # D21163. HF2E is a randomly separated cDNA from a cDNA library whose sequence is homologous to the elongation factor 2. Its function was not previously identified. The elongation factor 2 works in the translocation of mRNA in the ribosome. The bl20 polypeptide is referred to herein as the "elongation factor 2 homolog". It was identified that the peptide fragments of yl05 have identical amino acid sequences to the nucleolin sequences. Nucleolin is a resident nucleolar protein that is understood to function as a set of ribosomes. See, for example M. Srivastava et al. (1989) "Cloning and sequencing of the human nucleolin cDNA" (Cloning and sequencing of human nucleolin cDNA), FEBS Letters 250: 99-105 and M. Srivastava et al. (1990) "Genomic organization and chromosomal localization of the human nucleolin gene" (Genomic organization and chromosomal localization of the human nucleolin gene), J. Biol. Chem., 265: 14922-931. GENBANK acquisition record n ° M60858 J05584. The yl05 polypeptide is referred to herein as "nucleolin". IV. Uses of Telomerase Related Proteins Telomerase activity can be immunoprecipitated from nuclear fractions with antinucleoline antibodies. Because it binds to telomerase, nucleolin is a protein associated with telomerase. Nucleolin functions in vivo as a factor of telomerase activity. Accordingly, this invention provides methods for separating telomerase wherein telomerase is contacted with nucleolin and the telomerase-nucleolin complex is separated from other contaminating molecules. In one embodiment, an affinity matrix comprising nucleolin is contacted with telomerase, and contaminating molecules are washed out of the matrix. In another embodiment, nucleolin is first derived with a pof binders, such as GST / glutathione or biotin / streptavidin and then contacted with telomerase to form a complex. The complex is captured in an affinity matrix derived with the binder co-ingredient. In another embodiment, telomerase associated with nucleolin (either in its natural state or by prior contact with telomerase) is separated from a mixture using an affinity matrix comprising antinucleolin antibodies. Apparently, nucleolin functions in the telomerase complex process and the telomerase-mediated extension of chromosome telomeres in vivo. This invention provides methods for making natural or recombinant telomerase which includes expressing recombinant nucleolin in a cell expressing recombinant telomerase components. This invention provides methods for screening compounds to identify agents that alter the association of telomerase-associated proteins, such as nucleolin or HF2E, with telomerase. The methods generally comprise putting a compound in contact with the protein associated with telomerase and / or with telomerase, and determining whether the association between the protein associated with telomerase and telomerase is altered by the presence of the compound. An alteration indicates that the agent alters the binding of telomerase with the associated protein. The binding of a protein associated with telomerase can be determined by means of a variety of means, including the described methods that determine whether telomerase can be co-separated or co-detected with the associated protein. These methods are useful for identifying agents that can modulate the assembly or activity of telomerase in vivo. It is believed that nucleolin and HF2E function as telomerase activity factors in vivo. This invention provides methods for screening compounds to identify agents that alter the activity or function of telomerase, such as nucleolin or HF2E, in a cell that produces telomerase, by contacting the cell with the agent, and determining whether the activity or the function of telomerase is altered, for example, by measuring telomerase activity or telomere length. In another method, the compound is contacted with telomerase and / or telomerase activity factor in vi tro, and the ability of telomerase activity factor to alter telomerase activity is determined and compared with its capacity to alter this activity without the presence of the compound. This invention also provides methods for inhibiting telomerase assembly or telomerase activity in vivo by inhibiting the activity of a telomerase activity factor, such as nucleolin or HF2E. In one embodiment, the methods comprise the inhibition of telomerase assembly or activity by providing the cell with an antisensitivity molecule directed against the DNA or RNA encoding the telomerase activity factor. In another embodiment, the methods comprise the expression of non-functional mutant versions of a telomerase activity factor that acts as a decoy against the natural activity of these proteins. These mutants can be, for example, fragments of the native protein, mutants comprising several substitutions, additions, deletions and equivalents without amino acid preservatives. Both nucleolin and HF2E are useful for identifying other telomerase-related proteins that bind nucleolin or HF2E, as may be carried out, for example, using two-hybrid analysis. See, for example, Chien et al. (1991) Proc. Nati Acad. Sci. (USA) 88: 9578. This analysis identifies protein-protein interactions in vivo through the reconstitution of a transcriptional activator, yeast Gal4 yeast transcription protein. See Fields and Song (1989) Nature 340: 245. The method is based on the properties of the yeast Gal4 protein, which comprises separable domains responsible for DNA binding and transcriptional activation. Polynucleotides, generally expression vectors, which encode two hybrid proteins are constructed. A polynucleotide comprises the yeast Gal4 DNA binding domain linked to the polypeptide sequence of a known protein (eg, nucleolin or HF2E). The other polynucleotide comprises Gal4 activation domain linked to a polypeptide sequence of a second protein to be tested (eg, cDNA from a library). The constructs are introduced into a yeast host cell. In expression, the intermolecular binding between the joined moieties of the two fused proteins can reconstitute Gal4 DNA binding domain with Gal4 activation domain. This leads to the transcriptional activation of a reporter gene (e.g., lacZ, HIS3) operably linked to a Gal4 binding site. Variations of two hybrid systems were used to study the in vivo activity of a proteolytic enzyme. See Dasmahapatra et al. (1992) Proc. Na ti. Acad: Sci. (USA) 89: 4159. Alternatively, an interactive selection system of E. coli / BCCP can be used to identify reciprocal protein sequences (i.e., sequences of proteins that heterodiculate or form heteromultimers of higher order). See Germino et al. (1993) Proc. Nati Acad. Sci. (USA) 90: 933; Guarente (1993) Proc. Nati Acad. Sci. (USA) 90: 1639. Once the DNAs encoding the reciprocal polypeptides were identified, they can be used to screen a library of compounds (eg, small molecule libraries) to identify agents that inhibit the binding interaction. V. Polynucleotides Coding Telomerase Related Proteins This invention also provides polynucleotides having nucleotide sequences encoding telomerase-related proteins, including telomerase protein components, telomerase-associated proteins and telomerase activity factors. The nucleotide sequences encoding the telomerase-related proteins are inherent in the amino acid sequence of these polypeptides and the genetic code. These nucleotide sequences can be used in the preparation of recombinant polynucleotides used to express telomerase-related proteins in various expression systems. In one embodiment, the polynucleotide comprises the cDNA or genomic DNA sequence encoding a telomerase protein component. These polynucleotides are obtained, for example, in the following manner. The complete or partial amino acid sequences of the telomerase protein component are used to create degenerate sets of probes or polynucleotide primers that encode the amino acid sequences. As is well known to the person skilled in the art, it is preferable to select the amino acid sequences containing amino acids encoded by as few codons as possible (preferably single codons) to decrease the number of possible polynucleotides encoding the sequence. These probes or primers are then used to check or amplify polynucleotide sequences from cDNA or genomic DNA libraries. The naturally occurring polynucleotide sequences encoding telomerase-associated proteins are identified by sequencing positive clones or amplified sequences.

In another aspect, this invention provides probes and primers of polynucleotides of at least 7 nucleotides that specifically hybridize to a naturally occurring sequence encoding a protein associated with telomerase or its complement. Probes and primers are useful for detecting polynucleotides that encode telomerase protein components. Detection of the telomerase mRNA protein component is useful for detecting cancer in a cell because most cancer cells express telomerase. In another aspect, this invention provides methods for detecting a polynucleotide encoding a telomerase protein component in a sample, comprising steps (a) contacting the sample with the probe or polynucleotide initiator comprising a sequence of minus 7 nucleotides that specifically hybridizes to a nucleotide sequence selected from a nucleotide sequence of the telomerase protein component and (b) detects whether the polynucleotide specifically hybridized to the polynucleotide of the telomerase protein component. The specific hybridization provides a detection of the telomerase protein component in the sample. In another aspect this invention provides methods for inhibiting telomerase expression in a cell comprising providing the cell with an inhibitory polynucleotide that specifically binds to a polynucleotide of the telomerase protein component, eg, mRNA. The inhibitory polynucleotide may be an antisense molecule, a ribozyme, a sense molecule or a decoy encoding an inactive decoy telomerase analogue. Recombinant polynucleotides that include expression control sequences operably linked to a nucleotide sequence encoding a telomerase protein component are useful for preparing large amounts of recombinant telomerase. In one embodiment, the method comprises providing the cell with an expression apparatus for recombinant expression of the related telomerase proteins and the telomerase RNA component. Alternatively, a cell that already expresses one or more components of the ribonucleoprotein can be complemented with an expression apparatus to express the other components. SAW . Methods for generating an immunization response against telomerase Purified telomerase, proteins associated with telomerase or the natural or synthetic peptides derived therefrom are also useful for inducing an immune response in animals. Accordingly, this invention provides polynucleotide and polypeptide vaccines that elicit a humoral or mediated cell-mediated response against telomerase, telomerase-associated proteins or cells expressing telomerase. This invention also provides methods for producing antibodies that recognize telomerase or telomerase-associated proteins both polyclonal and uniclonal. The antibodies that specifically recognize telomerase or telomerase-associated proteins are useful affinity agents in methods for separating these proteins or as controls in the immunoassays for them. The methods comprise immunization of an animal with purified telomerase, a protein associated with telomerase or a fragment of any of the foregoing. Antibodies that specifically recognize telomerase or a protein associated with telomerase are also useful for detecting the presence of these proteins in a sample, such as a cell or tissue. Because telomerase is present in most cancers, the identification of telomerase helps in the diagnosis of cancer or pre-cancer states. Detecting the presence of telomerase with antibodies is cheap and offers speed and ease. Purified telomerase or immunogenic fragments thereof are also useful in vaccines to immunize a person. A polypeptide or polynucleotide vaccine for generating an immunization response against telomerase comprises an immunogenic analogue of telomerase polypeptide or a polynucleotide encoding the analogue. In an embodiment, the immunogenic analog has a MHC Class I binder or Class II MHC binder. The use of autonomous protein fragments as immunogenic is known in the art and is described for initiation of Cytotoxic T lymphocytes in International Publication WO 94/03205. Vaccines that comprise these materials are useful in treating diseases associated with over expression of telomerase, such as cancer. I. PROTOCOL FOR TELOMERASE ASSAYS. A. Essay of Elongation of Initiator, a) Essay. The primer elongation assay described in this example is the standard for determining the specific activity, for the purposes of determining the relative purity of the preparation of a telomerase. The telomerase reaction of 40 μl should have a final concentration of: 1. Telomerase IX buffer (see below). 2. 2 mM MgCl2. 3. 2 mM dATP, 2 mM dTTP, 8 μM dGTP. 4. 1 μM or 0.25 μg / reaction of oligomer-initiator M2 / TS. . 20 μCi / reaction of (a32P-dGTP (0.624 μM dGTP, specific activity 800 Ci / mmol, NEN).

To start the reaction, 20 μl of the 2X reaction mixture should be combined with an equal volume (20 μl) of enzyme extract. (For the control of RNase, add 1 μl of RNase A at 5 mg / ml to the buffer mixture just before adding the extract. (Final concentration of RNase is 125 μg / ml) Incubate at 30 ° C for 90 minutes (total volume = 40 μl) To stop the reaction add 50 μl of TE stop solution (10 M Tris pH 7.5 and 20 mM EDTA) containing 100 μg / ml RNase A (Existence = 10 mg / ml, dilution to 100X). (Final concentration is 55 μg / ml) Incubate at 37 ° C for 15 minutes (total volume = 90 μl) • To remove proteins add 50 ml of 10 mM Tris, pH 7.5, 0.5% SDS, and 300 μg / ml proteinase K. (Final PK concentration is 107 μg / ml and 0.18% SDS, replace with fresh material each time.) For 1 ml: 10 μl of Tris 1.0 M 25 μl 20% SDS 30 μl PK (10 mg / ml) 0.935 ml water Depc Incubate at 37 ° C for 15 minutes (total volume = 14 μl) Extract with an equal volume (140 μl) of PCIA (phenol: chloroform: isoamyl alcohol (25: 24: 1)). Transfer the aqueous phase to fresh tube. 8. Precipitate DNA by adding μL of NHOAC, 2.5 M (good for precipitating small oligos) containing 100 μg / mL of tRNA (10 mg / mL of stock, 100X dilution, 30 μg of carrier / tube) and 2 to 3 volumes ( 500 μl) of cold absolute ethanol. 9. Let stand at -20 ° C for 30 minutes or overnight. 10. Spin in microcentrifuge for 15 minutes at room temperature. 11. Discard supernatant and dry the pellet or granule in a vacuum apparatus with "speed-vac" speed or air dry overnight. Optional: rinse the granule with 50 μl of cold ethanol. 12. Resuspend the granule in 3 μl of charge dye in sequence (99.9% formamide, 0.05% xylene cyanol, and 0.05% bromophenyl blue). 13. Boil for one minute and cool on ice. 14. Load samples in a sequencing gel of 8% acrylamide / 7 M urea and run at 1500 V (50 W), (until the BPB is 2/3 to 3/4 of the descending path). 15. Transfer the gel to Whatman filter paper and dry at 80 ° C for 35 minutes, and cool for 15 minutes before removing the gel from the dryer. 16. Expose the gel to the screen of the Phosphor-Imager (Molecular Dynamics). Typical exposures are between 6 and 24 hours. a) Solutions with Human Telomerase Tests. 1. Telomerase Buffer 10X. Component amt / 10 ml (Stock) (10X) (IX) For 10 ml of 10X 1. Tris.Cl pH 7. .5 1 M 500 mM 50 M 5 ml 2. Espermii dina • • 3HC1 1 M 10 mM 1 mM 100 μl 3. BMED 14.3 M 50 mM 5 mM 35 μl 4. 1 M MgCl2 10 mM 1 mM 100 μL . K-OAc 5 M 500 mM 50 M 1 ml 6. EGTA 0.5 M 10 mM 1 mM 200 μl DEPC 3,565 ml D Alternatively, make (-) BME, aliquot 1 ml. Add 3.5 μl of BME to give 10X before use. 2. Stop the Component Solution (Existence) [Final] amt / 10 ml 1. Tris.Cl pH 7.5 1 M 10 mM 10C) μl 2. EDTA 200 mM 20 mM 1 ml 3. cold dNTPs Use for example Pharmacia (Uppsala, Sweden), 100 mM of stock. Combine 10 μl of dATP and 10 μl of dTTP in a tube and store at -20 ° C or -70 ° C.

Do not use the same tube more than 3x since the dNTP are unstable with repetitive freeze / thaw operations. When a solution is made, use 10 mM of Tris.Cl, pH of 7.5 (do not use water since the acidic pH will destroy the nucleotide). 4. Oli-Initiator Prepare the sequence in synthesizer or buy commercially (for example, Operon). Gel (10% acrylamide / 7M Urea), purify the crude oil and concentrate using the C-18 column. Dry in a "speed-vac" apparatus, and resuspend the granule in 10 mM of Tris.Cl with a pH of 7.5. Usually a yield of 30% to 45% is obtained. About half of the oligo is lost during the purification of the gel and 5% to 30% of the spine. Preferably purify 300 to 600 μg and resuspend the final granule in 40 μl of buffer. Determine the concentration by reading the absorption at 280 nm. Assume for an oligo that 1 DO = 30 μg / ml. A. Spot Spot Assay for Telomerase Activity (Dot Biot). 1. Combine components of the Assay Mixture: Volume Stock Final Concentration (in 40 μl) 4.0 μl HTB 10X IX 0.4 μl 100 mM of 1 mM dATP 0.4 μl 100 mM of 1 mM dGTP 0.4 μl 100 mM of 1 mM dTTP 1.0 μl 0.25 μg / ml Oligo 1 μM MS / TS 13.8 Water depc μL V = 20.0 ul 2. Add 20 μl of test extract, mix and incubate at 30 ° C for 90 minutes. 3. Add 160 μl of 0.5 M NaOH-, 12.5 mM EDTA (Final Concentration = 0.4 M, 10 mM), mix and let stand at room temperature for 5 minutes. 4. Transfer the samples to the Silent Monitor, Biodyne® B (0.45 μM), 96-well box, then place the box in the vacuum main tube to filter the sample. 5. Turn off the vacuum and add 200 μl of 0.4 M NaOH to the cavities, and apply vacuum until the filter is highly dry. 6. Peel the membrane filter, rinse in 2X SSC (to neutralize the NaOH), and place in 50 ml of preheated prehybridization mixture (6X SSC, Denhardt's IX, 20 mM NaPhos, pH 7.2, 0.4% SDS, water depc) during 1 hour at 65 ° C.

Do ribo-probe using the transcription kit for Estratagena RNA (AD? Template of pBLRep4 digested with HindIII, polymerase of AR? T3). To stop the reaction, add 1 μl PNase-free Dasease and incubate at 37 ° C for 15 minutes, PCIA extract (equal volume), add 1/10 volumes of 3 M aOAc and 2.5 volumes of ethanol for precipitate the RNA probe. Centrifuge for 10 minutes and dissolve RNA in 100 μl of TE in depc water (diethylpyrocarbonate). Hybridize the spot (biot) by adding 50 μl of the probe per filter and incubate overnight at 65 ° C. The next day, heat the wash solution (SSC IX, 0.1% SDS) to 65 ° C and transfer the filter to the washing solution. Rinse quickly, transfer the filter to fresh solution, discard the wash until radioactive waste and repeat. Wash four more times for 15 minutes each time at 65 ° C. Remove the filter from the washing solution and drain excess liquid. Seal in bag and expose to Pl screen for 1 hour. Track the screen and quantify, using the grid. C. Assay RT-PCR. to. Preparation of AR ?: The AR? it is extracted from the column fraction: 300 μl / reaction.

Prepare 1 ml of 10% SDS, 100 mM EDTA solution in fresh form before use: 200 μl of stock, 500 M EDTA, 800 μl of stock, 10% SDS up to 1 ml. In each reaction, add 30 μl of previous SDS, EDTA buffer and 5 μl of stock. Proteinase K (10 μg / μl) to each reaction, so that the final concentration will be: 1% SDS, 10 mM EDTA, 50 μg proteinase K. Incubate at 37 ° C for 10 minutes. Phenol extract: chloroform, twice (be careful not to take the material from the white interface). To the final supernatant, 30 μl of 3 M sodium acetate are added, and the nucleic acids are precipitated during the addition of 900 μl of 100% ethanol and incubation with -70 ° C or dry ice for 30 minutes. To turn the precipitate down, microcentrifuge at full speed for 15 minutes, remove all the liquid, and use 200 μl of 85% ethanol to rinse the granule, and then use the "speed-vac" apparatus to dry the granule. Resuspend the granule in 30 μl of water Depc. Take 1 μl of resuspension in 100 μl of water Depc, and read D026onm.

Use DO260 = 40 μg / ml to calculate the RNA concentration. b. First Band cDNA synthesis: 1. Take 0.1 to 1 μg of RNA made and form each of the telomerase fractions; it is mixed with 40 to 80 ng of random hexamer, up to 10 μl. Choose the random hexamer, for example from Pharmacia pd (?) ß, total 50 OD of powder units per bottle. Use 90 units of DO / thousand = 2.97 mg / ml, which is 1 unit DO = 33 μg. 2. Denature at 95 ° C for 10 minutes, cool on ice and rotate down the steam above the Eppendorf tube. 3. Prepare the reaction mixture: 1 Rx 13 Rx 5x, first place, sys buffer. (BRL): 4 μl 52 μl DTT 0.1 M (BRL): 2 μl 26 μl 10 mM d d TP (BRL): 1 μl 13 μl RNAguard® (Pharmacia): 1 μl 13 μl Water Depc: 1 μl 13 μl TOTAL: 9 μl 117 μl Add 9 μl / each reaction, and incubate in a water bath of 42 ° C 4. After incubation for 1 to 2 minutes, add 1 μl Superscript II RTase (BRL) to the mixture and incubate for 60 minutes at 42 ° C 5. Stop the reaction by heating the tube for 10 minutes at 95-98 ° C. Cool on ice and turn the steam down. b. PCR Amplification of cDNA with Specific Primer Set: PCR Reaction Buffer: 1 Rx 13 Rx Initiator 1: 1 μl 13 μl Initiator 2: 1 μl 13 μl 2.5 mM dNTP: 2.5 μl 32.5 μl μ / μl of Taq polymerase (BM): 0.4 μl 5.2 μl 5 mg / ml of the T4 gene, protein 32 (BM) 0.04 μl 0.52 μl lOx of TCR buffer (BM): 2 μl 26 μl 10 μCi / μl of a-32P ATPd: 0.5 μl 6.5 μl Depc water: 10.56 μl 137.28 IU TOTAL: 18 μl 234 μl In 2 μl of the first band cDNA, add 18 μl of the previous PCR reaction buffer. Then a drop of mineral oil is added to each PCR tube. Establish the condition for PCR amplification for clone hTR: 94 ° C for 34 seconds, 72 ° C for 45 seconds, 72 ° C for 1.5 minutes, 20 cycles. After the PCR, 5 μl of the product is mixed with a 10X DNA sequencing charge dye and the material is loaded onto 6% native polyacrylamide gel (there is no need to run the gel beforehand). Run at 250 volts for 90 minutes. Dry the gel and expose Pl. It will be clear that one can substitute the material for equivalents known in the previous protocols. D. Detection of Telomerase RNA by Northern Analysis. 1. Parameters of the gel 20 cm long gel 1 mm comb with 16 cavities 5% Acrylamide gel / 7 M Urea in lx TBE Run the O / N gel at 125 V for about 12 hours (the BPB and the XC they will have left the gel). The U2 is near the bottom, and the hTR will be about 1/4 part of the way down into the gel. The hTR is run at approximately 700 nt with respect to the DNA markers when 5% gel is used. It appears like a doublet. The RNA granules (made from 50 to 100 μl of a telomerase fraction) are resuspended in 15 μl of sequencing dye (dye-deionized formamide), boiled for a few minutes and cooled rapidly before loading: Note: Yes 1 mm of a 6% gel is used, thick, and run overnight, the hTR runs at approximately 1 kb. If an amount of 0.4 cm of a 5% thick gel is used, and if it is run at 1000 V (35-40 W), the hTR is run at approximately 450 nt. The hTR runs as a single band using this method, but if the fractions are not purified, the samples become dirty and the signal will be poor. 2. Staining of EtBr The gel is stained for 20 to 30 minutes in 0.5 μg / ml of EtBr in lx TBE to see the snRNP profile. 3. Electromancing Genie Transfer Device The transfer is performed on Hybond? +. Transfer: rT ° in lx TBE for 0.7 hours, using 0.95 A. 4. Fixation of AR? in the membrane Ultraviolet interlacing using a Stratagena interlayer; autofixation (120 mj). Intertwine with the AR? on the membrane, with its face up. 5. Probe Use either PCR (R3C and U3B primers) to make a 154 nt radioactive fragment, or label the 154 nt hTR fragment using Klenow with I hexamer.

Probe at 65 ° C and the final wash is done in O.lx SSC. Perform the stain using the Church protocol (indicated below). 6. Analysis in the Image Apparatus Phosphor-Imager Expose the spot for 5 hours on a phosphorus image creation plate (for example Fuji). The exposure time can be shortened by using concentrated extracts. Exhibition of the film: 2 to 7 days, dependent on the signal. CHURCH PROTOCOL Prehybridize in 50 to 100 ml of Church solution (500 mM Na2HP04, pH 7.2, 1 m MEDTA, 1% BSA, and 7% SDS). Prehybridize for a few hours at 65 ° C. Treat the probe with 0.1 μg of 32P-5 'of labeled label at the end (rxn forward) (specific activity of the probe should be -108-109 cpm / μg). Probe overnight at 65 ° C. Remove the stain from the bag at room temperature and rinse manually in 2x SSC (heated at T ° of hybridization) twice, for 1 minute per wash, 500 ml per wash. This serves to quickly detach from the hot label that could stick non-specifically to the membrane. Wash the stain 5x, in 2x SSC (rT °), 0.1% SDS, 5 minutes per wash, 500 ml per wash at rT °. (rT ° = room temperature). Wash spot lx, in O.lx - 2x SSC and 0.1% SDS at hybridization temperature (500 ml) for 30 minutes. Place the white spot on filter paper, wrap with Saran or bag, and expose to O / N film. Notes: 1. If more than one spot is made, start the rinse with the second spot after the first spot has been left in the second 5 minute wash. 2. Washes for 1 spot: 4 liters; 400 ml of 20x SSC and 3600 ml of ddH20. Remove 1 liter and add 15 ml to 20% SDS to 3 liters. (2x SSC = 300 mM NaCl, and therefore about half that salt concentration for hybridization). 3. Church solution (100 ml): 1 g of BSA to 50 ml of NaP04, 1 M, pH of 7.2, 15 ml of H20, dissolve, and then add 0.2 ml of 0.5 M EDTA, and 35 ml of SDS to the twenty%. II. PROTOCOLS FOR AN EXTRACT OF RAW CELLS FROM 293 CELLS. This preparation prefers a minimum of 107 cells, either suspension or adherent cells. It can be increased to achieve a proportionally larger number of cells. A preparation with such a large amount of 7.7 x 10 10 cells with excellent activity but with a slightly higher background has been achieved. In addition, the entire procedure should be carried out at 4 ° C and on ice. The cells must be cultured to a phase of medium logarithmic value. Additional cells suitable for counting must be provided: the calculation of buffer amounts depends on the number of cells and not on the volume. The CHAPS or CHPSO detergent can be used. These forms show few differences to obtain acitovs extracts. A. Wash Buffer Tampons Stock Final 20 mL lOOmL 1 M HEPES pH 7.5 10 mM 200? 1 L 1 M MgCl2 1.5 mM 30? 150? 1 M KCl 10 mM 200? 1 mL 1 M DTT 1 mM 20? lOO? H20 Type DEPC 19.55 mL 97.75 mL Lysis Buffer (0.5% CHAPS / CHAPSO) Stock Final 10 mL 50 mL 60 mL 1 M Tris Cl, pH of 7. 5 10 mM lOO? 500μl 600μl 1 M MgCl 2 1 mM 10? 50μl 60 μl 0 5 M EGTA 1 mM 20? lOOμl 120 μl 0 1 M of PMSF 0.1 M lO? 50μl 60 μl BME 5 mM 3? 15 18 μl % Detergent 0.5% 500? 2500μl 3000μl % Glycerol 10% 1 mL 5 mL 6000 μl H20 Type DEPC 8.36 mL 41.785mL 50.14mL Procedure For adherent cells, two sets of 15 cm boxes should be grown and one used for counting. Once the cell count is determined, the other set must be rinsed twice with 20 mL of cold PBS. Add 10 L of PBS and scrape the cells to enter a 15 mL centrifuge tube. Continue with step 3. For suspension cultures, cultivate the material at a density no greater than 10 6 cells / ml (about 7 X 105 / ml is preferred). After establishing the count of the cells, it is necessary to convert into pellets in cells centrifuges of 5 mL at 200 g / 4 ° C / 5 minutes. It is resuspended in cold PBS equivalent to the original volume. Granulate the cells in a clinical centrifuge apparatus at 200 g / 4 ° C / 5 minutes. The material is resuspended completely in washing buffer at a concentration of 10 6 cells / 100 [mu] l. and it is transferred to microcentrifugal tubes. Turn the cells down at 13 k rpm for 1 minute. Remove the wash buffer and resuspend the granules in lysis buffer with detergent at a concentration of 106 cells / 18.5? and transfer the material to a suitable ultra-safe tube (see below). Leave the material on ice for 30 minutes. In case of necessity you have to convert a Parafilm the upper part of the tube.

Volume Centrifugal tube from Beckmann Rotor < 1 mL thick-walled icro-polyalomer TLS 55 1.2 L thin-walled micro-polyalomer TLS 55 2 to 5 mL polyalómer SW 41 . Prepare the ultrafluous rotors of suitable oscillating cells and cool the appropriate ultracentrifuge at 4 ° C. For the SW41 rotor, place the XL-80 Ultra at 28.5 k rpm for 100,000 g. For the TLS 55 rotor, place the Ultra tabletop at a speed of 39k rpm. 6. Rotate the cells subjected to lysis for a period of 30 minutes. 7. Collect the clear aqueous portion of the supernatant (This part is the extract). Divide the extract into aliquots of 100? and freeze on dry ice.

Store at -80 ° C. III. FOUR STEPS METHOD FOR PURIFYING TELOMERASE A method is described for making human telomesara that is 3550 times more purified than the material found in the extracts of the crude cells. This method comprises four steps in succession: 1) S-100 with CHAPS detergent from 293 cells; 2) chromatography of the S-100 extract on a POROS® 50 HQ matrix; 3) chromatography of the active fractions of POROS® 50 HQ on a matrix of POROS® 20 Heparin HE-1; and 4) chromatography of the active POROS® 20 Heparin HE-1 fractions on a matrix with affinity for Oligo 5. The protocols are provided for each step. In the first step, 7.3 x 1010 293 cells were harvested from 128 liters of suspension culture and the CHAPS was extracted as described in Example II to yield 883 ml of the CHAPS S-100 extract. In the second step this extract was subjected to chromatography on a PROSO® 50 HQ column, and the fractions containing a telomerase activity were combined for a total active 72 ml pool material. This step was carried out as follows: The POROS® 50 HQ resin (PerSeptive Biosystems, Cambridge, MA, catalog number 1-2559-05) was resuspended in an equal volume of buffer (20 M Hepes pH 7.9, 2 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.1% Nonidet P-40, 1 mM Dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM Benzamidine, 1 mM Sodium Metabisulfite), equilibrated with 100 mM of NaCl (buffer A / 100 mM NaCl) and the aqueous slide was packed by gravity on a 26/20 XK chromatography column (Pharmacia, Uppsala, Sweden, catalog number 18-1000-72). The column was operated on a GradiFrac® system (Pharmacia, Uppsala, Sweden, catalog number 13-2192-01).

The column was equilibrated with 3 column volumes of the buffer A / 100 mM NaCl, followed by a wash in high salt content, applying 3 column volumes of buffer A / 2000 mM NaCl. Finally, the column was rebalanced with 3 column volumes of buffer A / 100 mM NaCl. The binder capacity of the column was determined by loading increasing amounts of the CHAPS extract until a telomerase activity was detected in the flow fractions. It was found that the capacity of the POROS® 50 HQ was in the range of 20 milligrams of CHAPS extract per milliliter of resin. 1.6 grams of CHAPS extract was loaded onto a column containing 80 ml of POROS® 50 HQ at a flow rate of 20 ml / min. The column was then washed with 3 volumes of buffer A / 100 mM NaCl. The first elution was carried out by washing the column with a step with a medium amount of salt, in an amount of 3 column volumes (buffer A / 480 mM NaCl). Telomerase activity was recovered by a step with high salt content (Buffer A / 1050 mM NaCl). The fractions from the elution with high salt content were dialyzed separately against buffer A, overnight. Fractions were scanned for telomerase activity by an elongation test of the initiator. Active fractions were collected. This procedure was repeated several times to handle the entire 6.8 grams of the CHAPS S-100 extract. To determine the relative specific activity of telomerase for the CHAPS S-100 extract and the pooled active material POROS® 50 HQ, protein concentrations were determined for each one (Reagent for Coomassie Protein Assay, Pierce Product # 23200, Rockford, IL), and the preparations were compared to establish their relative telomerase activity by the primer elongation assay as described in Example I (Figure 5). The dried gel was exposed to a phosphor imaging screen for 4 to 16 hours. The screen was scanned and the screen was adjusted and the gray scale was adjusted in order to produce an image that appeared in linear condition with respect to the corresponding assay titration. This usually occurred between 5 and 25 at the lower end and between 1000 and 2000 at the upper end. Telomerase activity was measured in arbitrary units and was derived from a visual evaluation of the signal resulting from a titration of the fractions in a range of 10 to 20 times greater, increasing by 2 times in each case. The relative comparisons of the activity between the fractions were determined from the linear range of each titration. Lanes 1 to 5 of Figure 5 show initiator elongation products from telomerase activity in 1.25 μl to 20 μl of extract S-100 with CHAPS; lanes 6 to 10 show the same result from a tritration of active pooled material POROS® 50 HQ. The amount of telomerase products increases in proportion to the amount of the preparation tested in lanes 3 to 5 and in lanes 6 to 10, providing a linear range for comparison between the two preparations. From the linear range for each preparation, it was estimated that an amount of 20 μl of the CHAPS S-100 extract (lane 5) generates the same amount of telomerase primer elongation products as 2.5 μl of the active pooled material POROS® 50 HQ (lane 7). Accordingly, it can be said that these volumes each contain an arbitrary unit of telomerase activity. This arbitrary unit is only important for this particular comparison. Using the measurements of volume, protein concentration, and volume per unit of telomerase activity, simple calculations are performed that provide the total of the units as well as the total amount of protein, from which the relative specific activity of the protein was derived. telomerase for the two preparations (Table 4). The specific activity of the active material POROS® 50 HQ (0.037 units / μg) is 5.8 times greater than that of the CHAPS S-100 extract (0.0065 units / μg). Therefore, the human telomerase in the active reactive material POROS® 50 HQ has a relative purity increased by 5.8 times compared to that of the CHAPS S-100 extract. The total units of telomerase activity in the active material POROS® 50 HQ (28,800 units) represents 65% of that of the CHAPS S-100 extract (44,150 units). Accordingly, POROS® 50 HQ chromatography has a 65% yield for telomerase activity. In the third step, 24 ml of a POROS® 50 HQ active pooled material was subjected to chromatography on a POROS® Heparin 20 HE-1 column, and the fractions having a telomerase activity were combined to achieve a total active pooled material of ß ml. Chromatography was carried out in the following manner. The POROS® 20 HE-1 was obtained from PerSeptive (Cambridge, MA, catalog number 1-5229-06). Resin handling, packing and column equilibration were carried out in exactly the same manner as described for the above resin, except that an XK 16/20 chromatography column was used (Pharmacia, Uppsala, Sweden, catalog number 18-8773-01). It was determined that the binder capacity was in the range of 15 mg / ml resin. 146.4 milligrams of pooled POROS® 50 HQ material was loaded onto a 25 ml column of POROS® Heparin HE-1 with a flow rate of 15 ml / min. The column was then washed with 3 volumes of buffer A / 100 mM NaCl. The first elution was carried out by washing the column with a medium salt pass with 3 column volumes (buffer A / 347 mM NaCl). Telomerase activity was recovered by a passage with high salt content (Buffer A / 1430 mM NaCl). The fractions from the elution were dialysed separately with salt in high quantity, against buffer A, overnight. Fractions were scanned for telomerase activity by initiator elongation assays. Active fractions were collected. The relative specific activity of telomerase for the pooled active material POROS® 50 HQ and the pooled active material of POROS® Heparin 20 HE-1 was determined exactly as described above for the previous step. The titrations of the two preparations in the primer elongation test are shown in Figure 6; the measured and calculated values are shown in Table 4. These data indicate that human telomerase in the pooled active material of POROS® Heparin 20 HE-1 is purified in 6.8 times higher compared to the level found in the active pooled material of POROS® 50 HQ. The cumulative purification after this third step is the product of all the previous steps. Accordingly, human telomerase has in the pooled active material of POROS® Heparin 20 HE-1 an increased relative purity of 6.8 x 5.8 = 39.4 times compared to that found in the CHAPS S-100 extract. The performance of the chromatography of POROS® Heparin 20 HE-1 is 75% for the activity of telomerase, and the cumulative yield after this third step is 49%. In the fourth step, 0.14 ml of the active pooled material of POROS® Heparin 20 HE-1 was subjected to chromatography in an affinity matrix with the use of Oligo 5 as the affinity ligature. Fractions from the affinity column containing the telomerase activity were combined to achieve a total active pool material of 0.4 ml. Affinity chromatography was carried out in the following manner. NeutrAvidin accounts immobilized with UltraLink®(Pierce, Rockford, IL, catalog number 53151) were pretreated with an equal volume of buffer A / 100 mM NaCl supplemented with 1 mg / ml BSA, 0.2 mg / ml tRNA and 0.2 mg / ml DNA testicles of salmon for 1 hour at 4 ° C. The beads were then rinsed with 5 volumes of buffer A / 100 mM NaCl. Oligo 5, an antisense DNA oligonucleotide (National Biosciences, Inc., Plymouth, MN) covering nucleotides 407-436 of human telomerase RNA was identified with the sequences 5 '- * gccgagtcct gggtgcacgt cccatagctc-3' ( in which * G is biotinylated) (identification of sequence number 4). Oligo 5 was resuspended in a concentration of 100 μM for use in subsequent experiments. 140 μl of pooled heparin material with a protein concentration of 9 mg / ml was supplemented, applying 1.6 mM of 5.2 mM oligo dTTP, 0.1 mM ddATP, 2 mM dGTP, 50 mM Tris HCl with pH of 7.5, 1 mM of Spermidine (spermidine), 5 mM of D-mercaptoethanol, 1 mM of MgCl2, 50 mM of Potassium Acetate, 1 M of EGTA and the material was incubated at 30 ° C for 1 hour. The reaction mixture was added to 400 μl of pretreated NeutrAvidin beads and mixed at 4 ° C for 1 to 4 hours. The aqueous paste was then emptied into a small disposable column and the flow was drained. The flow rate was returned through the column up to three times. The column was then washed with at least 4 column volumes of buffer A / 100 mM NaCl and eluted with buffer A / 500 mM NaCl. Fractions were tested for telomerase activity by initiator elongation assays. The relative specific activity of telomerase for these two preparations was determined exactly in the manner described above for the previous steps with one exception. Telomerase is released from the columns of POROS® 50 HQ, POROS® Heparin HE-1, and the affinity columns of oligo 5 in purification steps 2, 3 and 4 by increasing the salt concentration in the wash buffer. In view of the fact that salt concentrations beyond 100 mM inhibit telomerase activity (Figure 7, compare lanes 7 and 9), the assembled active materials from purification steps 2 and 3 were dialyzed to lower the salt concentration to level of the preparation loaded in the column, for such steps. In the purification step 4 (affinity of oligo 5), the assembled material of the active fractions was not desalted. To determine a precise relative activity, the salt concentration of the loaded preparation was adjusted on the affinity column (active pooled material of POROS® Heparin 20 HE-1) to be the same as that of the pooled active affinity material of Oligo 5. The titrations of the two preparations are shown in the primer elongation assay in Figure 7; the measured and calculated values are shown in Table 4. These data indicate that human telomerase in the active pooled material of Oligo 5 is 90 times more purified as compared to the degree obtained in the active pooled material of POROS® Heparin 20 HE -1. The cumulative purification after this fourth step is 90 6.8 * 5.8 = 3.550. Thus, human telomerase made with this method has a relative purity increased 3, 500 times compared to that obtained in the extract of CHAPS S-100. The yield of the affinity chromatography step is 29% for telomerase activity; The cumulative yield of telomerase activity for this method is 14%. IV. METHOD OF FIVE STEPS TO PURIFY TELOMERASE. A method for making human telomerase having a relative purity 32,660 times higher compared to that found in crude cell extracts is described. This method comprises five steps in succession: 1) preparation of the CHAPS detergent S-100 extract from 293 cells; 2) chromatography of the S-100 extract in the POROS® 50 HQ matrix; 3) chromatography of the POROS® 50 HQ active fractions on the POROS® Heparin 20 HE-1 matrix; 4) chromatography of the active POROS® Heparin 20 HE-1 fractions on the POROS® spermidine matrix; and 5) chromatography of the active POROS® spermidine fractions on the affinity matrix of the oligonucleotide 5. Protocols are provided for the preparation of the spermidine matrix and for the operation on the spermidine column. Protocols were provided for all other steps in the 4 step method according to Example III. In the 5-step method, the first three steps are the same, in a 4-step method. In the fourth step, 0.7 ml of the pooled active material POROS® Heparin 20 HE-1 was subjected to a chromatography on a freshly prepared (ie, not commercially available) column of POROS® spermidine, and the fractions containing a telomerase activity to achieve a total active material of 0.15 ml. Chromatography was carried out in the following manner. The POROS®-Spermidine material was prepared in the following manner. The dry POROS® 20 EP (PerSeptive 1-6129-03) was placed in coupling buffer (0.1 M sodium phosphate adjusted to a pH of 10.0 with KOH) to allow the beads to hydrate. The hydrated beads were transferred to a disposable column and rinsed with coupler buffer for a volume of 20 beds. Two bed volumes of a material consisting of 0.2 M spermidine tetrachlorhydrate (Sigma) and 0.1 M sodium phosphate were added. The solution of the spermidine coupler buffer was readjusted to a pH of 10. (20 ml sodium phosphate 0.1 M-spermidine 0.22 M + 1.5 mL of 1 N NaOH = 0.2 M Spermidine 0.1 M sodium phosphate, pH of 10.0). The beads were spun with the spermidine solution overnight at room temperature. The spermidine solution was removed by washing with a volume of 20 beads of coupling buffer. The remaining reagent groups of the POROS® 20 EP beads were quenched with a volume of 2 0.1 M ethanolamine beds constituted in a coupling buffer. The mixture was rotated for 2 hours at room temperature. The mixture was washed with equivalent volumes to 20 beds of coupling buffer. The mixture was washed with a volume of 10 beads of buffer A / 100 mM NaCl and packed in columns for chromatography. The POROS®-Spermidine column was equilibrated at 4 ° C with 10 column volumes (abbreviated VC) of Buffer A / 100 mM NaCl before sample application. The pooled active material of POROS® Heparin 20 HE-1 as a sample was dialyzed in buffer A to below 100 mM NaCl) which was applied to the column and chromatographed at a flow rate of 0.6 VC (column volumes ), per minute. The column was then washed with the same flow rate by applying 5 VC of Buffer A / 100 mM NaCl. The proteins were eluted with the same flow rate, in the following steps: -3 VC of buffer A, 100 mM of KCl, 90 mM of NaCl -3 VC of buffer A, 150 mM of KCl, 85 mM of NaCl -3 VC buffer A, 200 mM KCl, 80 mM NaCl -3 VC buffer A, 1000 mM KCl Proteins were eluted at each step, eluting telomerase primarily from the POROS®-Spermidine column with buffer A / 150 mM KCl and 85 mM NaCl. The relative specific activity of telomerase was determined for these two preparations. The titrations of the two preparations in the primer elongation assay are shown in Figure 8; the measured and calculated values are shown in Table 5. These data indicate that human telomerase in the pooled active material of POROS®-Spermidine is purified by 9.2 times more compared to the degree obtained in the active pooled material of POROS® Heparin 20 HE-1. The cumulative purification after this fourth step is 9.2 x 6.8? 5.8 = 363 times more than the level obtained in the CHAPS S-100 extract. The performance of POROS® spermidine chromatography is 30% for telomerase activity, and the cumulative yield after these four steps amounts to 14.6%. Affinity chromatography with Oligo 5 of the POROS® espermidine active fractions purifies human telomerase by a factor of 90 with a yield of 29% (as was the case with the active POROS® Heparin 20 HE-1 fractions). A) Yes, it can be said that this 5 step method produces a human telomerase having a relative purity increased by 90 363 = 32, 660 times higher compared to the level reached with the extracts of CHAPS S-100. The performance of this 5-step method is 4.2%. V. METHOD OF SIX STEPS TO PURIFY TELOMERASE. A method for making human telomerase that is purified at 65, 320 times higher than the level reached in crude cell extracts is described. This method comprises six steps in succession: 1) the preparation of the CHAPS-detergent-S-100 extract from 293 cells; 2) chromatography of the S-100 extract in the POROS® 50 HQ matrix; 3) chromatography of the active POROS® 50 HQ fractions on the POROS® Heparin 20 HE-1 matrix; 4) chromatography of the active POROS® Heparin 20 HE-1 fractions on the POROS® Spermidine matrix; 5) chromatography of the active fractions of POROS® Spermidine on the Superóse® 6 graduated column; and 6) chromatography of the active fractions of the graduated column with Superóse® 6 on the affinity matrix of Oligo 5. A protocol for operating the Superóse® 6 column is provided. Previously, the set of protocols for all the other steps. In the 6-step method, the first four steps are the same, as in the 5-step method. In the fifth step, 0.02 ml of material from the pooled active POROS® spermidine material collected in Example IV was subjected to chromatography on a Super6se® 6 grading column and the fractions containing a telomerase activity were combined to achieve a material Total active collection of 0.16 ml. Chromatography was carried out with Superóse® 6 in the following manner. The 2.4 ml column of Superóse® 6, PATIENTS 3.2 / 30 from Pharmacia is pre-equilibrated with 2 column volumes of buffer A and 150 mM NaCl with a flow rate of 40 μl / min. For each pass with Superóse® 6, 20 μl of an active POROS® spermidine fraction is loaded onto the column, which is operated with a flow rate of 20 μl / min in buffer A with 150 mM NaCl. The thickness of the absorbent proteins with D02so is eluted from the column between 1.00 ml and 1.4 ml. The peak of telomerase activity is eluted between 1.4 and 1.6 ml, on the trailing end of the protein peak. The relative specific activity of telomerase was determined for these two preparations. The titrations of the two preparations in the primer elongation test are shown in Figure 8; the measured and calculated values are shown in Table 5. These data indicate that the human telomerase in the assembled active material Superóse® 6 is purified with a 2-fold increase compared to the level reached with the active pooled material of POROS® spermidine . The cumulative purification after this fifth step is 2 x 9.2 6.8 x 5.8 = 725.7 times the relative purity obtained with the CHAPS S-100 extract. The yield of chromatography with Superóse® 6 is 20% for telomerase activity and the cumulative yield after these 5 steps is 2.9%. Affinity chromatography of Oligo 5 of the active fractions of Superóse® 6 can purify human telomerase to the same extent as for the active POROS® Heparin 20 HE-1 fractions. Thus, this 6-step method would produce human telomerase that is purified at 90 x 725.7 = 65,320 times higher than the level obtained in the CHAPS S-100 extracts. The performance of the method based on 6 steps is 0.85%. SAW. SECOND METHOD BASED ON SIX STEPS TO PURIFY TELOMERASE. A method for making human telomerase that has a purification degree of 60,000 times higher than the levels obtained in extracts with crude cells was implemented. This method comprises six steps in succession: 1) homogenization in a Dounce apparatus and preparation of the nuclear extract from 293 cells; 2) chromatography of the nuclear extract in the POROS® 50 HQ matrix; 3) chromatography of the active POROS® 50 HQ fractions in the POROS® Heparin 20 HE-1 matrix; 4) chromatography of the active POROS® Heparin 20 HE-1 fractions in the SOURCE 15Q® matrix; 5) chromatography of the active fractions of the SOURCE 15Q matrix in the affinity matrix of Oligo 14ab; and 6) chromatography of the active fractions of the affinity matrix 14ab in a sorting column of TSK-Gel * G5000PWXL. Protocols are provided for all steps. A. Preparation of Nuclear Extract. 1. Cells The 293 cells are constituted by a kidney cell line, human embryo transformed by adenovirus. These cells grow without problem in suspension culture with an optimum harvest density of 0.5 X 109 cells per liter and with a reduplication time of 24 hours. Cellex cells were obtained in the form of wet cell pellets on dry ice. 293 suspension cultures were grown in spinner bottles and were provided in batches of 2 X 10 11 cells (from 400 liters of culture). The 293 cells are harvested, the material is made twice in PBS (free of Ca / Mg), frozen in a rough form as a wet cell granule, and placed on dry ice. (Frozen cells can be stored at -80 ° C). 2. Crude Extracts. to. The cell granules are rapidly thawed, the volume of the packed cells is measured, and then the cells are suspended in an equal volume of a cold, ice-like buffer. The cells are stored on ice and interrupted using a dounce-type homogenizer equipped with a crusher or shredder B with 10 ascending and descending runs. b. The cytoplasm and nuclei are separated by a rotation at low speed: the homogenized material is centrifuged for 10 minutes at 4 ° C with a speed of 2,500 r.p.m. (revolutions per minute) in a Beckman JS4.2 rotor (1780 Xg). The supernatant is decanted into clean bottles and the granule is stored on ice for the preparation of the nuclear extract (see below, step j). Alternative Step b. If there is not enough time for a preparation of a nuclear extract, you can store the nuclear granule but first you have to supplement the buffer with glycerol. The volume of the homogenized material from step a (to be assigned to the variable and in the following equation) must be measured. Add 80% ice cold glycerol to the homogenized material to obtain a final concentration of 10%. Mix well. The volume of 80% glycerol that must be added, that is, x, can be determined by the equation: x = O.ly/0.7. Centrifuge exactly as in step b. The nuclear granules are abruptly frozen and stored at -80 ° C. The addition of glycerol does not change any of the following extraction steps. c. Measure the supernatant volume (to which the variable is assigned and in the following equation). 5 M NaCl, cold as ice, is added to the supernatant to obtain a final concentration of 150 M. It is mixed well. The volume of 5 M NaCl that must be added, that is, x, can be determined by the equation: x = 0.15y / 4.85. d. The supernatant is centrifuged for 1 hour, at 4 ° C and with 40K r.p.m. in a Beckman 45Ti rotor (100K Xg). The supernatant is decanted carefully in a clean container. and. The volume of the supernatant is measured. 259.5 g of solid ammonium sulfate (45% final concentration) are gradually added. Mix gently in the cold room for about 30 minutes. F. The precipitate is collected by centrifuging the mixture for 30 minutes at 4 ° C with a speed of 10K r.p.m. in a Sorvall GSA type rotor. The supernatant is decanted and the granules are drained very well. g. The granules are resuspended in buffer A containing 50 mM NaCl. One-fifth of the volume measured in step e (before the addition of ammonium sulfate) is used and the granule is resuspended using a dounce type homogenizer, equipped with a type A sucker. H. It is dialyzed in buffer A containing 50 mM NaCl overnight with a single buffer change. i. The dialyzed material is collected and centrifuged for 30 minutes at 4 ° C with a speed of 15K r.p.m. on a Sorvall SS-34 rotor. The supernatant is filtered through a Mira cloth. This is the "crude cytoplasmic extract". It can be frozen abruptly and stored at -80 ° C or can also be loaded directly on the TosoHaas super Q column. The crude cytoplasmic extract is typically 230 ml with 25 mg / ml protein and a quantity of 10 μl is sufficient to establish telomerase activity in the conventional primer elongation assay. j. From step b, the volume of the nuclear granule is measured. The nuclei are resuspended in 0.5 volume of Buffer C. k. A quantity of 0.5 packed nuclear volume of buffer C containing 1.2 M NaCl is added slowly and while stirring. 1. It is homogenized in the dounce apparatus (5 to 10 runs) with the forage A. m. Transfer the material to a glass beaker on ice and stir for at least 30 minutes. n. Centrifuge for 75 minutes at 4 ° C at 18K r.p.m. on a Sorvall SS-34 rotor. or. The supernatant is transferred to dialysis bags and dialysed overnight or 2 X 2 hours with the "hypo" buffer containing 100 mM NaCl. p. The dialyzed material is collected and centrifuged for 30 minutes at 4 ° C at 15K r.p.m. on a Sorvall SS-34 rotor. The clarified supernatant is the "crude nuclear extract". It freezes abruptly and is stored at -80 ° C. The crude nuclear extract is typically 650 ml with 7 mg / ml protein and a quantity of 10 μl is sufficient to establish the telomerase activity in the conventional assay. B. First Chromatography with Ion Exchange. 1. The material of the nuclear extract is subjected to the next chromatography with ion exchange. A POROS® 50 HQ resin from PerSeptive Biosystems is resuspended in an equal volume of the hypo buffer containing 100 mM NaCl and the aqueous paste is packaged by gravity on an XK 50/30 chromatography column (Pharmacia). 2. The column is operated in a GradiFrac system (Pharmacia) after having been equilibrated with 3 column volumes of hypo buffer containing 100 mM NaCl followed by a high salt wash with 3 column volumes of hypo buffer containing 2000 mM NaCl. The column is then rebalanced with 3 column volumes of the hypo buffer containing 100 mM NaCl. The capacity of the resin for telomerase is in. the range of 20 mg of nuclear extract per milliliter of resin. 3. In a typical preparation that starts with 2 X 10 cells (600 grams of cells), 650 to 700 ml of crude nuclear extract or approximately 4.7 grams of proteins are loaded onto a 300 ml column with a flow rate of 20 ml / min. The column is then washed with 3 column volumes of hypo buffer containing 100 mM NaCl. A washing step with average amount of salt is carried out with 3 volumes of hypo-buffer columns containing 404 mM NaCl. Telomerase activity is recovered by elution with high salt content with hypo buffer containing 1050 mM NaCl. The high salt content elution fractions are dialyzed against the hypo buffer containing 50 mM NaCl overnight and it is screened for telomerase activity through a conventional assay before meeting and undergoing the next step of purification. C. Chromatography with Heparin. 1. An amount of 100 to 120 ml of combined active fractions from the previous step, that is approximately 660 milligrams of proteins is loaded onto a 60 ml column of POROS® Heparin 20 HE-1 from PerSeptive Biosystems with a flow rate of 8 ml / min. Resin handling, packing and column balancing are performed in the same manner as described for the above resin, except that a column is used for XK 26/20 chromatography (Pharmacia) and because the binding capacity for this column is in the 15 mg / ml resin range. The column is then washed with 3 column volumes of hypo buffer containing 100 mM NaCl. A washing step with a medium salt level is performed with 3 column volumes of hypo buffer containing 290 mM NaCl. The telomerase activity is recovered by an elution with high salt content by applying a hypo buffer containing 1430 mM NaCl. The high salt content elution fractions are dialysed against the hypo buffer containing 250 mM NaCl overnight and screened to establish telomerase activity by a conventional assay before being pooled and subjected to the next purification step. D. Second Chromatography with Anion Exchange. 1. An amount of 20 to 40 ml of combined active fractions from the previous step, ie approximately 240 milligrams of proteins are diluted in a ratio of 1 to 1 with hypo buffer before being loaded onto a column with 16 ml of Source 15Q from Pharmacia. The column is usually operated with its maximum binding capacity with a flow rate of 5 ml / min in an FPLC device (Pharmacia). According to the number of fractions gathered from the previous step, consecutive passes are needed. Resin handling, packing and column equilibration are carried out in the same manner as described for the above resin except that a 16/10 HR chromatography column (Pharmacia) is used and because the capacity agglutinating agent for this column is at the level of approximately 10 mg / ml resin. The column is then washed with 3 column volumes of hypo buffer containing 100 mM NaCl. A wash step is carried out with an average salt level with 3 column volumes of hypo buffer containing 307 mM NaCl. Telomerase activity is recovered by elution with high salt content by applying a hypo buffer containing 1000 mM NaCl. Elution fractions with high salt content are scanned for telomerase activity by conventional assay before meeting and subjected to affinity chromatography. Starting with the nuclear extract of 2 X 1011 cells, this partial purification program provides 10 ml with 10 mg / ml of protein and 2 μl is sufficient to achieve telomerase activity in the conventional assay. The relative purity of telomerase is 100 times more than the crude extract with a yield of approximately 30%. The concentration of telomerase, through a quantitative RNA analysis, is 0.3 pinoles / ml. This material is suitable for affinity chromatography. Buffer Buffer H - 10 mM HEPES-KOH, pH 7.9; 2 mM MgCl2; 1 mM EGTA; 10 mM KCl. Add just before use: 1 mM DTT; 1 mM Sodium Metabisulfite; 1 mM Benzamidine and 0.2 mM PMSF. Buffer A - 20 mM HEPES-KOH; pH of 7.9; 1 mM MgCl2; 1 mM EGTA; 10% glycerol. Add just before use: 1 mM DTT; 1 mM Sodium Metabisulfite; 1 mM Benzamidine; 0.2 mM of PMSF; and 5 μg / ml of Leupeptin. Buffer C - 20 mM HEPES-KOH; pH of 7.9; 1.5 mM MgCl2; 0.5 mM EGTA; 20 mM NaCl; 25% glycerol. Add just before use: 1 mM DTT; 1 mM of Sodium metabisulfite; 1 mM Benzamidine; and 0.1 mM of PMSF. Hipo Buffer - 20 mM HEPES-KOH; pH of 7.9; 2 mM MgCl2; 1 mM EGTA; 10% glycerol; 0.1% of NP-40. Add just before use: 1 mM DTT; 1 mM Sodium Metabisulfite; 1 mM Benzamidine; and 0.2 mM of PMSF. E. Affinity Chromatography. 485 μl of active pool material was supplemented Source 15Q with a protein concentration of approximately 7 mg / ml with 100 μg of dT oligonucleotide (30-mer), 125 μg of polyuridyl acid (Sigma, St. Louis, MO, catalog number P-9528) and 500 picomoles of 14ab oligonucleotide as ligation (2 '-O-methyl, biotinylated, disulfide-linked, RNA oligonucleotide, Yale University, New Haven, CT) in a total volume of 500 μl. Oligo 14ab has the sequence 5 '-cgttcctctt cctgcggcctt-3' (Oligo 14ab) (sequence identification number 7) and covers nucleotides 361-380 of human telomerase RNA. After incubating for 30 minutes at room temperature, the mixture is added to 4-6 x 106 particles of MPG®-Streptavidin beads (CPG, Lincoln Park, NJ, catalog number MSTR0510) material that was equilibrated with buffer A and 700 mM NaCl and the beads were allowed to settle for approximately 10 minutes. Then the beads were subjected to a magnetic separation, the supernatant was discarded and the beads were washed with 100 μl of buffer A / 700 mM NaCl. The beads were resuspended in an elution buffer of 20 μl (Buffer A supplemented with 500 mM Tris HCl, pH 8.3 and 100 mM DTT) and the material was incubated at room temperature for 20 minutes with frequent resuspension. Again the beads were subjected to a magnetic separation, the supernatant containing the telomerase activity was collected and used as such for the subsequent purification. F. Gel Chromatography and Applying Exclusion Classifications. A telomerase tracing activity can be performed with the use of a chromatograph to the exclusion of certain sizes, in material purified by affinity. The eluted material (150 μl, 2 fmoles of telomerase / μl) from an affinity chromatography of 14ab is applied to a column containing TSK-Gel * G5000PWXL (30 cm x 7.8 mm, TosoHaas), this material already equilibrated in the buffer? B '. The column is eluted in the same buffer as a flow rate of 250 μl / min. The profiles of the activity of the telomerase, as well as the quantities of hTR are determined for the different fractions. The enzymatic activity as well as the peak of the hTR concentrations are between 7.0 ml and 7.4 ml of eluent. Buffer? B ': 0.2 M of Triethylamine-C02; 1 nM of EGTA; 1 M DTT; 1 mM MgCl2; 1% Glycerol. G. Analysis of Amino Acid Sequences. The amino acid sequences of peptide fragments of bl20 and yl05 are determined in the following manner. Acrylamide gels were prepared using the standard protocols and stained with silver stain. In gel reduction, acetamidation and tryptic digestion were similar to published procedures (JenD et al., Analyt, Biochem 224, 451-455 [1995], Rosenfeld, et al., Ana-Zyt. Biochem. , 173-179 [1992]). After washing with 100 mM NH4HC03 and acetonitrile, the gel pieces were swollen in the digestion buffer containing 50 mM NH4HC03, 5 mM CaCl2 and 12.5 ngμl-1 trypsin (Boehringer Mannheim, degree of sequencing) at 4 ° C. After 45 minutes, the supernatant was aspirated and replaced by 5 to 10 μl of the same buffer without trypsin to keep the gel pieces moist during enzymatic dissociation (37 ° C, overnight). Peptides were extracted by three changes of 5% formic acid and acetonitrile and dried in a downward direction. Approximately 10 or POROS R2 sorbent (PerSeptive Biosystems) were placed on the tip of a capillary apparatus with traction ° C 100F-10 (CEI, Pangbourne).

Note that the resin is not packaged and that the presence of some frit or other LC-type assembly is not necessary. A new capillary apparatus and a new resin portion are used for each analysis in order to avoid cross contaminations even at the femtomole level. The mixture of the dry peptides was dissolved in 10 μl of 5% formic acid, the material was loaded into the pre-equilibrated capillary apparatus, washed and eluted with 60% methanol in 5% formic acid to introduce it into the capillary sprayer The volume of elution is 10 times greater than the volume of the resin which results in a good recovery of peptides. A nano-electrode is carried out in an API III mass spectrometer (Perkin-Elmer Sciex, Ontario, Canada) as described (Wilm et al., Analyt.Chem. 68: 1-8 [1996]; Mann et al. ., Analyt, Chem. 66, 4290-4399 [1994]). For the selection of the precursor ions, quadruple 1 was established at a level suitable for transmitting a window of mass of 2 Da. The step size for the spectra of the tandem mass was 0.2 Da, and the resolution was adjusted in such a way that the masses of the fragments could be assigned to a level better than 1 Da. We searched the gene bank to identify the polypeptides that contained the amino acid sequences of the fragments yl05 and bl20. Nucleolin (gene bank M60858 J05584) contained sequences of yl05 and the elongation factor homolog 2 (gene bank D21163) contained bl20 sequences. The present invention provides novel methods for purifying telomerase. While specific examples have been provided, the above description is illustrative only and does not contain a restriction element. Many variants of the invention would be experienced by those skilled in the art once the present text has been studied. The scope of the invention should be determined, therefore, not so much with reference to the foregoing description, but rather based on the appended claims in combination with its full set of equivalences. All publications and patent documents cited in this application are incorporated herein as a reference material in its entirety and for all purposes and to the same extent as if each of such individual patent documents or publications had been transcribed as such. .

Table 4: PURIFICATION TABLE FOR THE 4-STEP METHOD Purification Performance Faso Volume Conc. Prot. Μl by Total Ac. Sp. Step Acum. Step Acum. Prot. Total "unit / unit unit activity" and g CHAPS 883 ml 7.7 mg / ml 6799.1 mg 20 μL 44.150 0.0065 100% 100% SS OO HQ 72 ml 10.7 mg / ml 770.4 mg 2. 5 μl 28, 800 0.037 5. 8x 5. 8x 65% 65% I 24 ml 6.1 mg / ml 146.4 mg 7 .5 μl 3200 0.0219 Hep 6 ml 2.7 mg / ml 16.2 mg 2. 5 μl 2400 0.148 6. 8x 39. 4x 75% 48. 8% Table 5: PURIFICATION BY POROS® ESPERMIDINA AND SUPERÓSÉ® 6 Volume Conc. Prot. Prot. Total pl by Total Act. Es. Purification Performance unit activity Unit / pg unit Hep 700 μl 7. 5 g / ml 5250 μg 1 μl 700 or 0.133 Spd 150 μl 1. 13 mg / ml 169. 5 μg 0.72 μl 208.3 or 1.23 9.2x 30% 20 μl 1. 1 mg / ml 22 μg 0.5 μl 40 u 1.82 Sup6 160 μl 0. 014 mg / ml 2.24 μg 20 μl 8 u 3.57 2x 20%

Claims (22)

1. Claims 1. A method for purifying telomerase from an impure composition containing organic biomolecules comprising contacting telomerase with an affinity agent having specific affinity to telomerase, separating telomerase from other organic biomolecules that do not bind to the affinity agent and gather telomerase. The method of claim 1 comprising: (a) contacting telomerase with a matrix that binds positively charged molecules or a matrix that binds molecules with a negative charge, separating telomerase from other organic biomolecules that are not join the matrix and gather the telomerase; and (b) contacting telomerase with an affinity agent that has a specific affinity for telomerase, separating telomerase from other organic biomolecules that do not bind with the affinity agent and bind telomerase. 3. The claim method comprising: (a) contacting the telomerase with a first matrix that binds molecules that are negatively charged, separating telomerase from other organic biomolecules that do not bind to the matrix and bind telomerase; (b) contact of telomerase with a matrix that binds molecules that are positively charged, separating telomerase from other organic biomolecules that do not bind to the matrix and bind telomerase; (c) telomerase contact with a second matrix that binds molecules that are negatively charged, separating telomerase from other organic biomolecules that do not bind to the matrix and bind telomerase; (d) contacting telomerase with an affinity agent that has specific affinity for telomerase, separating telomerase from other organic biomolecules that do not bind to the affinity agent and bind telomerase. (e) separating telomerase from other organic biomolecules according to molecular size, shape or floating density. The method of claim 2 further comprising the step for contacting telomerase with a matrix of intermediate selectivity and pooling telomerase. The method of claim 3 wherein the first and second matrices that bind negatively charged molecules are anion exchange resins, the matrix that binds positively charged molecules comprises heparin, the affinity agent is an oligonucleotide having a sequence complementary to a sequence of the RNA component of telomerase, and the separation step comprises separation according to size. 6. The method of claim 3 wherein the first matrix that binds the negatively charged molecules is POROS 50 HQ, the matrix that binds the positively charged molecules is POROS® Heparin 20 HE-1, the second matrix that binds molecules with negative charge is SOURCE 15Q®, the affinity agent comprises an oligonucleotide comprising the sequence 5 '-cgttcctctt cctgcggcct-3' (SEQ ID NO: 7), and the step to separate comprises the separation according to size in a column of Size classification TosoHaas TSK-Gel * G5000PWXL. The method of claim 4 wherein the intermediate selectivity matrix comprises a polynucleic acid, a polyamine, a divalent metal ion, a positively charged proteinaceous material or aminophenyl boronic acid. 8. The method of claim 6 wherein the impure composition is a nuclear extract of 293 cells. The method of claim 6 which further comprises the separation of telomerase from the other organic biomolecules by gel electrophoresis. 10. A composition comprising human telomerase having at least 2,000 times increased relative purity compared to the crude extract of 293 cells. The composition of claim 10 wherein the telomerase has at least 60,000 times increased relative purity. 12. A composition comprising human telomerase with at least 60,000 fold of increased relative purity compared to the crude extract of 293 cells produced by the following steps: (a) contacting telomerase, from an impure composition containing organic biopolymers, with a first matrix that binds negatively charged molecules, separating telomerase from other organic biomolecules that do not bind to the matrix and bind telomerase; (b) contacting telomerase with a matrix that binds positively charged molecules, separating telomerase from other organic biomolecules that do not bind to the matrix and bringing telomerase together (c) bringing telomerase into contact with a second matrix that binds negatively charged molecules, separating telomerase from other organic biomolecules that do not bind to the matrix and bringing telomerase together (d) bringing telomerase into contact with an affinity agent that has specific affinity with telomerase, separating telomerase from other biomolecules organic that do not bind to the affinity agent and bind telomerase; (e) Separate telomerase from the other organic biomolecules according to the molecular size, shape or floating density and gather the telomerase. 13. The composition of claim 12 wherein the impure composition is a nuclear extract of 293 cells, the first matrix that binds the negatively charged molecules is POROS 50 HQ, the matrix that binds the positively charged molecules is POROS® Heparin 20 HE- 1, the second matrix that binds negatively charged molecules is SOURCE 15Q®, the affinity agent comprises an oligonucleotide comprising the sequence 5 '-cgttcctctt cctgcggcct-3', and the step to separate comprises separation according to size in a size classification column, TosoHaas TSK-Gel * G5000PWXL. 14. A polypeptide fragment of a human telomerase protein component that, when presented to an animal is an immunogenic, elicits a humoral or cell mediated immunization response. 15. A composition comprising an antibody or antibody fragment that specifically binds a human telomerase protein component. 16. A recombinant polynucleotide comprising a nucleotide sequence encoding a polypeptide having at least 5 consecutive amino acids of a human telomerase protein component. 17. The recombinant polynucleotide of claim 16 further comprising an expression control sequence operably linked to the nucleotide sequence. 18. A polynucleotide probe or primer that specifically mixes with a polynucleotide that encodes a human telomerase protein component. 19. A recombinant cell comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding a mammalian telomerase protein component, wherein the cell produces the RNA and the enzyme protein components medullary telomerase 20. The recombinant cell of claim 19 further comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding the AR component. of telomerase. 21. A method for making recombinant telomerase comprising the culture preparation of a recombinant cell comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence encoding a mammalian telomerase protein component, in Where does the cell produce AR? and the components of the medullar telomerase enzyme protein. 22. A composition comprising human recombinant telomerase prepared by the method of claim 21.