title: One-step affinity purification protocol for human telomerase.
abstract: Human telomerase is a ribonucleoprotein (RNP) enzyme, comprising protein components and an RNA template that catalyses telomere elongation through the addition of TTAGGG repeats. Telomerase function has been implicated in aging and cancer cell immortalization. We report a rapid and efficient one-step purification protocol to obtain highly active telomerase from human cells. The purification is based on affinity chromatography of nuclear extracts with antisense oligonucleotides complementary to the template region of the human telomerase RNA component. Bound telomerase is eluted with a displacement oligonucleotide under mild conditions. The resulting affinity-purified telomerase is active in PCR-amplified telomerase assays. The purified telomerase complex has a molecular mass of approximately 550 kDa compared to the approximately 1000 kDa determined for the telomerase RNP in unfractionated nuclear extracts. The purification protocol provides a rapid and efficient tool for functional and structural studies of human telomerase.
authors.affiliation: Department of Oncology Research, Boehringer Ingelheim Pharma KG, Birkendorfer Strasse 65,88397 Biberach an der Riss, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Schnapp
authors.forename: H P
authors.initials: HP
authors.lastname: Rodi
authors.forename: W J
authors.initials: WJ
authors.lastname: Rettig
authors.forename: A
authors.initials: A
authors.lastname: Schnapp
authors.forename: K
authors.initials: K
authors.lastname: Damm
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D017931:DNA Primers; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D019098:Telomerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D019098:Telomerase