title: Telomerase repeat amplification protocol (TRAP) activity upon recombinant expression and purification of human telomerase in a bacterial system.
abstract: Telomerase biogenesis is a highly regulated process that solves the DNA end-replication problem. Recombinant expression has so far been accomplished only within a eukaryotic background. Towards structural and functional analyses, we developed bacterial expression of human telomerase. Positive activity by the telomerase repeat amplification protocol (TRAP) was identified in cell extracts of Escherichia coli expressing a sequence-optimized hTERT gene, the full-length hTR RNA with a self-splicing hepatitis delta virus ribozyme, and the human heat shock complex of Hsp90, Hsp70, p60/Hop, Hsp40, and p23. The Hsp90 inhibitor geldanamycin did not affect post-assembly TRAP activity. By various purification methods, TRAP activity was also obtained upon expression of only hTERT and hTR. hTERT was confirmed by tandem mass spectrometry in a ∼120 kDa SDS-PAGE fragment from a TRAP-positive purification fraction. TRAP activity was also supported by hTR constructs lacking the box H/ACA small nucleolar RNA domain. End-point TRAP indicated expression levels within 3-fold of that from HeLa carcinoma cells, which is several orders of magnitude below detection by the direct assay. These results represent the first report of TRAP activity from a bacterium and provide a facile system for the investigation of assembly factors and anti-cancer therapeutics independently of a eukaryotic setting.
authors.affiliation: Medical University of South Carolina, Department of Biochemistry and Molecular Biology, 173 Ashley Avenue, Charleston, SC, 29425, USA.
authors.forename: Debra T
authors.initials: DT
authors.lastname: Hansen
authors.affiliation: Medical University of South Carolina, Department of Biochemistry and Molecular Biology, 173 Ashley Avenue, Charleston, SC, 29425, USA.
authors.forename: Thirumagal
authors.initials: T
authors.lastname: Thiyagarajan
authors.affiliation: Medical University of South Carolina, Department of Biochemistry and Molecular Biology, 173 Ashley Avenue, Charleston, SC, 29425, USA.
authors.forename: Amy C
authors.initials: AC
authors.lastname: Larson
authors.affiliation: Medical University of South Carolina, Department of Biochemistry and Molecular Biology, 173 Ashley Avenue, Charleston, SC, 29425, USA; A.T. Still University, School of Osteopathic Medicine in Arizona, 5850 E. Still Circle, Mesa, AZ, 85206, USA; Barrow Neurological Institute, Phoenix, AZ, 85013, USA. Electronic address: JHansen@atsu.edu.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Hansen
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D019098:Telomerase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D018076:DNA, Complementary; D011994:Recombinant Proteins; C422952:telomerase RNA; D012313:RNA; C509186:TERT protein, human; D019098:Telomerase
keywords: Escherichia coli; TRAP assay; Telomerase; hTERT; hTR