Study for activation of human telomerase reverse transcriptase, as major component of cancer, expressed in E. Coli

Abstract

Telomerase is a specialized RNA-directed DNA polymerase that extends telomeres of eukaryotic chromosomes in most human cancer. To date, little is known about how telomerase is activated and controlled in cancer, although activation is thought to be involved in cancer cell immortalization. Telomerase is composed of two main subunits, the ratelimiting catalytic subunit, human telomerase reverse transcriptase (hTERT), and the integral template of hTERT, human telomerase RNA components (hTR). Also, the hTERT are phosphor-proteins and its phosphorylation is a prerequisite for the activation of telomerase. Thus, phosphorylation of hTERT by protein kinase represents an elemental and essential step in maintenance of telomerase activity. To regulate activation of telomerase, hTERT gene amplified from FLAG-hTERT cDNA was expressed with 6xHis residues in E. coli. The expressed hTERT was identified using hTERT antibody and purified by His-bind resins. Also, hTR gene was amplified from genomic DNA of HeLa cell and gained hTR by in vitro reverse transcription methods. After the purified 6xHis-tagged hTERT and the hTR was reconstitution, the reconstituted complex was phosphorylated. Then, the activated hTERT by protein kinases was detected by PCR-based modified TRAP assay. This study will be useful for developments of drug design targeting hTERT in anticancer therapy. © Springer-Verlag Berlin Heidelberg 2007.