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Soluble domains of telomerase reverse transcriptase identified by high-throughput screening

Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado 80309-0215, USA

(RECEIVED April 21, 2005; FINAL REVISION May 20, 2005; ACCEPTED May 20, 2005)

Telomerase is a ribonucleoprotein complex responsible for extending the ends of eukaryotic chromosomes. Structural and biophysical studies of this enzyme have been limited by the inability to produce large amounts of recombinant protein. Here we perform a high-throughput screen to map regions of the Tetrahymena thermophila TERT (Telomerase Reverse Transcriptase) protein that are overexpressed in a soluble form in Escherichia coli using a GFP-fusion system. Many of the soluble protein domains identified do not coincide with domains inferred from multiple sequence alignment, so screening for fluorescent colonies provided information not otherwise readily obtained. The method revealed an essential, independently folded N-terminal domain that was expressed and purified with high yield and found to be suitable for structural analysis. These results provide a tool for future structural and biophysical studies of TERT.

Keywords: telomerase; GFP; high throughput; TERT

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051532105.

Reprint requests to: Thomas R. Cech, Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA; e-mail: Thomas.Cech{at}colorado.edu; fax: (303) 492-6194.

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