Soluble domains of telomerase reverse transcriptase identified by high-throughput screening.
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.
Jacobs
Steven A
SA
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Podell
Elaine R
ER
Wuttke
Deborah S
DS
Cech
Thomas R
TR
D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D049408:Luminescent Agents / Q000032:analysis; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012995:Solubility; D019098:Telomerase / Q000737:chemistry* / Q000235:genetics; D016808:Tetrahymena thermophila / Q000201:enzymology
D004268:DNA-Binding Proteins; D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019098:Telomerase