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Soluble domains of telomerase reverse tr ... ed by high-throughput screening
Soluble domains of telomerase reverse transcriptase identified by high-throughput screening.
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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 SA, Podell ER, Wuttke DS, Cech TR
Protein science : a publication of the Protein Society
2005-08-01 00:00
14
8
2051-8
Amino Acid Sequence,Animals,DNA-Binding Proteins,Escherichia coli,Green Fluorescent Proteins,Luminescent Agents,Molecular Sequence Data,Mutation,Nuclear Magnetic Resonance, Biomolecular,Protein Structure, Tertiary,Recombinant Fusion Proteins,Solubility,Telomerase,Tetrahymena thermophila,DNA-Binding Proteins,Luminescent Agents,Recombinant Fusion Proteins,Green Fluorescent Proteins,Telomerase
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA
Protein Sci.
0961-8368
10.1110/ps.051532105
14/8/2051
272
True
16046627
