Related Experiment Videos
Soluble domains of telomerase reverse transcriptase identified by high-throughput screening
Steven A Jacobs1, Elaine R Podell, Deborah S Wuttke
1Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Protein Science : a Publication of the Protein Society
|July 28, 2005
Summary
Researchers identified soluble protein domains of Tetrahymena thermophila Telomerase Reverse Transcriptase (TERT) using a high-throughput screen. This method revealed an essential N-terminal domain suitable for structural studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Telomerase is a crucial enzyme for eukaryotic chromosome maintenance.
- Structural and biophysical studies of telomerase are hindered by difficulties in producing sufficient recombinant protein.
Purpose of the Study:
- To identify soluble and overexpressed domains of Tetrahymena thermophila TERT in E. coli.
- To develop a method for facilitating structural and biophysical studies of TERT.
Main Methods:
- A high-throughput screen using a GFP-fusion system was employed to map soluble protein domains.
- Escherichia coli was used for recombinant protein expression.
Main Results:
- Several soluble protein domains of TERT were identified, some not predicted by sequence alignment.
- An essential, independently folded N-terminal domain was discovered.
- This domain was expressed and purified in high yield and is suitable for structural analysis.
Conclusions:
- The high-throughput screening method effectively identifies soluble protein domains for structural studies.
- The identified N-terminal domain is a promising target for future structural and biophysical investigations of TERT.