Protein Expression of Tetrahymena Telomerase Catalytic Core With a TER-tRNA Construct

Telomerase is a ribonucleoprotein (RNP) which extends and protects the ends of linear chromosomes. The catalytic core of telomerase in the model organism Tetrahymena thermophilia is comprised of an RNA component (TER), a reverse transcriptase subunit (TERT), and a protein cofactor called p65. Investigations of telomerase structure and function have been hampered by challenges associated with telomerase protein purification, such as the insolubility of TERT. Our lab hypothesized that we could improve TERT solubility in E. coli by coexpressing a chimeric TER-tRNA molecule together with the telomerase catalytic core protein subunits. The purpose of using a tRNA scaffold to express TER is to improve the stability of TER expression in E. coli. Preliminary experiments have demonstrated the feasibility of producing catalytically active telomerase RNP complexes in E. coli using the TER-tRNA scaffold approach, and we are currently trying to optimize this result. Unlike standard telomerase reconstitution methods which employ commercially available rabbit reticulocyte lysates, our E. coli expression system requires the p65 protein cofactor, suggesting our approach more accurately reflects the native telomerase biogenesis pathway. Optimization of our procedure will establish a cost-effective and p65-dependent method for telomerase RNP reconstitution, providing a critical tool for future studies aimed at elucidating the structure and mechanism of telomerase which may contribute to advancing cancer research.